314****************AO3k - ERIC

ED 209 115

TITLE

INSTITUTION

%--d, REPORT NOPUB DATENOTE

AVIILABLE.FR

4r

DOCUMENT RESUMR-

/s. SE 035.924. . . .

Bacteriological Methods'in Water QualityloControlPrograms. Traihing Matual..-1 .-

Office of Water Program Operaticds (EPA) ,. Cincinnati".1 .

Ohio. National Training and Operational.Technology ,

-.---Ceiter. . 'r '.EPA-430/1-80-004Apr 801590.; For related document, see SE 035 925.'Contains,Occasional small, li4ht, 'aid broken type. ',,

ON EPA, InstruCtiOnal Resources Center, 1200 Chambers ilk

Rd.e3rd.Floor,"Coluhbus, OH 43212 ($1.00 plus $0.031 9 per Page).

.%

.

. .. ....

EDRS PRICE liF01/PC07 Plu.s Postage.'DESCRIPTORS

..,.

IDENTIFIERS

*Biological Sciences; Data Analysis; *Eaviron4ental.-Standards ;*Laboratory Procedures; Postsecondary

Education; *Water Pollution; Water ResourcesAnalytical Methods; *Bacteriology; *water Quality;

-Water Sampling Standards .

ABSTRACT r .. .! 1

.

'-4/ This.training,manual presents gaterial on,basicbacteriologiCaklaboratory proceAures as required by Federal Register"Vetter .QuillIJGuidelines. Course topics include: characteristics, -

occurrences, And significance of bacte4lat indicators of poirution;bacteriological rater quality standards and criteria; collection and'handling of samples; 2aborattory test, procedures; and data analysis.The material 'is designed for students who can perform basicbacteriological...laborktory'procedvres such as: simple inoculations,transfers, leighings, and.related,skills. Chapters include reading,materials and laboratory>actiiities. (CO) . .

.

0

S

1

f

O

Ic

ff.

J ,4

****314**********************AO******3k********;4**#***4*314****10**.1g% 40Pre4uctiows supplied by EDRS are the hest that 'can be lade- ,'

Ag7;:-,. .':'. froi-ihe7Originai dochmeni: *11,44******Iivrt******4ii******t****!4*************ig***#***1!**,***4i*****.******.

,.

. .

, , ., , ,.

4

United States National TrainingEnvironmental Protection and OperationalAgency Techncilogy Centergency

Cincinnati OH 45268

EPA,430/1-80-004April 1980

Water

0

BacteriologicalMethods in WatprQuality controlPrograms P'

Training Manual

q

ti

U S DEPARTMENT OF EDUCATIONNATIONAL INSTITU1'E OF EDUCATION

EDUCAtIONAL RESOURCES INFORMATIONCENTER IERIC/

Xthis document has been` reproduced asreceived from the perS0r, or ORprIlEattonoriginating it

Minor changes have bev41 mode to irrIprove.rer1e0dUCttOrl QUalita,

Points ofvieW or opinions on his docudienr do not necessarily represent official NIEOositIon or policy

7, s-4--IM C/

a1%. kil.----..1111.----alini."1_ 112111-- j11.11_ rIe CC) a._ .1111111' __= _.4-_ __._ __.11111,_ _....1_ .111111- JIIMIll--' __...

L-111(:) --_ 1111111--

11.. _.01.11111--__ 111.=_ ,_..ml-- __IIIIII-- __111111 _:11111_ _...1111- .111._ _.

,

' EPA-430/1-.80-001,'April 1980

1011$1,"

O

4

Bacteriological Methods in Water Quality Control Programs

'1

This course is for labotatory personnel who canperform basic bacteriological laboratory proceduressuch as sample inoculations, transfers, weighings andrelated skills.

After successfully completing the .course the °student will have increased knowledge of allaspects of sampling, analysis and data handlingfor bacteriological samples as required by FederarRegister Guidelihes for effluent Monitoring antiother niter quality program's.

The training incorporates, classroom instructionand activity sessions, student performance of.laboratory assignments and follow-up discussions.

, .

11'. EI4VIROZ141ENTAL PROTECTION AGENCOffice of -Water ProgramOperations

National.' Training and Operational Technology genter

.

010

FOREWORD0

16

These manuals Are prepared for reference use of students en oiled in sRheduledtraining courses-of the Office of Water Program Operations, U. S. EnirironmentalProtection Agency.

Due to the limited .availability of the manuals iris not appropriateto cite them as technical references in bibliographies, on other forInsof publication.

a

References to praciuct6 and manufacturers are for illustration onlY;,such references' do not imply product endorsement by the Offi Ce ofWater Program Operations, -U, S. Environmental Protection Agency.

A- -

The reference outlines in this manual have been selected and developed with agoal of providing the student with a fun.d of the best available current informationpertinent to the subject matter-of the course. Individual instructors may provideadditional material to coverspecialaspects of their own9lesentations.

This manual, will beuseful to anyone who has need for information on the subjectscovered. However, it should be understood that the manual will have its greatestvalue as-an adjunct to classroom presentations, The inherent advantages ofclassroom presentation is in the giye-and-take discussions and exchange of infor-

-mation between and among students and the inst.ructional,staff.

Constructive suggestions for improveMent in the coverage, content, and formatof the manual are solicited and will be given full consideration,.

r,

0.

.9

-

60.

P o

,

r

s

Title or Description Outline Number

11/.

Federal Register qUidelifies Establishing Text Procedureel;for theAnalysis of Pollutant:I * 1

Bacteriological Indicators of Water Pollution 2

Examination of Water for Coliforrri and Fecal StreptococcOs Groups 3

Media and Solutions for Multiple Dilution. Tube Methods 4

.JJse of Tables of Most Probable Numbers

The Membrar Filter inWater Bactericflogy 6

Membrane Enter Equipment and its preparation for Laboratory Use 7

Membrane;Fhtent Equipment for Field Use4

8

-Principles or Culture -31LteaVfor Use with Membrane Filters . 9or

Selection of Sample Filtration Volumes for Membrane Filter. Methods 10

Detailed Membrane Filter Methods 11

Colony Counting on; embrane Filters'-1 t. o.

12

Verified Membrane Filter Tests 13

Collection and Handling of Samples for Bacteriological Examination 14

Testing the suitability'of Distilled Water for the Bacteriology Laboratory 15

Residual Chlorine and Tlerbidit\y------: " 1,6

ti

I'

O

t

Is

'

p

FEDERAL REGISTE GUIDELINE S FOR CHEMICAL ANALYSES

I FEDERAL REGISTER GUIDELINES, .

A Authority

1 In 1972, section 304(g) of Public Lb.w92;500; required the EPA Administra--for to promulgate ,guidelines establishingtest procedures for the analysis of pol-lutants that would include 'the factors.that must be provided in any state certi-fication (section 401)-; or NationalPollutant Disiharge Elimination System(NrPIDES) permit application (section

2

402).

These test procedures are to be usedby applicants to demonstrate that efflu-ent discharges meet applicable pollutantdischarge limitations, and by the statesand other enforcers in routine or randommonitoring of effluents to verify effective-ness of pollution control measures.,

B Establighmenta

Following a proposed listing there was aperiod for reply by interested parties.The first rulemaking was published inthe Federal Register on October 16, 19731

C., CUrrgnt Guidelines

. Proposed amendments and update wei-epublished in .1975. The cUrre t guide-lines were issued in the D ber 1, 19762i -,Federal Register.

D, Format

The "Approved Test Prooedures"a,regiven in a table which lists 115 parameters, 'the methodolpgy to be used to detexminethem and either the page number it standardreferences or else a source v4iere the aria-

a# lytical procedure can be found.' -

4, 1:

r .

II

1 Divisions.

2

The parameter's are listed alphabeticallyincluding four'subcategories of relatedtests:

a bacteria

b metals

c radtoliogical..

d residue

Standard References

Those citedmos.t often as sources of .analytical procedures for the listingsare the EPA Chemical Methods Manual,"Standard Methods4 ASTM and U. S.Geological SurveyP Other sources ofprocedure's are given in footnotes to theTable.

EPA CHEMICAL METlibDS ,MANUAL

A Analytical Procedures .

The EPA Chemical Methods Manual wasdeveloped for their water qua).ity laboratories,using Standard Met,IV and ASTM as basic ;references. In many cases, EPA modifiedmethods.from these sources or else develop-ed methods suitable for their own laboratories.

B Sampling and Preservation Techniques. '

The manual also contains a s ctiorampiing and preservation. Thin is in tabular'form and contains information on'volumesreqtfired for analysis, the type.Of containerthat can be.used, preservation measures andholding times.. The current Federal RegisterreferenCes this Table for recommendationson these*aspects of saniple.tialldling torNPDES/Cirtificatibh purposes.

It

I 7 6

.

r

11

'Federa:1`:Register Guidelines Tor Chemical Analyses

C PreciSion and Accuraey Data .

Predision and accuracy data from inter -laboratory qualityquality colitrol stpdies aregiven for most of Olt methods -cited.

III MgTHODS NOT IN 1976 GUIDELINES

A Application to Use

A system has-been established for' permitholder's to apply for approval to usemethods not listed in the December 1, 1976Federal Register. One supplies reasonsfor using an alternative method to the EPA/Regional Administrator through the stateagency which issues certifications and/Or-permits. If the state doesamot have such,_.an agency, the application is submitteddirectly to the EPA Regional Administfato;.

B Order a Procasing

Befor approving such applications, theRegional. Administrator sends a copy to

'the Director of the EPA Envir'onmentalMonitoring and Support Laboratory (EMSL)for review and recommendation. If theRegional Administrator rejects any appli-cation, a copy is also sent to EMSL. Within90 days the appliCant is to be notified (alongwith the appropriate state agency) of approvalor rejection. EMSL also ..receives a copy ofapproval or rejection notifications for purpOsedof national coordination.

IV REQUIRED. ANALYSES

Which measurements are to he dohe andreported dependon the specifications, ofthe individual certffications or permits.

A Mandel OrY-for Secondary Plants

By ,July 1, .1977all municipal secondary'w.astewater treatment plants will be re-quired to measure1/4;and report pft, BOD5(biochemidal oxygen:demand), suspendedsolids and flow. Many plants are requiredto,repoffifiebe rim).

-1

. A

IN. f

B -Additional. for Secondary Plants

C

Measurements which also may be requiredof secondary treatment plants are fecalconform bacteria, residual chlorine,settleable solids, COD (chemicaloxygendemand), total phosphorus, anrthe nitrogenseries (total Kjeldahl N, NH3-N, NO,; -N,NO 2-N). : .

Municipalities and IndustrieS

Other required analyses depend on localfactors for a municiNlity. Each industryhas requirements pertinent to the processesinvolved.

1 Non-specificgi>

Non-specific measurements to a ssessoverall waster quality might be requiredlike",a,cidity, alkalinity, color, turbidity,specific cdhduc.tance.

2 Organics

Va.Fious organfc"analyses might be relevantsucb,,as totar.organic carbon, organic niiro-gen,phenols, oil and grease, surfactants,pesticides.

IMetals

Specified metals may be of interest.Currently, the Federal Register lists.35 trace metars.tri the.test procedureguidelines.

a-° 4 Others

Cyanide; bromide, chloride, fluoride andhardness are other Measurements thatmight be _required. %

70

V METHODOLOGY AND'SKILS

A Methodology.

Th& analytical methods specifieein theFederal Register for these measurements:range ffom 'rwet" probedures using equip-ment commonly, found in most laboratories,

procAures.re'quiring sophisticatedinsiruments, such as'an organic carbon

.. analyzer or an atomic absorption unit.

0 J

, B Skills

-Federal_ Register GuidelinesFor Chemical-Analyses

-The, degree of' analytical skills requiredto perform the analyses likewise varies,-as does the cost of haying such analysesperformed by service laboratories.

VI OTHER ANALYTICAL CONSIDERATIONS

A Sample

The importanceof securing a represent-ative sample of the type (grab or compo-site) specified by the permit cannot beover-stressed..

B Record Keeping .

Keeping complete and permanent recordsabout the sample is also essential. Suchrecords include conditions when the samplewas collected, chain of custody.signa,tures.and detail's and results of analyses.

C' Quality Control

. Whether the_analyses are done in-house. or by a seevice"labora:tory, an AnalytiCal

Quality Control Program should be estab-lished. Fifteen to twenty percent ofanalytical time (cost) should be given tochecking standard curves for-colorimetry,analyzing, duplicate ,Samples to check pre-cision and analyzing -"spiked samples tocheck accuracy. Recording precision andaccuracy data on quality control charts isan effective method of using such data as ,a daily check on:analytieal performance..This can also be done with'riumbersreported on "blind" samples sent toservice labs.

WI SUMMARY'...

The December 1, 1976 Federal Registerpromulgates guidelines establishing testprocedures for the analysiS of pollutantswhich might be required for certification(PL 92759,0, section 401) or for-NPDESpermits"(PL 92-590, section 402)., Theissue lists page, numbers in standardreferences whererocedFief; can be

4,fotirrd to measurirthe 115 parameters

I

listed.' ft also updates the .regulations forapplication to use methods not cited'in theguidelines. The measurements which mustbe made are -specified by either a stateagencyor by, U. S. EPA. Apparatus andprofessional skills to do the measurementswill vary. Representative samples, corn-plete records and analytical quality controlmeasures are,all necessary elements forpPoducing reliable data.

REFERENCES

1 Federal Register, Vol 38; No 199, Tuesday,Octobei 16, 1973, Title 40, Chapter 1,Subchapter DPart 136, page 28758.

2 Federal Register, Vol 41, No.?232, Wednes-day, December 1, 1976, Title 40, Chapter 1, 'Subchapter D, Part 136, page 52780.

3 Methods for Chemical Analyses of Wafer ..and Wates, 1974, EPA, EMSL, Cincinnati,Ohio. '

Standard Methods for the Examination of. Water and Wastewater, 14th ed., 1976

)* AlItHA, Washington, D. C.

5 Annual Book of Standards, Part 31, Water,1975, ATM, Philadelphia, Pennsynia.

6. Methods' for Collection.andAnalysis ofWater Samples for Dissolved Minerals .and Gases, p. S. G. S. Survey 'Technique of Water - ResourcesInv'entory, Book 5, ChapterU.S. GPO, Washington, D. C.

Thisoutline was prepared by A. I?; Kroner,Ch4mist, 'National Training and OperationalTechnology Center, MOTD, OWN:), USEPA,dincinnati, Ohip 45268

Descriptors: ohemicar analysis, chemicalguidelines, self-monitoring requirements,non-approtred analytical rriethods, NPDES

J1 -3

a

I

4.

a

WEDNESDAY, DECEMBER 1,1976

k

PART II:

ENVIRONMENTALPROTECTION

AGENCY

1 #

WATER PROGRAMS

'Guidelines Establishing Test Procedures

. for the Analysis of Pollutants

a

Amendmads

.44

52780

Title 40Protection of EnvironmentCHAPTER 1ENVIRONMENTAL

PROTECTION AGENCYSUBCHAPTER D-WATER PROGRAMS

[FM 630-41

FART" 136GUIDELINES ESTABLISHINGEST PROCEDURES FOR THE ANALYSIS

OF POLLUTANTSAmendment of Regulations

On June 9,1975. proposed amendmentsto the Q elines Establishing Test Pro-cedures for e Analysis of Pollutants140 CFR 136) w re published in the FED-ERAL REGISTER ( FR 24535) as requiredby section 3021(g of the Federal WaterP011ution Control Act Amendments of1972 (86 Stat. 816, et-seq., Pub. L. 92-500,19720 hereinafter referred to as the Act.

Section 304(g) of the Act requtes thatthe Administrator shall promulgateguidelines establishing test proceduresfor the analysts of pollutants that shallinclude factors which must be provided,in : el) any certification pursuant to sec-?Lion 401 of the Act, or t2) any permit ap-plication pursuant to section 402 of theAct. Such test procedures are to be usedby permit' applicants to demonstrate thateffluent discharges meet applicable pol-lutant discharge limitations and by theStates and other enforcement activitiesin routine or random monitoring of ef-fluents to verify compliance with pollu-tion control measures.

Interested person s were requested to.submit written comments, suggestions, or

objectfons to the proposed amendmentsby September 7. 1975. One hundred andthirty-five letters were received fromcommenters. The following categories oforganizations were represented by thecommenters: Federal agencies` accountedfor twenty-four responses; State agen-cies accounted for twenty-six responses';local agencies accounted for seventeenresponses: regulated major dischargersaccounted for forty-Seven responses;trade and professional organizations ac-cottneed for eight'responses; analyticalinstrument manufacturers and. vendorsaccounted for seven responses: and an-alytical service laboratories accountedfor six res ones. _

All c meats Were carefully evaluatedby a chnical review committee. Basedupon e review-of comments, the follow-ing principal changes to the, ploposedamendments were made:

IA Definitions. Section 136.2 has beenamended to update references. Twentycommenters, representing the entirespectrum of responding groups pointedout that thereferences cited in II 136.2f 136.2(g), and 136.2(h) were out-of-

eittt 44 138.2(f), 136.2(g ),-and 136.2(h),respectively, have been amended to shqwthe following editions of The standardreferences: "14th Edition of StandardMethods for the Examination of Waterand Waste Water;" "1974 EPA Manualof Methods for the Analysis of Water andWaste;" and "Part 31, 1975 Annual Bookof ASTM-Standards."

(B) Identifica.timp of Test Procedures.Both the content and-format of I 136.3,

-.Table I, List of -Approved Test Prom--,

4% .

RULES 5ND REGULATIONS

dui " been revised in response toy-o comments received from

S and oval governments, major regu-la dischargers, professional and tradea ocilitiont; and analytical laboratories.

Table I has been revised by:(1) The addition of a f ourtit. column

of references which includes proceduresof the United States Geological Surveywhich are equivalent to previously ap-proved methods.

(2) The addition of a fifth column ofmiscellaneous rderetiLes to procedureswhich are equivalent to previously ap-proved nietliods.

t3) Listing generically related param7eters alphabeticillly within four subc4te-gories. bacteria, metals, radiological andresidue, and by listing these subcafegorYheadings in alphabetic sequence rel-ative to the remaining parameters:

t4) Deleting the parameter "Algicides"and by entering pie single relevant'algi-cide, ' Pentachlorophenol" by lts chemi-cal name.

(C) Clarification of Test Paraneters.The. conditions r analysis of severalparameters ha een more specificallydefined as a r It of comments receivedby the Agency:

(1) In respon,e to five commentersrepresenting State or local goverrunents,major dischargers, or analytical instru-ment manufacturers, the end-point for.the alkalinity determination is specii1-tally designated as pH 4.5.

(2) Manual digestion and distillationare still required as necessary prelimi-nary steps for the Kjeldahl nitrogen pro-cedure. Analysis after such. distillationmay be by Nessler color comparison,titration, electrode, or ahtoinated pheno-late procedures.

(3).- In response to eight commentersrepresentative of Federal and State gov-ernments, major dischargers" and ana-lytical instrument manufacturers, man-ual distillation at VII 9.5 is now specifiedfor ammonia measurement.

(D) New Parameters and AnalyticalProcedures. Forty-four new parametershave been added to Table I. In additionto the designation of analytical proce-dures for these new parameters, the fol-lowing modifications have been made inanalytical procedures designated in re-sponse to comments.

(1) the ortho-tolidine piocedure wasnot approved for the measurement of

,residual chlorine because of its poot ac-curacy and precision. Its approval hadbeen requested by seven commenters rep-resenting major dischargers, State, Orlocal governments, and analytical instru-ment manufacturers Instead,' the N,N-diethyl-p-phenylenedlaniine (DPI))method is approved as an interim pro-cedure pending more Intensive laboratorytesting. It has many of the, advantages

the orthotolidine procedure such aSlow cost., ease of operation, and also is ofacceptable precision and accuracy.

(2) The Environmental ProtectionAgency concurred with the American DyeManufacturers' request to approve Itsprocedtire for measurement of color, andcopies of the procedure are now availableat the Environmental Monitoring and

Support Laboratory Cincinnati (EMSL-CI;

(3) In response to ree requests fromFederal, State gove ents, and dis-chargers.."hardness," may be measuredas the sum of calcium and magnesiumanalyzed by atomic absorption and ex-pressed as their carbonates.

(4) The proposal to limit measure-ment of fecal coliform bacteha in thepresence of chicane to only the "MostProbable Number" (MPN) procedure hasbeen withdrawn in response to requestsfrom forty-five commenters includingState pollution control agencies, permitholders, analysts, treatment plant op-erators, and a manufacturer of analyt-ical supplies The membrane filtei (MP)Procedure will continue to be an ap-proved technique for the routihe meas-urement of fecal coliflorm in the pre-sence of chlorine. However the MPNprocedure must be used to resolve con-troversial situations, The techniqueselected by the analyst muat be reported,with the data. '

(5) A total of fifteen 4jections, re-presenting the entire spectrum 'cif com-menters, addressed the drying tempera-tures used for measurement of residuesThe use of different temperatures in dry-ing of total residue, dissolved residue andsuspended residue was cited as not alldw-ing direct intercomparability betSteenthese measurements. Because the intentof designating the three separate residueParameters Is to measure separate wasteCharacteristics (low drying temperaturesto measure volatile substances, high dry-ing temperatures to measure anhydrous"'inorganic substances), the difference indrying, temperatures for these. residueParameters musj be preserved.

(E) Deletion of_ Measurement Tech-niques. Some masurement techniquesthat had been proposed have been ode-leted in response to objections raisedduring thepublic comment period.

-(1) The proposed infrared spec-trophotomItric analysis for oil andgrease has been withdrawn. Eleven com-menters representing Federal or. Stateagencies and major dischargers claimedthat ,tha parameter is defined by the

' measurement procedure. Any alterationin the procedure would change the ,clef-inition of the parameter. The Environ;mental Protection Agency agreed.

for sulfide at concentra ihns belo 1(2) The proposed param ter

mg/1, haS been with di. . Meth eneblue spectrophotomotry is now dedincin Table I as an approved procedu forsulfide analysis. The titrimetrlc odineProcedure for sulfide analysis may onlybe used for analysis of sulfide at concen-trations ih excess Of one milligram perliter. ,

(F) Sample Preservation and RoldivgTim Criteria for sample preservationan ple holding times were requestedby sever commenters. The reference forsample preservation and holding timecriteria applicable to the Table I param-eters is given in footnote (1) of Table L

(G), Alternate Test Procedures. Com-ments pertaining to fi 13644, Applicationfor Alternate Test Procedures, includedobjections to various obstacles within

FEDERAL REGISTER, VOL. 41, NO. 2 3/.-WEONESDAY, DiEgEit 1, 1976

these ,procedures for expeditious ap-proval of alternate test procedures. Fouranalytical instrument manufacturerscommented that by limiting of applica-tion for review and,' or approval Of alter-nate test procedures to NPDES permitholders, § 136.4 became an impediment to .the commercial developnYent of new orimproved measurement devices based opnew measurement principles. Applica-tions for such review and/or approvalwill now be accepted ,from any person,The intent of the alternate test pro-cedure is to allow the use of measure-ment systems Aluch are known to beequivalent to the approved test proce-dures in waste water discharges

Applications for approval of alternatetest procedtires applicable to specific dis-

, charges will continue to be made onry byNPDES permit holders, and approval ofsuch applications will be. made on acase-by-case basis by the Regional Ad-

- ministrat,or in whose Region the dis-' charge is made. -

Applications for .oval of alternatetest procedures which) are intended fornationwide use can now be submitted byany person directly to the Director of theEnvironmental Monitoring and Support,Laboratory in Cincinnati. Such applica-tions should include a complete methodswrite-up, any literature references, com-parabtlity data between the proposed al-ternate test procedure and those -alreadyapproved by the Administrator. The ap-plication should include precision andaccuracy data of 'the proposed alternatetest procedure and data confirming thegeneral chpplicability of the test proce-dure to the-industrial categories of wastewater for which it is intended. The Di-rector of the Environmental Monitoringand Support Laboratory, after review ofsubmitted information, will recommendapproval or rejection of the applicationto the Administrator, or he will returnthe application to the applicaht for moreinformation. Approval or rejection of ap -plications for test prikedures intended`for nationwide use will be made by.theAdministrator, after considering the-rec-ommendation made by the Director bfthe Environmental Monitoring and Sup-port Laboratory, Cincinnati. Since_ theAgency considers these'procedures forapproval bf alternate test procedures fornationwide use to be interirti procedures,we will welcome suggestions for criteria

. for approval of alternate test proceduresfor nationwide use. Interested personsshould submit their writtehcomraents intriplicate/on or before June 1, f917 to:Dr. Robert B. Medz. Environmental Pro-tection Technqlogist, Monitoring QualityAssurance Standardization, Office ofMonitoring and Technical Support (RH-680) Environmental Protection Agency,Washington, D.C. 20460.

(II) Freedom of Information. A copyof all public comments, an analysis byparameter of those comments, ancrclocu-

. merits providing further information onthe'rntionale for the changes made inthe final regulation are available forinspection and copying at the Environ-mental Protection Agency Public Infor-mation Reference' Unit, Room 2922,

e

RULES AND REGULATIONS 52781

Waterside Mall, Street, SW.,Washington, D.C. 20460, during normalbusiness hours. The EPA informationregulation 40 CFR 2 prbvides that a rea-sonable fee may be charted for copyingsuch documents;

Effective date; These amendments be-Some effective on April 1, 1977. .

Dated: Naember 19, 1976.JOHN Qt7ARLES.

Acting Adininistrator,Environmental Protection Agency..

Chapter I, Subchapter D; or Titles40,Code of Federal Regulations is amendedas follows:

1. Ili § 136.2, paragraphs tf ), (g), and( h) aye amended to read as follows:

§ 136.2 Definitiont.i.

al "Standard Methods" means Stand-and Methods for. the Examination ofWater and Waste Water, 14th Edition,1976. This publication ,is available fromthe Americana Pul?lic Health Association,1015 18th Street, R.W., Washington, D.C.20030.

(g) "ASTM" means Annual Book' DIStandards, Part 31, Water, 1975. Thispublication is available front the Ameri-can Society for Testing and Materials,1916 Race Street, Philadelphia, Pennsyl-vania 19103.

( h) "EPA Methods" means Methods4for Chemical Analysis ep, Water andWaste, 1974, Methods Develop/Cunt andQuality Assurance Research Laboratory;-

TABLE of app

rarametrand =PA Meth

National Enthronniental Research Cen-ter, Cincinnati, Ohio 45268; U.S. Envi-ronmental Protection Agency, Office ofTechnology Transfer, Industrial Envi-ronmental Research Laboratory, Cincin-nati, Ohio 45268. This publication isavailable' from the Office of TechnologyTransfer.

2. In 1 136.3, \the second sentence ofParagraph (b) is amended, and a newparagraph (c) is added to read as _fol-..lows:

§136.3 Identificaiion of te:t proctsdures.,(b) Under, such circumstances,

additional tea procedures for analysisof pollutants may be specified( by the

Regional Administrator or the Directorupon the recbinmendation of the Direc-tor of thcEnvironmental Monitoring andSupport LabOrab5rY. Cincinnati.

. (c) Under certain circumstances, theAdministrator may approve, upon rec-ommendation by the Director, Environ-mental Monitoring and Support Labora-tory, Cincinnati, additional alternate testprocedures for nationwide use.

3. Table I of § 136.3 is revised by listingthe parameters alphabetically: by adding44 new parameters; by adding a fourthcolumn under references flaking equiva-lent United States Cieologfcal Surveymethods: by adding a fifth column underreferences listing miscellaneous equiva-lent methods; by deleting footnotes 1through 7 and adding 24 new footnotes.to read as follow's:

, .roved teat deciures

Referinces1it74 14th ed, (page nos.) OtherEPA standard approved

methods methods Pt. 31 UBOB inethodx1976 methods 9 7r

AMA

1, Aidity; per CaC4) 9, Eketrometrie ehd point -grams perpter, (ph of L2) or phenol-

..leepitbalsin sad ptabst.2. Alkalinity, as CaCO 1, ZMetrumetric titration, grams per liter. (auk, to pll 4.5) manual

' or automated, or squire-lent automated methods.'

_____

273(4d) 11% 40 "(007)

3 278 111 41 1(407)..

3, Anirnonia Sas N). milligrams Manual distillation (4_01per liter.

1falba, tilratlek9.5) followed by nmakri

ekc 165 .150, 412 237 116

trode. Automated phe-uolate.

BACTERIA

4. OM:7 (fecal)+, nuinber pef T.IPN,; nriliul.rano, filter.137922 ,.

' (45) ... ... .... ..'(eal)(

p

Mtediform. In presenee . . (+ - :of chlorine, number per 100ml.

L Coliform (total); t ntunber per do. s...,

7. Coliform (tota/en in piesenee MPN; membrane Bike., )03 la. of dhlorine, number ba 100 with endeliment.

'a. Fecal streptneoccl,t number 31PN,t membrane Alter;

pez 100 mi. plate count. .

10. 11131acelinitCaTIAAI9g4dtegt7-, Winkler (Aside modifies-Oxidationoolorlmetrit .

5-d (BOW, milligrams Pet llea).er electrode method,' liter.

11. nke, ligrams per liter_ Titrimetrieodin-Ip dafe _-12 Chemical demand Dichromatetflus.

(CO D), mill !grains per liter13. Chloride, milligrams per liter:: Silver nitrate; TIMM* ni-

trator automated oolorimetric-ferriermide.

not.7 at nd of table.A

1420

2931

tFEDERAL REGISTER, VOL 41; NO, 232 -- WEDNESDAY, DECJEMIER I, 11074P

92292e,937

916nes916933

943944947

643

. ..150.

KS100.413

-

s

223472

267365

Vaiii . 8.. ,

-s

' (60)sts

(50)

13458

-.....

.II (46)

-,..

(17)

s onto(17)

(4100-

n

52782

o o

j

0

a,

;'

iRULES AND REGULATIONS

,14.4.2

16. 0 'tor. plaunttm cobalt unitsor dominant wave length,, hue, luminance, purity.

17. Cyanide. total," Mililfcrall'OSPer liter.

ParLeter and units Method

ItSierenees1114 1941s ed. (pegs nos.) \ OtherIPA. alowlsed Wel**

methods methods it 31 t7808 methodsWM methods I

s '-ABTM i

14. Chlorinated organic corn. Car chromatography Itpounds (except pes(icides),milligrams per liter.

15. Chlorine-total residual, milli-grams per liter.

a.

0

18. Cyanide amenable to chiori it-salon. milligrams per liter.

. Diseoived- oxygen. milligrams. per liter. .

30. Pluoride, milligrams per liter .

31. Hardness-Total, as CaCO3,mllligr5m s per liter.

32. 11ydrogen ion (OenIL p unH

33. INdalii nitrog (as N),milligrams per liter.

MICTALI

31. Altuniuttm-Total, milligratnsPee liter.

36. Aluminum-. pissolvod, mlilli grams per liter.

W.' Antimony-Total, milligramsper Mar.

If. Antimony-Dissolved, m1111.-grams per liter.

lodoine trio titration, am per.°metric or starch-iodineendpoint; 1)PD color!metric or Titrimetricmethods (these last 2 areinterim-methods pendingAboratory testing).

Colortmetric; spectrophoto.Metric: or At&Sif pro-cedure.to

llistillation followed bysilver nitrate titration orpyridine pyratolone .torbarbiturie acid) color,-metric.

. .2

Winkler (Aside !noddle*.lion) or electrode method.

Distillation , followed byloll electrode: SPA1)NS;or aUtontatedromplesone,

E DTA titration: auto.Milted colorimetne. orate:Mile atx..orptlOn (sumof ,Ca and Mg as theirrespective.carbonatesb

Etectrotnetrio measurement .Digestion and distillation

followed by netulerizatlon,titration, or , electrode;tuireemited digestion auto-

y ,

digestion to followed ' byatomic absorption to by

'

'

35

36a,

40

49

5156

65.59616870

175165

92

318,,2W9

6466

361

376

443aso

393891

393614202

......

444 .=

-

278

503

505 _

368

807 '

306

161

'".

se

65 .

126

93

94

Al

11 (Il9

.. .... -aP

_0

NA

s(603).

.

- s(617)

........

846121)

of _.,eolorimetrie (EririworulCyanide R). ... 7 -

0.45 micron filtration q fol.'''owed by referenced silent-

0.45 micron filtration IT fol.

Digestion 11 followgd byatomic absorplion.q \

\..meth-

ods for total aluminum.

...

36 Aristlio-Tothl, milligramsPe:4iter.

Ir 39. Arsiiic-oDlasol Ted, milli-grams per liter.

30. Barium-Total, milligramsper liter.

. 31. Barium-llesolved, milli-grams pet liter.

32. Beryllium-Total. milligramsper liter.

33: Iler;lium-..Dissolved, milli-grams Per 1191. ;i

34..Botssen-Total, milligrams per

35. Ytilbstri-DIssidved, milligramsperllter.

36. Cadmium-Total, milligramsper

CadmInin-Diseofved, milli-grams per liter.

IL Calilurn-Total, milligramsper liter.

Caleinm-DiMaed, m1111-

grams per liter. -

40. Chronsitun VI, milligrams per

41. Cluominnt VaeDiasolved,milligrams per.ifter.

42. ChieMlum-Total, milligramsWitter.

10.

lowed by referencedmethod for total antimony.

1"

Digestion followed by silver INdiethylififhiooarbamatego - 9 IN ll illOr atomic &WWI/WM.9 II 66 `190 r

0.45 Micron filtration " f01- .,--

lowed by referencedmethbd for total arsenic.

Digestion " followed byMonde absorption."

0.45 micron filtration " followed,- by referenced

97 152 ... 521

Method foe total bariunt. c

Digestion to folloWed by ' 97atomic absorption n Of WV

Chromium-Dissolved, milli-grams per littr;

See footnotes. at end or tittle.

- colorimetgle (Altfinthon).0.45 micro ri filtration " fol-

lowed by referencedmethod for total beryllium.

Colerimettio (Cureumln)....

0.45 micron filtration ro fol-lowed by referenced-meth.

',o4 foe total boron.

-atomic absorption W or by "4Digestion to followed by 4101 148 345 6:3 4 0119) 0 IX)183

oolorimetrie (Dithleona).0.45 micronliltralion " fol- a1 lowed bkrefereneedmeth-

od foe tdialleidmium.1)1gestion lo i followee-ty 103 .. 148 , $16arc Ion: or -. 186 ,

E lion.0.45 micron Ittation l' fol-

lowed by re rented meth-od for total dun).

Extraction an atomic air .89, 105sorption; co mettle (DI- 193 _phenylear de). . 5

0,45 micron fit lion " AA.lowed by ref , need meth-od for chromitim Vi.

Digestion? fo lowed 'by 105 148 745 , 7$atomic 41.4:ory loaner by ,,,,,,,..2 16$ In 77.

' colotimetric Diphenyi.earbmide).

0.45 micron filtration tf fol.lowedby refers Teti.-od for total ehrol hint,

162177

..... 4

11 387

tM

76TS

12

MEM ItEGISTiii, VOL. 4i, 4-117. 232-WIONISOAi, DECEMUR ), 1976.

T

._

RULES AND REGULATIONS

raipmeter and units,

Itelefeeees1974 14th ed: Owe nos.) . OtherMethod " XPA standge4 Improvod

methods methods Pt. Ill v8 08 methods1971 methods

ASTI(

44. Cobalt-Total, milligrams perliter.

45.,C4balt-Dissolved, mllii-grams per liter.

46. Lipper- Total, milligramsPer liter.

47. Copper -Dissolved, mllligrams per liter.

48. Cold-Total, milligrams perliter.

49. Iridium-Total, milligramsper liter.

60. Iron-Total. milligrains perliter.

51. 4ron-1ssolved, milligram=per liter.

52. Lead-Tool, milligrams perliter.

53. Load- Dissolved, milligramsper liter.

54. Magnesium- Total, milli-grams per liter. ,

55. Magnesium-Dissolved milli-grams per liter.

50. Manganese-Total milligran5sper liter. /

57. Manganese-Dissolved mll11-.grams per liter.

58. 3.fercury-Total, 'milligramspfr liter.

59. Merepry-Dissolved, mill -grffins peril ter.

110. Molybdenum-Total, nal-grams, perUter.

61. Mmybdentunr-Dlssolved,milligrams per liter.

62. atilligrarus,per liter.

Nickel-Nbsolved, mill-'s

'grams per Mee.

K. Osmium-Total, Milligramsper liter.

35. Palladium-Total, milligrams'per liter: t r-

06 PM/intim-Total, millIgratasper liter.

67. Foismitun-Total, milligramspet4iter.

08. Potassium - Dissolved.grants per liter.

.0O.. Ritodir -Total, milligiamspee I ter.

70. Ruthenium - Total, milli-Intl" per liter.

71;Selenium-Total, milligrams

is14. eMlittmLDittsolved, milli-ma per.liter.

fillic&-DlisolVed, milligrants- per liter. . .

74. 8ilver:-Total,14 milligramsper liter

75. Silver.-DimalVed,14 mill.grams per liter.

74. Sodium-Total, milligramsper Met.

, 77. Sodium-Dissolved, mill-grams per liter;

Bee foOtfioten nentl:pf table

"Digestion 11 followed by 107 143 346atomic absorption."

0.4.5icron filtration" fol-lowed by referenced Math-od for total cobalt.

Vigeation 11 followed by 108 .148 345 83 (619)0.(31)atomic absorption 1 or by 106 293 .... ... ....... ...-. .....colortinetric ..(Neocu-Wm I10). -

0.15 micron filtratidn " fol-lowed by referenced meth-od for total copper.

Digestion " followed byatomic absorption."

Digestion 0 followed byatomic absorption," -

Digestion" followed by 110 148 34S al- tomatomic absorption "Or byoolorimetric (Phentintitro- VSline). .

045 micron filtration " fol.'lowed by referenced methdel for total iron.

Digestion" followed by 112 148 / 345 103atomic absorption I, or byoolorimetric (Dithisone). 215

0.45 micron filtration U fol-lowed by referehced meth-od for total lead.

1)Igestion 11 followed by 114 148 345 109 2(619)atomic absorption; or 221gfavtmetrlc.

0.45 micron filtration 11 dollowed by referencedmethod for total magne-sium. ' a

Digestion 11 followed by 116 148 346 111 s (6111)atomic absorption 11 or by 225.227 '. .......... ........ .... .... -oolorimetr)c il'ersulftte orpertodatel.

045 micron filtration" fol-lowed by referencedmethod for total minas- ...IWO.

Flamelem atomic absorp: 118 134 33$ M (51)tion,

,0.45 micron filtration " fol. _lowed by referenced `.1method for total mercury.

Digestion" followed by 139 .......atomic absorption."

0.45 micron filtration 11 fol.lowed by referenced ,method for total molybde-num.

Digestior114- followed'' by -.1.4V - 146 343L atomic absimption " or by

oolorimetric (Heptoxlme).045 'micron. filtration "'fol-

lowed by - referencedmethod for total nickel.

Digestion " followed by ,.. -...".,-..'

atomic absorption."Digestion " followed bye_

..,atomic absorption." . ,rDigestion" followed by

atomic absorption."Digestion 0 followed by 141 tool

atomic absorption. colori . 135metric (Cobsitinitrite), or J14 4041 - ,.. .by flame photometric.

40.45 micron filtration " fol-lowed by referenced meth.of for-total potassium.

Digestion 11 followed byatomic aloorption."

Digestion 11 followed byatomic absorption." e

Digestion " followed. by 14.5.atomic absorption." 0 .

0.45 micron filtration" fol..lowedby referenced meth- .od for total selenium.

0.45 micron filtration" fol- 274 461 VSlowed by- calorimetric(Molybdosilleate).

atomic absorption" or by 241la sow sonDigestion Is, followid by . WI 148

gplorimetrio With/sone),0.45 micron filtration n fol-

lowed by referenced meth=od for total sliver.

Digestion" -followed by 147 . ; 14$ (ON) .atomic absorntitm-,or; by 233 -flame photometric: ...

0.45 micron filtration fol.lowed by referenced meth. ,od for total soditno.

4

OHO

1/1: ......

115

. .

4

150

-r-

.

MOIRA if.GliTit, .VOL. 41, NO. 232-WW41.FSDAY, DitilAtililt 1, On4.

tf

52783

s,

46*

"

1

=

52784 RULES AND REGULATIONS

1

.

Parameter and units

,

79. Tim 11hun-Total, utUllgroinsper liter.

29.grams per liter.

SO. Tin-Tulal, milligrams ixrliter. .

el. Tin-I iia..0-1414.1, Ii1111.ruoislierJiter. S. 7

62. 'fiLanions--Tot..1. nil lgr.,tustom niter.

63. Titaitlitin-1164....14.(1, willt-grum 4.1 liter.

64.Per lit;r.

Vntiatliom- 11141%gramn per liter,

kne Total. milligrams prtiter.

pr*m !s: n \ tburatu. 11.

Men

69 Nitr ite N per

o0. 1111 and cr. .4.e. :Wirt:ran* per

91. (Itr111.6 tOUlt 'Till '1,millignom per liter.

V.:. Organic hitrogengrutns per liter.

:1 ntlbnpllntpfmte te I, twillgrams per liter.

l'entat Illorophotiol,grnmr tier

9.1. l'tst b ales. Mill tgriois Iwrliter.

,e, 1111111grant. per97. 1'1,am/torus reletrtental), ttJlli-

grants per liter.ist l'Iagiphorus: total as

aulligrams per liter.

1141.101J...1C L

119. A Whit -Total, pl. i pt.r .

1974 114th ed.EPA standard

methods methods

Ite6ere1114.4(Pagejlos.) Other------ approval!,

Pt. 14 tlitt hi methods197. methods:

AMIN

-0.4a5t° nnilliiumnnbsqlrligtohnitr .M1-

Digiatio1414 'followed by

laried by referencednteth.oil for total thallitnn.

_149 ...

IngeStiodJs followed by 11465)

atomic alworption.ft 4045 mirror filtration II 101.

hat ed by'refereneed meth:oil for total th i.

Ingestion It !Ohm eel by 151

aWinie sibwaption,It r0.41 nib ion filtration 1/ fol- . . ..'.. ........... ....- ... -

lowetiv referenced meth- ..Oil for total titanium.

I 1 tgect lost is tollowol by 133 ' .;donde altot 110011 It or by . .1. rot 441 11 167).. . ....

... *44.1 uo 111:Olio addl. , 1

041 aileron !titration It tolt . . .... '--10%4140 1)3 irbrvot4.4 israb.0:1 tot !mid vanadium.

ingestion :II, followed by 155 345 159 41619)44(r)Monne absorpt ion Is or by

7Z"

.

I clot Intel de t Ditiliroind.0 45 tnieron filtration II fol. ..-

lowed by h4lretit ,1 meth-od for total ru. '

radinitint reduction. bro.me .111fale; automated. aft ii or hydraYine re.

Mama! sr.0 it., i 1, at ..1401ori. 213 434 .. .. .. 121 ' . ..iii.ty14-41.p.0441 Union)). A

1.1401141114141441 .qtr.s. tom rrs l' 515a Ph int Illormtrillutametlittot,grat mtetrit ,

r 'GmbH:moo- I ohar,.1 236 I 531 467 (I (4). . ..

Kij.n1:11.:17111titrov,11 moms 175,159 :s 431 122 t (612, 414). r

i .

SI WIMI or au(tnituted.44 or.ammonia nitrogen.

249 i 481 384 131. 3 1621)

iljea. it rednef ion. 236 y 624I ;Kt 1 Ilrolotrmrtillit5 I? i 366

4

I 'olurinietra...4AAPi. ... 2411 682 545t f.n. I )1711111.114apItY.144; .. .4 -i

Itote.1 1,.> manual Or IMO.Pormiltate date:410k fol- W 476,481 364.

634133 r (621)

nailed I... .aid, neidrtituo.,iiii. (

I. ........... ,

201 1123

e 197 - y427 35907 :620 ...

. ......119 4(614) 14(24)

100. Alpha-Coupling error, netper liter.

101. Beta-Total, pt per liter_...102. Beta-Com ii.g error, pet per

liter.103. 'a) Ita.111011;Total, pel per

liter.41.1 3,3 Htt, VI per liter.. ..

ttr. to(

104. Total. milligrams per liter ...105. Total dissolved itilterable),

Milligrams pellter.106. Total suspended (nonfilter.

able), milligrams per liter.107. SettleaMt, milliliters per liter

or n11114E411,11.4 per Ilter.108. 'rots.' volatile, milligroms per

liter..-16). $4404.111e ertiolitetance, mhro.

nth% per centimeter at 23°*

110. smut. (as 81),1, milligram*ler liter.

11). Suit'ete rty. it', milligrams Perr.

.4

112. bugle. (as bit;',,

per liter.113. Surfactants, mIllIgrathe per

114. Teninenttur., :141'24% C .....

113. Turbidity, NT(.7

I'loportoo 44. MI 111O1:4 /II ... . 448r.

l'roport bond eoutiter.......

...........oniter.

:rat inietrie. '(ato 1064 I!...Oliva nil. r littratioo, ler C.

flier litiration;103 to10.14

Volitiii.tric or graInietrie ....-1,ravista tro. 550,1% 172

270266

262

39111*(73+75)

648, 694 e

646 )646 6061

"(73+78

661 641

667 st (II) . .

91 -'92 .94

93

'93

It lota.too,4 bridge twinkle. :76 - 71 120 148 4(606)Jiateirl.

4;r1111..trir; turbitlitnetrie; .4 493 424or automated calorimetric 277 490 425

:gig.

(barium chloranilatt). 2794

Titrimetrio-lorline for lev. 284els gnoute Hum 1 tug per 503

154

met; MetItyletto blue plus. .tomot r1c.

Thrlinetrle, lodinelotiate... 285 304 433

Colorillietric (1441bylena 137 600 494 38(11)

blue).ralibrated glass or electro- 286 . 125 *(11).--..

mettle thermometer. 1Nephelometrie. A 295 132 221

Rfeoninivolatfoint for Sampling am! proservailan of Aniples aecortling to parameter Melearod MY be found N'Method: for rhumba' Apaly.849 of Water and Waste., 1574' U.13. Environmattal Protection Agency, table 3, pp.viiisit: , -

)

4,

MIRA REGISTER, VOL. 41, NO. 232-- WEDNESDAY, DECEMBER 1, 1974

14

9

a

r dap

4

A

1

ti RULES AND REGULATIONS 52785

I MI pale references for UsGS methods, unless °Merit notedrare to Brown, E., Skougstad, M. W and Fish-man, M. "Methods for Collection and Analysis of Water Samples for Dissolved Minerals and (lases," U.8. Ocologl-cal SurveiTechniques of Water-Resources Inv., book 5, oh. Al, (1970).

s EPA comparable method may. be found on ludicated page of "Official Methods of Analysis of the Association ofOfficial Analytical Chemists" methods manual, 12th ed. (1975).

Manual distillation is not required if comparability data on reprnsentritIve effluent samples are on company fileto show that this preliminary distillation step is not itecsary, however, mama' distillation willbe required to resolve

any controversies.method used must be specified. . '

6 The 5luboMPN is used.o I Slack, KW. and others, "Methods for (*oiled eau and Analysis of Aquatic Biological and atircoblological Samples;

V S Geological Survey Techniquee of WaterResources Inv. bodta ch. A4 (1973)."Since the membrane niter technique usually yields low and variable recovery from chlorinated wastewaters, the

.11P.,t4 method will be required to resolve any controversies.Adequately tested methods for banalities are not available. Until approved methods are available the following

interim method can be used for the estImanon of benzidillk (I) "Method tor Iteauldine and its Salts in Wastewaters,"available from Environmental Monitoring and Support Laboratory, U.S. Environmental Proteetion Agency, Clip

§ 136.5 [Amended]5. In § 138.5,paragraph (a) is amended

by inserting the phrase "proposed by theresponsible person or firm making thedischarge" immediately after the words"test procedure" and before the periodthat ends the paragraph.

8, In f 138.5, paragraph (b) is amendellby inserting in the first sentence thephrase "prOposed by the responsible per-son or fl4m, making the discharge" int-mediately after the words "such

Oltio 45288.o American NationabStaiidard on Photographic ProRessiug Effluents, Apr. 2, 1975. Available front ANSI, 1430 tion" and immediately before the comma.

Broadway, New York, N V.II Fishman. M. J. and 13rdwn. Eugene:, 'Selected Methods of tho U.S. Ceologuall Survey for Analysis of Waste-

waters," (1978) openIlle report 76177.n l'rpoedures for pentachloropheual. dike mated.organic compounds and isestleides can be obtained from the E n-

vironnfental Monitoring and Support Lbaomtory, U.S. EnvirounmatalProtection Agency, f incilinall, Ohio 4.5288IS Color method ()LAM( procedure) available from Environmental Monitoring and Support Lbaoratory, U.S.

Environmental Protection Agency, Offichtnati, Ohio 45388,is Igor samples suspected of having thlocyanate interference:mem:Slum chloride is used as the digestion catalyst

lit the approved teat prdeedure for mnddes:the reconunended catalysts are replaced with 20 ml of &solution of 5100magnesium Chloride (MgC1,411:0). This substitution a ill eliminate tide( yunate interference for both total cyanideand cyanide amendable to ehlarluation measurements.

Is For the determination of total metals the sample Is not filtered before 'processing. Because vigorous digestionprocedures may result in a loss of certain metals through preciptation. n less vigorous treatment is reconunended asgiven on p. 83 (4.1.4) of "Methods for Cliomical.Annlysis of Water and Wastes" (1974). In those instances where amore vigorous digestion Is desired the procedure on p.82 (4.1.3) should be followed. For the measurmnont of the noblemetal series (gold, iridium, osmium, palladiuth, platimum. rhodium and ruthenium), an aqua regla digestion Is to besubstituted as follows Transfer a represeffittive aliquot of the weimIxed sample to a Uritlin beaker and add 3 nilof concentrated rethstilled HMOs Place the beaker on a steam bath and evaporate to diyness Cool the beaker andcautiously- add a 5 ml portion of aqua ulna. (Aquaregiats prepared immediately before use by carefully adding 3volumes of concentrated lilt to one voluble ofemacentrated HNO,.) Cover the beaker with n watch glass and returnto the steam bath. Continue heating the coveredteaker for 50 min Remove cover end evaporate to dryness. Cooland take up the residue in a small quantity of 1 1 MCI Wash down the beaker walls and watch glass with distilledwater and alter the sample to remove silicates and other Insoluble ma erial that could clog the atomizer Adjust the

cell%voila e to some predetermined value based on the expected metal con hoe The sample Is now ready for analysis.

to he approved test methods. Methods of standard addition are to I fellowedas not ed to p. 78 of "Methods for .Clieni-1. As the various furnace devices (nameless Atilisre easentiallyilionil, bsorpt ion techniques, they are considered

teal Analysis of Water and Wastes," 1974.r Dissolved metals are defined as those constitutents which will pass Moult n 0.45 pm rim maw filter A pre.

nitration is permissible to free the sample from larger suspended solids Filter the sample . s $0011 as practicalaftereallectIon a.ag the first 50 to 100m1 to rinse the alter flask. (Glass or plastic filtering aPpanitus are recommendedto avoid possible contamination.) Discard the portion used to rinse tbf flask and collect the required volume offiltrate. Acidity the filtrate with 1.1 redistilled IINO, to a pll of 2. Normally. 3 nil of it,: li acid per liter should besufficient to preserve the samples.

Is See "Atomic Absorption New stet ter," vol. 13, 73 09741. Available (rout Perkin-Elmer Corp., Maid Ave , Norwalk,Conn. 05852.

Is Method available from Environmental Monitoring auel,Support Laboratory, U.S. Environmental ProtectionAgency, Cincinnati, 01116 45288.

,

r* Recommended methods for the analysis of silver in Industrial 'wastewaters at concentratiens of 1 ing/1 andabove are inadequate where silver exists as an inorganic halide. Silver halides such se the bronlide and chlorideare relatively insoluble In reagents sttch as nitric acid but are readily Soluble in an aqueous buffer of sodium t °-sulfate and sodium hydroxide to a pH of 12. Therefore, for levola of silver above 1 mg /1 20 int of sample should be -, person or firm making the discharge,"diluted to 100 ml by adding 40 ill each of 2M Naffis()) and 2M NaOH. Sterulards should be prepared in the same

The second sentence of paragraph (b)is amended by deleting the phrase"Methods Development and Quality As-surance Research Laboratory" immedi-ately after the phrase "State PermitProgram and to the birector of the" atthe end of the sentence, and inserting inits place the phrase "EnvironmentalMonitoring and Eiupport Laboratory,Cincinnati."

7. In § 136.5, paragraph cc) Is amendedby inserting the phrase Iproposed by theresponsible person or firm making thedischarge" immediately after the phrase"application for an alternatetest pro-cedure" and immediately befgre thecomma; hnd by deleting the phrase"Methods Development and Quality As-surance Laboratory" immediately afterthe phrase "application to the Directorof the" and immediately before thephrase "fbr review and recommenda-tion" and inserting in its place the phrase"Environmental Monitoring and SupportLaboratory, Cincinnati."

8. In § 136.5, the first sentence of para-graph id is amended by inserting thephrase, "proposed by the responsible

manner. For levels of silver below- 1 mg/1 the recommended method Is satis &dory.xi An automated hydrazine reduction method Is available from the Environmental Monitoring and Support

Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268.11 A number of such systems manufactured by variants companies are considered to be coMparable in their lid.,

(armistice. In addition, another technique, based on combustion - methane detection is also acceptable.Goerlits, D. Brown, E.. "Methods for Analysis of Organic Substances in Water", I' Cleologleal,Survey Tech-

'nestles of Water-Resources Inv., book 5, A3 (1972).zt F. Addison and R. O. Ackman. "Direct Determination of Elemental Phosphorus by Cas Liquid Chrome

tography." "Journal of Chromatography," vol. 47, No. 3, pp. 421-426, 1970.U The method found on p. 75 &attires only the dissolved portion aisle the umtlwd oil p. 78 measures only in:.

'pended. Therefore, the 2 results input be added together to obtain "total."w Stevens, H. 11., Ficke, J. F., and Smoot, (7'. F.. "Water Temperature.-Infinential Factors, Field aleasuremeat

and Data Presentation. l S. Geological Survey Techniques a1N aka Itetannees I mmv,, beak 1 t1975) "

4. °In § 136.4, the' second sentence ofParagraph. (c) is amended by deletingthe word "subchapter" immediately fol-lowing the phras "procedure under this"and immediately ' preceding the word"shall" and replaced with the phrase"paragraph c;". and § 138.4 is amendedby adding- a new paragraph (t) to read

%Its follows:

1 136.4, Application for alternate testprocedfires. .

(0 4 Any application for an alter-nate test procedure under this paragraph(c) shall:

(d) An application for approval of analternate test procedure for nationwideuse may be,made by letter in triplitateto the Director, EnvironmentAl Monitor-ing and Support Laboratory, Cincinnati,Ohio 45288. Any application'for an alter-

s

nate test procedure under this 'Paragraph

11) Provide the name and address ofthe responsible person or firm makingthe application.

(2) Identify the pollutant(s) or pa-rameters) for which nationwide ap-,f)roval of an alternate testing 'procedureis being requested.

(3) Prdvide a detailed description ofthe proposed alternate procedure, to-gether with references to published orOther studies confirmitg the general ap-plicability of the alternate test procedureto the polltitant(s) or parameter(s) inwastewater discharges from representa-tive and specified industrial or Othercategories.

(4) Provide comparability data for theperformance of the proposed alternatetest procedure compared to the perform-ance. of the approved test procedures.

15

immediately after the phrase, "applica-tion fpr an alternate test procedure,"and immediately before the comma.

The second sentence of paragraph (d)'is amended by deleting the phrase,"Methods 'Development and Quality As-surance Research Laboratory," imniedi-ately after the phrast, ':to the RegionalAdministrator by the 'Dli.ector of the,"and. immediately preCeding the periodending the sentence and inserting in itsplace the phrase, "Environnierital-Moni-taring 'and Support. Lahqtatory,' CI ill-cinnati."

The third sentence of paragraph (d)is amended by deleting the phrase,"Methods Development and Quality As-surance Research Laboratory," immedi-ately after the phrase, "forwarded to theDirecto; and immediately before 'thesecond comma and by inserting in itsplace the phrase, "Environmental Moni-toring and Support Laboratory, Cin-

ecinnatl."9. Section 138.5 is 'amended by the

addition'of a new paragraph (e) to readas follows:

, .

MENAI. MISTER,, VOL. 41, NO, 232 - WEDNESDAY, DECEMSER 1, 1976

"

I.,

_RULES AND REGULATIONS

1136.5 'Approval of alternate test pro:eedures.

(e) Within ninety days 'of the, receiptby the Director of the EnvironmentalMonitoring and Support Laboratory,Cincinnati of an application for analternate test procedure for .nationwideuse? the Director of the Environmental

... Monitoring and Support Laboratory,Cincinnati shall notifyrthe applicant ofhis recommendation to the Adininis-trator to approve or =reject the applica-tion, or shall specify additional inforraa-tion which is required to determinewhether to approve the proposed test A

procedure. After such notification, analternate method determined by the Ad-ministrator to satisfy the applicable re-quirements of this part shall be approvedf or' nationivide use to satisfy the require-ment,s of thii.subchapter; alternate testProcedures'/, determined by the AdminisAtrator not to meet the applicable require-irrents of t his part shall be 'elected.Notice of these determinations shall be .subinittedioI publication in the FEDERAL t,

REGISTER no later than 15 days aftersuch notification. and determination ismade.

[PR Doc.78 -35082 Filed 1140-76;8:45 am]

;

.11111111AL IiGi

16

232.N.WIDNISDA; WM* 1. iq4

1

r-

, ar

13Ap)TER,IOLOGIC.AL INDICATORS OF WATER POLLUTION

Part 1. Cleperal Concepts

t

A BacterialIndication of Pollution

1 In the broadest sense, a bacterial, indicator of pollution is any Organism

.-t4.4..which, by, its,presence; Wouldstrate that pollution hts occurred, andoften suggest the source of the pollUtion.

2 In a more restreetilig sense, bacterialindicators of pollution are associated,primarily with demonstration of con-tamination of water, originatingfromecreta,of warm-blooded.animals

. man, domestic and wildanimals, and birds). -

B Implications of Pollution of Intestinal

'1 Intestinal wastes from warn- bloodedanimals regularly include a widevariety of genera-and Species ofbacteria. Among these the coliformgroup may lob listed, and species ofthe genera Streptococcus, Lactobacillus,Staphylococcus, Proteus, Pseudomonap,certain spore4orming bacteria, andothers. ,

2__In addition, many kinds of path' ogenic"baCteria and other microorganismsmay be released in wastes on 'arilnier-

.raittent basis, varxing withAhe geo-"grEiph4c area, state of community

nature and degree of wiete"itres.txnent, and other factors. These

1.may include the 'following:.

a Bacteria: Species Of Salmonellae'Le2tosgira, Brucelia,

Mycobacterium,, and Vibrio'comina.'0,V40)-

Viruses: A wide variety, includingthat of infectious hepatitis,- Polio-viruses, COntsaekie virus; ECHO.viruses (enteric cytopathogenichuman orphan -- "viruses,in search ,

of a disease"), and unspecifiedviruses postulated to account foroutbreaks of diarrheal and upperrespiratory diseases of unknownetiology, apparently infective bythe water-borne route.

Protozoa: 'Endamoeba histolytica

3 As routinely practiced, bacterialevidence of water pollution is a teatfiar thelpresence and numbers ofbacteria-in wastes which, by their

. presence, indicate that intestinalpollution has occurred. In this con- .

indicator groups discussed insubsequent parts of this outline areas follows:

a Coliform group and certain sub-. 'groupings .

--13 Fecal streptococci and certainsubgroupings

c Miscellaneous indicatolof water'___ qdaelity .

4 Evidence, of water contamination byintestinal wast s' of warm-blooded 'animals i's reg ded as evidence ofhealth hazard the water being tested.

II PROPERT4F AN IDEA L INDICATOROF POLLUTI

A An "ideal" bacterial indicator of pollutionshould:

applicable in all types of water

2-,1

Bacteriological Indicators of Water Pollution

2 Always be pie,sent in water'whenpathogenic bacterial constituents afecal contamination are present.Ramifications of this include --

a Its density should have some directrelationship to the degree of fecalpollution.

b It should have greater survival timein water. than enteric, pathogens,throughoUt its course of 'naturaldisappearance from the water body.

a It should disappear rapidly from,water following the didappearanceof pathogens, either through naturalor man-made processes:It always should be absent in abacteriologidally safe water.

33 Lend itself to routine quantitative,testing procedures without interferenceor confligion of results due to extra-,

neous bacteria '.

4 Be Harmless to man and other animals

B In all probability, ah,1a31ear bacterialindicator does not exist: The discussion

-"Jr-r of.bacteriai indict tais ofpollution in thefollowing parts of his outline i cludeconsideration of the merits an limitationsof each group, with their applio 'ons in,evaluaticterial quality of--water.

III .APPLICATIONS OF TESTS FORPOLLUTION INDICATORS

A Tests for Compliance with BIcterialWater 'Quality Standar.ds 1

1 Potability tests on drinking water tomeet Interstate Quarantine or other.standards of regulatory agencies.

Z7 Determination of bacterial quality ofenvironmental,Water for which qualitystandards may exist-, such ea.-shellfish,waters, recreational waters,' water

_- resources for mun icipal or'other, supplies.

¶5

3 Tests for complianch.with eiitablished'standards in cases involving the iSio- ,

tection or prosecution of municipalities,industries, etc. Si

B Treatment Plant Process Control

1 Water treatment plants

Wastewater treatment plants

C -Water Quality and Pollutant Souice Monitoring

1 Determination of intestinal pollutionin surface. water to determine type andextent of trRatment required for. com,pliance with standards

2 Tracing sc4rces of pollution

3 Determinat n of effects on bacterialflora, due t cldition of organic orother wastes

4

D Special Studies, such as

1, Tracing sources of intestinal pathogensin epidemiological'investigations

2 Investigations of problems due to theSphaerotilus group .

3 Investigations of bacterial' interferenceto.certain industrial procesdes,,vithrespeqt to such organisms as Pseudo-monas, A chromobacter, or others

IV - SANITARY SURVEY-

The laboratory bacteriologist is not alone Inevaluatien of indication of water pollution ofintestinal origin. ,.,Onrsite study (SanitarySur-Vey) of the aquatid environment and ;.adjacent areas, by a qualified person, 'is anecessary collateral study with the laboratorywork and frequently will reveal'informationregarding.potential bacteriological hazardwhich may or may not demonstratedthrough-laboratory findings fitoln a' single %1

sample or short series of samples. ..

'It


I

Part 2.. The Coliform Group and Its Constituents

4

I ORIGINS AND DEFINITIONA

A Background.

1 In 1885,. Escherich, a pioneer ba cteri-ologist, recovered certain bgrcteria fromhuman feces, which he found in suchnumbers and consistency as:to lead himto term these organisma 'Ithe charac-

t,teristic organism of human feces. ".

. He named these organisms Bacteriutncoli-commune and B. lactis aerogenes.

. In 18iW',7).nother bacteriologist,Mitula, renamed B:. coli commune as

&Eserichia cola, which today is theofficial name for the type species.

2 Later Work has substantiated much ofthe origfnal conceptof Escherich, but.has shown_that the:above species are .in fact a heterogeneous complex ofbacterial species and species variants.,

0 a This heterogeneous group occurs not-only in human feces but representativesalso are to be found in many environ-mental media, inclirding sewage;surface freshwaters of all categories,

-in and on soils, vegetation, etc.',b. The ,group may be subdivided into

various categories on the basis of-numerous biobhemicall and other .

differential tests that may be applied.

B Composition of the COliform Group

1 Currant definition..

'Ai defined in "Standard Methods for theExamination of Water.and.Wastewater"(14th ed): "The coliform group: includesall of the aerobic and facultativeanaerobic, Gram-negative; nonspore-forming rod-shaped bacteria whichferment lactose with gas formationwithinwIthin 48 hours 'at 350C. "

2' The term vcoliforms" or "coliformgroup" is an inclusive one, includingthe ibllowing bacteria which maymeet the definition above:

a Escherichia coli; lilk"riurescens,E. freundii, E..infermedia

b Enterobacter aerogenes, E. cloacae

c- biochemical ,intermediates betweenthe genera Escherichia and Entero-bacter

3 These is no provision in the definitionof coliform bacterila for "atypical" or"'aberrant" coliform strains.a An individual strain of any of ihp above

species may fail to meet one:Of thecriteria of the coliform group.

b Such an organism, by'Aefinition, isnot a member of the coliform group,even though a taxonomic bacteriologistmay be perfectly correct in classifyingthe strain in one of the above species.

\ID SUBDIVISION OF COLIF6RMS INTO

"FECAL" AND "NONFECAL" .CATEGORIES

.

. A Need,"

Single-test differentiations betweencoliforms Of "fecal" origin and those of

--"nonfecal" origin are based on the.as.sumptioh that typical E. Colkandclosery related strainsiiie of fecal-origin while E. aerogenes and its closerelatives are not of direct fecal origiti:(The latter /assumption is not fully borne'out by, invesSee Table.number of

ons at-this 'Center:IMViC Type --H).' Agle differential tests have

been proposed to differentiate between"fecal" and "nonfecal" coliforms.

'1

tiBacteriological Indicators of Water Pollution

Without discussion of their relative Merits,.several 'may be cited here: 1

.

, c

B 'Typed of Single-Test Differentiation

Determination of gas ratio

Fermentation of glucose by E. coilresults in gas production, withhydrogen and carbon dioxide beingproduced in equal amounts.Fernientation of glucose by E.

,aerogenes results in generation oftwice as much carbon dioxide ashydrogen.

Further studies suggested absolutecorrelation between H2/CO2 ratiosand the terminal pH restating fromglucose fermentation. This led to thesubstitution of the methyl red test.

2 Methyl red'test

Glucose fermentation by E. colitypically results in a culture mediumhaving terminal pH in the range 4.2 -4. 6.(red color ayositive test with theaddition of methYl.reci indicatorl)..EE. aerogenes typically result's in -aculture mecjium having pH 5..6 orgreater (yellow color, a negative test).

3 Indole1

When tryptoishane, an amino acid, isincorporated in a nutrient broth,typical E. colt strains are capable ofproducing indole (positive test) *Ongthe end products, whereas E. aerogenesdoes not (negative test).

.

In ievieNsiring technical literature, theviorker should be alert to the methoduselo detect .indole formation, as'theresults may be reatly influencedbythe analytical procedure.

..-

4 'Voges-Proekauer test (acetyltnethylcarbinol test)

The test is for detection of dcetylmethylcarbinol, a deriyative of 2,3, butylene-.

glycol,f as a ref:alit pf glucosefermentation in'thepresence ofpeptone. E. aerogenes producesthis end uct (positive test)wh . coli gives a negative test.

a, .Experience with conform culturesgiving a positive test has shown aloss of this ability with storage. onlaboratott media for 6 months to21years, in 20 -*25% of cultures(105 out of 458 cultures).

b Some workers consider that allcoliform bacteria produce acetyl-,,

a methyl Ca'rbinol in gl cose metab-olism. These worker regardacetylmethyl carbinol -n' gativecultures as those which have ,

enzyme systems capable of furtherdegradation of acetylmethylcarbine to other end products *r.-

which -db not give a positive -testwith the analytical procedure".Cultures giving a positive test foracetylmethyl carbinol, lack thisenzyme system.

c This reasoning leads to allypothesis(not experimentally proven) that the'change of reaction noted in certain*,'cultures in 4.a above is.cfue, to the .activation of a latent enzyme system.

5 Citrate utilization41-1;

Cultures of E. colt are unable to usethe carbon oin their rnof E. aero

citrates, negative test)s?h, whereas cultures

enes are- capable of usingthe carbon of citrates in their metab-olism (positive test).

Some workers (using Simmons-Citrate:?Agar) incorporate.a pH indicator(brom thymol blue) in the culturemedium in order to demonstrate thetypical alkaline reaction -(pH 8.4 - 9.0)resulting with citrate 'utilization. '

;6 levated temperature (Eijkman) teat

a The test is basecron-eVidence thatE. soli and other coliforms of fecal

r14.,L

4 0,

.4

v

K

4origin are capable of growing andfermenting carbOhydrates (glucoseor lactose) at temiferatures signif-Jean higher than the body tem-perature of warm-blooded animals.Orgaiisms not associated with-directfecal origin would give a negativetest result, through their inabilityto grow at the elevated temperature.

b While many media and techniqueshave been proposed., EC pro% amedium deVelopecl by Perry andfiajna, used as a confirmattir4 -

medium for 24 hours at 44.50,2°C.. are-the current standardmedium and method.

'While the "EC" terminology' of themedium suggests "E. -colt' theworker should not regard this as a

./ specific procedure tor isolation ofE. coli.

o A similar medium, Boric AcidLactose Broth, has developedby Leyine and Ms associates, Thismedium gives results virtually'id ntical with those obtained fromEC roth, but requires 48 hours of,inc tion.

cl Elevated temperature tests requireincubation in a water bath. StandardMethods 14th Ed. requires thiiitemperature to.be 4.5 ± O. 2el&.Various workers have urged use of

,temperatures ranging between'4340C and 46.00 Most of theserecommendations have provided atolerance of l 0.50C from th-e'rec-onunended l ,However`,. someworkers,

ofin the Shellfish

Program of the Puhlic Health Service;stipulate a tenipprafure of 44.5. t0.2crc. This requires use qfst waterbath with fOrced circulation-to main-

"4

Z.

Bacteriological Indicators of Water Pollution.. e

S.

.

fain this close tolerance. Thistolerance range was institutedin the 13th, edition of Standard Method

d the laboratory worker shouldorm to these new limits.

e The relidleility of .elevated temper-ature tests is influenced 6ythetime required for the newly-

.. inoculated cultures to reach thedesignated incubatiOn temperature.

4.Critical workers insist on place-ment of the cultures in the water

.bath within 30 minutes, at most,after inoculation! . '

(117 Other tests

. ,eNumerous other tests,for differentiation

petween.coliforms of fecal vs. nonfecalorigin have been proposed., Currentstudies suggest .little promise for they,following tests in, this application:uric acid test, cellobiose fermentation,gelatin liquefaction, production of

. hydrogen sulfide, sucrose fermentation,and tthers,

C IMViC Clasaificatio;

1 In 1938, Parr reported on a.review of **

'a literature survey on biochemical testsused to differentiatebetWeen coliformsof fecal vs. nonfecal origin. A summaryfollows: .

sTest

Voges-Prosrcauerreaction

Methyl red testCitrate utilizationIndole test .

Uric'acid test

Q

CellObioie fermentation

Gelatin liquefaction

f Eijkman test t

Hydrogen sulfide\production

" Sucrose fermentation.a-Methyl- d- glucoside

fermentation

s

No. of timesused airfer eiitiatia

22

20

20

15--

6.

4

3

2

'11

<

4.


2 Based on this summary and on his ownstudies, Parr recommended utilizationof a combination of tests, the indole,methyl red; Voges-Proskauer, and the,citrate utilization tests for this differ-entiation. This series of reactions isdesignated by the mnemonic "IMViC",:.Using this scheme, any conform culturecan be described by an "IMViC Code"according to thp reactions for eachculture. Thus, a typical culture ofE. coli would have a code and atypical E. aerogenes culture wouldhave a, code -1-14.

3 Groupings of coliforms into fecal,nonfecal, and intermediate groups, .as shownsin "Standard Methods for theExamination of Water and Wastewater"are shown at the.bottorn of this page.

D Need for Study of Multiple Cultures

All the systems used for differentiation ,

between coliforms of fecal vs. those ofnonfecal origin require isolation and study°of numerous pur cultures. Many workersprefer to-study qt least 10;0, cultures from

_.--any-environmental source before. attemptingto cattgbilizeth(e probable source of theconform*

O

. 2-6

.

tai

0

4

'OIL" NATURAL DISTRIBUTION.OF COLIFORM.BACTERIA

A Sources of Background Information

Details of the'NchumKota Icground oftechnical inforpiation on conform bacteriareCovered'4om one or more environ-mental m&dia are beyond the scope of

dfscussion: 'References of thisoutline are suggested routes of entryfor workers seeking-0, explore thistopic. °/>,,,,

B Studies do Coliforin1)istribution

Since 1960 numerous workers

\.1. ; .

have engaged in a continuing study ofthe natural distribution of conformbacteria and an evaluation of pro-cedures for differentiation betweencolifoims fecal vs. probable non-fecal origin. Resulti of this workhave special significance because:,

a Rigid uniformity of laboratorymethods 'have ;been applied through-out the series of studies

b , Studies'are based on massivenumbers .of cultures , fir beyond

'any, similar-ittidies heretoforereported

- INTERPRETATION OF IMV14C REACTIONS .

organism Indole ReYl 1YrWiltsailer Citrate

Escherichia coliCitrobacter freundiiKlebsiellaEnterobacter group

0+ pr -

+ Or - +

00

r

22;

.0

1

Li*


c A wider variety of environmental andbiological sources are being studiedthan in any previous series of reports.

d All studies are based on freshlyrecovered pure culture isolatesfrom the designated sources.

e All studies 'are bayed' on culturesrecovered from the widest feasiblegeographic range, collected at allseasons of the, year.

2 Distribution of coliform types_ .

Table 1 shows the consolidated resultsof conform distributions from variousbiological and environmental sources.

a The.results of itiese studies -show a- high order of correlation between

known or probable fecal origin: andthe typical E. coli IMViC code(++---). On the other hand,human faces also includesnumbers of E . aerogenes and otherIMViC types, which some regard as"nonfecal" segments of the coliformgroup. (Figure 1)

b The majority of coliforms attributableto excretal origin tend to be limitedto a relatively small number of'thepossible IMViC codes; on the other .

hand,= conform bacteria recovered'from undistnrbed soil, vegetation

.a ect life represent a widerra e of IMViC codes than fecalso ces, withauf clear dominance? ofany one type. (Figure 2)

c The most prominent IMViC code.from nonfecal sources is the inter-mediate type, -+-+, which accountsfor almost half the coliform-cultures-recovered from soils, and a sigh

. percentage of those recovered froinvegetation and from insects. Itwould appear that if any coliformsegment could be termed a "soil'type" it would be IMViC code -4-+.

_

r

P-

d It should not be surprising thatcultures of typical E. con arerecovered in relatively, smallernumbers from sourcesludged,on the basis of sanitary survey,to be unpolluted. There is noknolkn way to exclude the influenceof limited fecal pollution from smallanimals and birds in such environ-ments.

.

e The distribution of. coliform typesfrom human sources should beregarded as a renresentative valuefor large numbers of sources.Investigations have shown that therecan be large differences inthedistri ution of IMViC types fromperso to person, or even from an

;ual.

3 Differentiation between coliforms offecal vs. nonfecal origin

Table 2 is a summary of findingsbased on a number of different criteriafor differentiating between coliformsof fecal origin and those from other 00sources.

(-IMViC type ++-- is a measurementof E. col. Variety and, appearsto '0.ive reasonably. good' correlationbeiween known or highlyprobablefecal origin and daubtful fecal origin.

a

&

b The combination of IMViC types,++;-, +--1, and givesimproved identification.qf probable-fecal origin, and appears also toexclude mdst of the conforms not'found in eXcrets, of warm- bloodedanimals in large numbers.

c While the indole, methyl-red,Vow Proskauer, -and citrate .utilitzation tests, each used alone,appear to give useful gnawers whenApplied only to samples Of knownpollution from fecal'sources, einterpretetionds not as ear henapplied to coliforms fiom sourcesbelieved tobe remote from directfecal pollution.

2-7

9

o

-?Table 1. COLIFORM DISTRIBUTION BY IMViC TYPES AND ELEVATED TEMPERATURETEST FROM ENVIRONMENTAL AND BIOLOGICAL SOURCES

co.

1

-IMViC_type

Vegetation"--

InsectsSoil' Fecal sources 1_22.1__.thry

Undisturbed polluted Hu a -'v o kNo.

strains% oftotal

`Ns:, J.-.4strains ;

7ce Of

totalNo.

strains% oftotal

No. I % of1strains total

No.strains

\.,% oftotal

14o.strains

% of.total

No.strains

% oftotal

+4--

' o

128

237

10. 6

19. 7

9

I

134

113 I.0

12.4

4

4134

443

5.

18.

_

6

8

3

536 80. 6

13 2.,0

I

3932

.245

87. 2 4

5. 4

,2237'

0

95. 6 I

<0. 1

b.

1857

1

97.9.

0. 1

-+-H-4--

,,

++-+ '.

- ++ + -.

23

2

;168

116

32

291

88

r5

19

2

1203

169*

14. 1.*

1.

0. 2

14. 0

9. 6

2. 7

24.2

7. 3

7. 2

0. 4

1. 6

0. 2

0.40. 1

0

332

118

28

254

46

42

0

0

0

8

9'1084

162*

14. 9*

<0. 1

(<0. 1

30. 6

10.9 '

2. 6

23.4

4. 2.-3. 9

<0:1

<0. 1

<0. 1,

0. 7

O. 8

-.,78

7

1131

87'

181

. 159

67

4

'1

53

6

0

0

2348

216

9. 2

3.

0.

48.

3.

7.

46.

2.

0.

<0.

2.

0.

<0.

<0.

3

1

7

7

8

9

2

1.

3

3

1

1

1

0

87

22

5

0

0

1

0

0

0'\

0

0

665

551

82. 9

O. 2

<0. 1

.13. 0

3. 3

-0. 7a. 1<0. 1

0. 2

<0. 1

<0. 1

10. 1

<0..1

<0. 1

99

106

80

35

21

&

14

. 2

0

0

0

0

2

4512

4349

96. 4

2. 2

2. 4

1. 1

0. 8

0. 5

0. 1.0. 2

<0. 1

<0. 1,

<0. 1

0. 1

<0. 1

<0.,1

''

14

. -59

1

27

0

0

0

0

0

0*

0

0

0 .2339

2309

. 98. 7

6

2.5

<0. 1

1. 2

<0. 1

<0. 1

<0. 1

<0. 1

<0. 1

<0. 1

<0. 1

<0.1

O. 1

-

20

0

5

11

0

0

0

0,.

0

0

: 0

0

2

1896

1765

a"' 93. 0

1. 1

<0. f

0, 3

o. 6

<0. L

<0. 1

<0.4<0. 1

.

<0. 1

<0. 1

<0. i

<0. 1

<0. 1

-H-4-4;

+-++

-+-14

--+-4--+-

+--++---TotalNo. EC +% Et +.

24

*120 of these.-were ++--, were ++--,15 - . 27 -+-+,11 -+-- - 5 ++ -+

*129 of these

./

.1

sr.

-1-+--1


4

HUMANEC a) 96..4

. BA LB (6094.7

SUMMARYType Percent positive++ 91.8

+-- 1.5

d'1---++ 2.8

EC 4 96.1BALB 1 95.3

.6

'LIVESTOCKdo 987

BALB 98.6.

++

1.1 97.9

POULTRYEC ED 93.0

BALB ED 92.5 .

FIGURE 1 .

COLIFORNIS'67 `Soil Samples(6ejdreich. et. al.)

z

-+

+++

,

UndisturbedSbil.

U

FIGURE 2

9

"-PollutedSoil

V

11`

B cteriolo cal Indicators of Water Pollution

Table 2. COMPARISON OF COLIFORM STRAINS ISOLATED-FROM WARM-BLOODED ANIMAL

ALECES, FROM UNPOLLUTED SOILS AND POLLUTED.SOILS WITH USE OF THE

iViG REACTIONS AND THE ELEVATED TEMPERATURE TEST IN EC MEDIUM

A Tr 44.50 C ( O. 50 ) (12th ed. 1965; Standard Methods for the Examination of Water

and Wastewater)

I.

Test Warm-bloodedanimal feces

Soil:Unpollutell

Soil:Polluted

Vege-tation Insects

+ + - - . .. 91.8% 5. 6% 80. 6% 10. 6% 12.4%

.

+ - - - and, .

-+ --93. 3% 8. 9% . 80.7% 12.5% 13.2%

_

#

.

Indole positive.

94.0% 19. 4% . 82.'7% 52. 5% 52.4%

M. tle_yl--red positive 96,9% 75. 6% 97. 9% 63. 6% 79. 9%

o.V ges-Proskauer positivei k

5.1% 40.7% 97.3% 56.3% 40.6%

Citrate utilizers 3. 6% 88. 2% 19. 2% 85. 1% 86. 7%

Elevated temperature (EC).'positive .

96.4% 99 2% 82.9% 14.1% 14.9%

Number of cult resstudied

,

_1&, 747 2,348 665 1,203 1,084

Total Pure Cultures

d The elevated temperature test givesexcellent correlation with samplesof known or highly probable fecalorigin. The presence of smaller,but demonstrable, percentages ofsuch organisms in environmentalsources not interpreted as being,polluted could be attributed largelyto the warm-blooded wildlife in thearea, including birds, rodents, andother small mammals.

e The elevated temperature test yieldsresults equal to.those obtained fromthe total IMVIC code. It has markedadvantages in speed, ease andsimplicity of performance, and ltfoquantitative results for each watersample. Therefore, it isthe official standard method fordifferentiation coliforms ofprobable direct fecal origin from those .

which may have become established ;le

in the bacterial flora of the aquaticor terrestrial habitat. "

Studied: 14,047

1'..,c11, EVALUATION OF COLIFORMS ASPOLLUTION INDICATORS

'et

A The Conform Grobp as a Whole

1 Merits

a' The absence of conform bacteria isevidende of a bacteriologically safewater.

b The dens* of coliforms is roughlyproportional to the amount ofexcretal pollution present.

c If pathogenic bacteria of intestinalorigin are present, conformbacteria also are present, in much .

greater numbers.+NW

, d Conforms are always present in the .

intestines of humans and other warm-blooded animals, and are eliminatedin large numbers in fecal wastes.

r


p

e Conforms are moreersistent inthe aquiatic environment than are.pathogenic bacteria of intestinalorigin.

f Conforms are generally harfnles ,

to humans and can be determined'.quantitatively by routine laboratoryprocedures.

2 Limitations

a Some of the constituents of theconform group have a wide environ-'mental distribution in addition totheir occurrence in the intestinesof warm-blooded animals.

6b Some strains of theconform groupmay multiply in certain pollutedwaters ("aftergrowth"), of high

. nutritive values thereby adding tothe difficulty of evaluating a pollutionsituation in the aquatic environment.Members of the E. aerogenes sectionof the coliform are commonlyinvolved in this kind of problem.

c Because of occasional aftergrowthproblems, the age of the pollutionmay be difficult to evaluate undersome circumstances.

d Tests for criliforms are subject tointerferences due to other kinds ofbacteria. False negative resultssometimes occur when species ofPseudomonas axe present. Falsepositive results sometimes occur .

when two or more kinds lof non-conforms produce gas from lactose,when neither can do so alone(syner.gism).

B 'The Fecal,Coliform Component of theConform Group (ad determined by elevatedtemperature test)

4

1 Merits

a The majority (over 95% of the con--form bacteria from intestines tiofwarmrblooded animals grow at the

-elevated temperature.

b These ortanisms are of relativelyinfrequent occurrence except inasaociation with fecal pollutiop<7

c Survival of the fecal coliform groupis shorter in environmental watersthan for the coliform group aswhole. It follows, then, that highdensities of fecal conforms isindicative of relatively recentpollution.

d Fecal conforms generally do notmultiply outside the intestines ofwarm-blooded animals. In certainhigh-carbohydrate wastes, such asfrom the sugar beet refineries,exceptions have been noted.

In some wastes, notably those from .piilp and paper mills, Klebsiella hasbeOn found in large numbersutilizing the elevated temperaturetest. There has, been tmuch contro-versy about whether the occurrenceof Kiebsiella is due to afte.Nrowthdue to soluble carbohydrates in such

.wastes. The signifier:dee ofKlebsiella as an indicator of directdischarge of intestinal wastes thusis under challenge. The issue isstill further' complicated by questionsover whether Klebsiella is in and ofitself a pathogenic organism or ispotentially pathogenic. This is aserious problem which is the subjectof intensive research 'efforts.

.1

2 Limitations

a Feces from warm-blooded animalsinclude some (though proportionatelylow) numbers of coliformtr which donot yield a p&sitive fecal coliformtest when the elevated temperaturetest is used as the criterion ofdifferentiation. These organismsare-E. con varieties by presenttaxonomic classification,,

23

b There is at present no establishedand consistent correlation between

41.

2 =1.1

to

Bacteriological Indicators Of Water Pollution-

ratios of total coliformsifetalconforms in- interpreting sanitaryquality of environmental waters.

In domestic sewage, the fecalconform density commonly isgreater than 90% of the totalconform density. In environmentalwaters relatively free.,from'recentpollution, the fecal conform densitymay range from 10-30% of the totalconforms. There are, however,toe many variables relating to ,

water-bOrne wastes and surfacewater runoff to perinit sweepinggeneralization on the numericalrelationships between fecal- andtotal conforms.

o

c Studies have been made,regarding the survival of fecalconforms in polluted waterscompared with that of entericpathogenic bacteria. In recentpollution studies,' species ofSalmonella have been Sound in thepresence of 220 fecal conforms per

44 100 ml (Spiro), and'110 fecalcolifOrms per 100 ml, (Brezenski,Raritan Bay Project).

3 The issue of the*Klebsiella problemdescribed in an earlier paragraphmay ultimately be resolved as amerit or as a limitation of the valueof the fecal coliform test.

V APPLICATIONS OF COLIFORM TESTS

A Ctirrent Status in Official Tests

1 The coliform group is designated, in"Standard Methods for the Examinationof Water and Wastewater" (14th ed.,1970, through...the Completed TestMPN procedure'as the official test

, for bacteriological potability pi water.

The Confirmed Test MPN irocedure . °

is accepted where it has been demo ,strated, through -comparative testi,to:yield results equivalent to theCompleted Teat. The membrane filterthethocl also is accepted for examinationof waters subject to interstate regulation.

2 The 12th edition of Standard Methodsintroduced a standard test for fecalconform Bacteria. It is emphasizedthat this is to be used in pollution

v studies, and does not apply to theevaluation of water for potability.This procedure has been continued inthe 13tti and 14th Editions.

B Applications

1 Tests for the conform group as awhole are'used in official tests tocomply with interstate drinking waterStandards; state standards for shell-fish waters, and in most, if not all,cases where bacterial standards ofwater quality have been establishedfor such use as in recreational orbathing waters, water supplies, orindustrial supplies. Laboratorypersonnel shouldie aware of possible'implementation of the fecal coliformgroup as the official*st for-recreationaland bathing waters.

2 Theecal conform test has applicationinwater quality surveys,. as an adjunctto determination of total conformdensity. The fecal/coliform test isbeing used increasingky in all waterquality.surveys.

3 It is emphasizedAhat no responsibleworker advocates subStitution of a; .

fecal conform test for total, conformsin evaluating drinking water quality.

-

e

I INTRODUCTION

Investigations regarding streptococciprogressed from the streptococci of medicalconcern to those which were distributed indiffering environmental conditions which,again, related to the w4lfare of man. Tlfestreptococci were originally reported by Lawsand Andrews (1894), and Houston 41899, 1900)considered those Streptococci, which we nowcall "fecal streptococci, " as ... "indicativeof dangerous pollution, since they are readilydemonstrable in waters recently pollutedbandseemingly altogether absent from waters abovesuspicion of contamination.

From their discovery to the present time thefecal streptococci appear characteristic offecal pollution, being consistently present inboth the feces of all warm - blooded animalsand in the environment associated with animal,discharges. As early as 1910 fecal strepto-

, cocci were proposed as indicators topeMetropolitan Water Board cf London. ;.However, little progress resulted in theUnited States until improv7ft methods ofdetection and enumeration appeared afterWorld War II.

BacteriolgLical Indicators of Water Pollution

Part 3. The Fecal Streptococci

Renewed interest in the group as indicatorsbegan with the introduction of azide dektrosebroth in 1950, (Mallmann.igt Seligmann, 1950).The method'which is in the current editionof Standard Methods appeared soon after.-(Litslcy, et al.- 1955).

With the advent of improved methods fordetection and enumeration of fecal"24trep-,tacocci, significant body of technical

%literatur s appeared.

This outline will consider the, finding's ofvarious investigators regarding the 'fecal.-streptococci and the significance of dischargesof theq organisms into the itquatic environment.

6

, U FECAL MATERIALS

A DefinitiOn

The terms "enterococci," "fecalstreptococci, " "Group D streptococci, ""Streptococcus fecalis, " and even"streptococci" have been used in a looseand interchangeable manner to indicatethe streptococci present in the enterictract of warm-blooded animals or.of thefresh fecal Material excreted therefrom.

a

Enterococci are characterized by specifictaxonomic biochemistry. SerOlogicalprocedures differentiate the Group Dstreptococci from the various groups.Although they overlap, the three groups,

. fecal streptococcus, enterococcus; andGroup D streptococcus, are not synonymous.Because our emphasis is on indicators ofunsanitary origin, fecal streptococcus isthe more appropriate term and will includethe enterococcus as well as other groups.

Increasing attention is being paid to certainstreptococci found in humans and certainbirds whic were, at one time, consideredto be biotypes of Str. faecalis orStr. faeciumancrtherefore legitimatefecal13.t_reptococci. These are now con-sidered to be in a separate group in their

own right, theGroup Q streptococci.

A rigid definition of the fecal streptococcusgroup is not possible with our presentknowledge. The British Ministry of Health(1956) defines the organisms as "Gram-positive" cocci, generally occurring inpairs or short chains, growing in th4presence of bile salt, usually capable ofdevelopment at 45° C, producing acid butnot gas in mannitol and lactose, failing toattack raffinose,, failing to reduce nitrateto nitrite, prbducing acid in litmus milkand precipitating the casein in the form ofa loose, but solid curd, and exhibiting agreater resistance to heat, to alkalineconditions and to high concentrations ofsalt than most vegetative baCteria."However, it is pointed but that "streptococcideparting in one or more particulars fromthe "type species cannot be disregardedin water."

2.13

4

11'

Bateriological Indicators of Water Pollutign

Standard Methods (14th ed., 1975)describes the fecal streptococci as +pureculture iselective medium organismsWhich are Cita lase negative and capableof originating growth in BHI broth(45°C. for 48 hours) and Bile brothmedium (35° C. for 3 days).

For the proposes of this outline, and in linewith the consensus of most' water micro-biologists in this country, the generaldefinition of the fecal streptococci is

. "..The group composed of Group Dand Qspecies consistently present insignificant numbers in fresh fecalexcreta of warm-blooded animals,which includes all of the enterococcusgroup in addition to other groups ofstreptococci. "

B Species isolated

1 Findings

. .

a Human feces

Examination of human fecal specimens'yields a high percentage of theenterococcus group and usuallydemonstration of the S. salivariuswhich.is generally considered amember of-the human throat floraand to be surviving in human fecalmaterials rather than activelyMultiplying in the enteric tract,.Also present would be a smallpercentage of variants or biotypesof the enterococcus group.

b Nonhuman Feces

1) Fecal material which are fromnonhuman and not from fowl willyield high percentages of the'S, bovis and/or S. equinusorganisms with a concomitantlyreduced percentag6 of theenterococcus group.

.

2) Fowl excreta

Exc ment friim fowl characteris-tic yields a large percentageof ent rococcal biotypes,(Grou Q) as well as a.significant

t

percentageof enterococcusgroup.

2 Significance

Species associations with particularanimal hosts is an established fact andleads to the important laboratorytechnique of partition counting of coloniesfrom the membrane filter or eget' rpour plates in order to establish orconfirm the source of excretalpollution in certain aquatic investi-gations.

,It is important to realize that a suitablemedium is neceSsary'in order toallow all of the streptococci whichwe consider to be fecal streptococcito grow in order to give credence tothe derived opinions. Use of liquidgrowth m5dia into which direct

-- inoculations from -the sample aremade have not proven to be succeasfulfor partition counting due to the differinggrowth rates of the various species ofstreptococci altering the original:percentage relationships. Due to thelimited survival capabilities of someof the fecal streptococci it is necessaryto sample fresh fecal material or watersamples in close proximity to the -pollution source especially whenmultiple sources are contributing to areach of water. Also the pH range,must be within the range of 4.0-9.0.

Standard,Methods (14th ed., 197,5) nowincludes a schematic allowing for theidentification of fecal streptococci typespresent within a given sample.

III FECAL STREPTOCOCCIIN THEAQUATIC ENVIRONMENT

A General

From the foregoing it is appears thatthe preponderant human feeal streptococciare composed of the enterococcus group,and, as this is the case, several media arepresently available which will detect onlythe enterococcal group will be suitablefor use with aquatic samples which areknown to 'be contaminated or potentiallycontaminated with purely domestic

./

4-1

(human) wastes, On the other hand,whn it is known or suspeCted that other-than-human wastes have potential egress.to the aquatic environment under investi-gation, it is necessary to utilize thosemedia which are capable of quantitatingthe whole of the fecal streptococci group.

B' Stormwaters and Combined Sewers

1 General

Bacteriological Indicators of Water Vollution

Storm sewers are a heries of pipesand conduits which receive surfacerunoffs from the action of rainstormsand do not include sewage which areborne by a system of sanitary sewers.Combined sewersreceive both the stormrunoff and the water-bornewastes ofthe sanitary system.Both storm water and combinedsewer flows have been found tousually contain large quantities offecal streptococci in numbers whichgenerally are larger than those ofthe fecal coliform indicator organisms.

2 Bacteriological Findings

Table 1 represents, in, a modified form,some of the findings of Geldreich andKenner (1969) with respect to thedensities of fecal streptococciwhenconsidering Domestic sewage in contrastto Stormwaters:The Retro FCC /FS is that of theFecal conform and Focal streptococciand it will be noted that 'in each case,when considering.the -DomesticSewage) it is 4. 0 or grealer whileit'is less than 0. 7ofor stormwaters.The use of this ratio is useful toidentify the source of pollution asbeing human or nonhuman warm-blooded animal pollut When the ratiois greater than 4.0 it is sidered to b"e"human waste contaminated hile a ratioof less than 0.7 is considered to be

. nonhernan." It is evident that the storm-waters have-been Primarily pdlluted.byexcreta of raff"and-other rodenls andilbsiably'domestic and/or farm animals.

1

Species differonces are the main causeof different fecal' coliform-fecalstreptococci ratios. Table 2 comparesfecal streptococcus and fecal conformcounts for different species. Eventhough individuals vary widely, massesof individuals in a species have charac-teristic proportion of indicators.

C Surface Waters

In general, the occurrence of-fecal'streptococci indicates fecal pollution andits absence indicates that littlp or nowarm-blooded fecal contribution. instudies of remote surface waters the fecalstreptococci are infrequently isolated andoccurrences of small numbers can beattributed to wild life and/or snow meltsand resultant drainage flows.

Various examples of fecal streptococcaloccurrences are shown in Table 3 inrelation to surface waters of widely, varyingquality. (Geldreich and Kenner 1969)

IV FECAL ,STREPTOCOCCI: ADVANTAAND LIMITATIONS '

A General

Serious studies concerning the streptococciwere institutedtwhen it became apparentthat they were the agents responsible orsuspected for a wide variety of humandiseases. Natural priority then focuseditself to the taxonomy of these organismsand this, study his still causing consternationas more. and more microbiologAl techniqueshave been broughtto bear on these questions;

The sanitary-microbiologist is,concertied,with those streptococci which inhabit theenteric trktt of warm-blooded animals,thdir detection, and utilization in develop.:ing a criterium for water quality standards.

-1111ti%

322-15

.

.0

Bacteriological Indicittors of Water Pollution

Table 1 Table .3

DISTRIBUTION OF FECAL STREPTOCOCCIIN DOMESTIC SEWAGES AND STORMWATER

RUNOFFSFecal Streptococci

per 100 ml RatioWater:Source median values FC/FS

INDICATOR ORGANISMS IN SURFACE ,WATERS

Demities/100 ml

40'Fecel Fecal

Water Source conform streptococci

Prairie WatershedsDomestic SewagePreston, ID 64, 000 5.3 Cherry Creek, WY 90 83Fargo, ND 290, 000 _ 4.5 Saline River, KS 95 180Moorehead, MN 330,-009 4.9 Cub River, ID 110 160Cineinnati, OH 2,470, 000 4;4 Clear Creek, CO 170, 110Lawrence, MA 4,500, 000 4. 0Monroe, MI 700, 000 7.9 Recreational WatersDenver, CO 2,900, 000 16.9'

-Lake Mead 2 444Stormwater Lake Moovalaya 9 176Business Distfict 51, 000 0.26 Colorado River '7"--"`:4 256Residential 150, 000 0.04 Whitman River 32 88Rural 58, 000 0.05 MerriMack River 100 96

Public Water Intakes

Missouri River (1959Mile 470.5 1 , 500 39, 500

( ' Mile 434. 5 , 000 79, 000Mile 408.8 14, 000 59, 000

Table 2. ESTIMATED PER CAPITA CONTRIBUTION OF INDICATOR MICROORGANISMSFROM SOME ANIMALS*

_J

Average indicatordensity per gram

of feces

Avgrage. 'contributionper capita per 24 hr

Avg wt of 'FecalAnimals Feces/24 .hr, conform,

wet wt, g million -*

Fecalstreptococci,

million

Fecalconform,million

Fecalstreptococci,

millionRatio

FC/FS*111

Man 190 , 13.0 3.0 7-2 2, 000 450 4.4

Duck 336 33.0 54.0 11, 000 18;060 0.6

Sheep 1,30 _16.0 38.0 18;000 43, 000 0.4

'Chicken. 182 1.3 2,40, 620- - 0.4:Cow 0.2323, ir 1.3 5, 400 31, 000 0.2

Turley 0.29 2.8 130 1, 300 0. 1,

Pig 2,700 3.3 84.0 8, 900 230, 000

. *Publication WP-20-3, P. 102 z

3

OW,

Kabler (196?) discussed the slow acceptanceof the fecal streptococci as indicatois ofpollution resulting from:

1 Multiplicity and difficulty of laboratoryprocedures

2 Poor agreement between methods ofquantitative enumeration_

3 .Lack of-systematic studies of . . .

a sources

b 'survival, and

interpretations, and 4

4 Undue attention to the S. faecalis group.

Increased attention to the fecal streptococci,esRecially during the last deGade, haveclarified many of the.earlier cloudy issuesand have elevated the stature of theseorganisms as indicators, of,Court precedents establishyht legal statusand recommendation's of various technicaladvisory boards have placed the fecalconform group in.a position of primacyin many rater quality applidatiqns..Thefecal streptococci have evolved from aposition of a theoretically useful indicatorto one which was ancillary to the conformsto one which was useful when discrepanciesor questions evolved as to the validity ofthe colifOim data to one where an equalitystatus was achieved' in certain applications.In the future it iwticipated that, forcertain applications, the fecal streptococciwill ac:hisim a position of primacy foruseful data, and, as indicated by Litsky(1955) "be taken out of the /palm of step?children and given their legitimate placein the fieicof santiary bacteriology asindicators of sewage pollution."

B Advantages and Limitations

1 = Survival

In general, the fecal streptococci havebeen observed to have a more limitedsurvival time in the aquatic environmentwhen compared to the conform group.

"T"

Bacteriolo: ical Indicators of Water Pollution_

.

They are rivaled'in this respect onlyby the fecal conforms. Except fdr casesof persistence in waters of high electro-lytic content, as may be common to'irrigation waters, the fecal streptococcihavelibtbehn observed to multiply inpolluted waterslis stray sometimes beobserved for some of the coliforms.Fecal streptococci usually require agreater abundance of nutrients for sur-vival as compared to the coliforms andthe conforms are more dependent uponthe oxygen tension in the waterbody.In a number of situations it was concluded)

at the fecal streptococci reached aninctiop point more rapidly in warmer

aters hile the-reverse was true in thecolder situations as the coliformg nowwere totally eliminated sooner.

2 Resistance to Disinfection

In artifidial pools thesource ofcontamination by the bathers isusually limited to throat and skin-flora and thus increasing attentionhas been paid to indicators other'than those traditionally from theenteric tract. .This, one of theorganisms considered to be-a fecalstreptococci, namely, S. salivarius,can be a more reliable infficatorwhen detected along with the otherfecal streptococci especially since

' studies have confirmed the greater'resistance of the'fecal streptococci.to chlorination. This greaterresistance to chlorination,Nvhencompared to the fecal pliffrrhs, isimportant since the dieoff curvedifferences are insignificant when:the curves of the fecal coliformsare compared t9 various Gramnegative pathogenic bacteria whichreduces their effectiyeness asindicators.

3 Ubiquitous Strains

Among the fecal streptococcus aretwo organisms, one a biotype andthe other a variety of the S. faecalis,which; being ubiquitous (omnipresent)have. limited sanitary significance.

2-17.

,

O

,

/

'.Bacteriological Indicators/of Water Pollution

I

The tiotype, or atypical, S. faecalisis characterized by its ability-tohydrolyze starch while the varietalform, liquefaciens4 is nonbetainwmolytte and capable of liqdefyinggelatin. Quantitation of these organismsin anomalous conditions is due to their

'"4''capability of survival in soil or highelectrolytic waters 09.2d in waters witha temperature of less than 12 Degrees C.

Samples have been encountered whichhave been devoid of fecal coliforms .

and yet "contain a substantiil numbed of"fecal streptococci" of whi thes.ubiquitous Strains constitutor all of the isolations wheli yzed

. biochemically.

V STANDARDS AND CRITERIA

rity

O

Acceptance aAd utilization of Totaltoliforrncriteria, which must now be considered apioneering effort, has largely been supplantedin concept and in fact by the fecid coliformsin establishing.standards for recreational

Iwaters.

The first significant approach to the utillza#,?)tion of the fecal streptococci as a criterium'foi recreational water standards occurred in1966 when a technical commilfee recommendedthe utilization ofthe fecal streptococci with thetotal coliforms as criteria for standards

itertaining to the Calumet River and lower

.0 p.

44,U.

t

j

0 8

k

p

If

Lake Michigan waters. Several sets of4riteria were established to fit the intended

-uses for this area. The uai 91 the fecalstreptococci assa critertnn is indicated to.be tentative pending the "cumulation ofexisting densities and could be modified infuture standards.

With the existing state-of-the-art knowledgeof the presence of,the fecal streptococci inwaters containing-low numbers of fecalcoliforms it is difficult to est fish a eReCificfecal streptococcus density.li t of below100 organisms/0100 ml when u d alone orin conjunction with the taal do orms.

The mos seful application of the fecalstrep occus test is in the .deielopmentof the fecal conform: 'fecal streptococcus,ratio as previously described.

0a

c°-

V.

rr

0

e

t ;e.. . BacteriblogicS1 Indicators of Water Pollution

-

Part 4. Other, Bacterial IndicatOreof Pollution

TOTAL BACTERIAL COUNTS

A Hibtorical

The early studies of Robert Xo'ch ledhim to deVelop tentative standards ofwater quality based on a limitation ofnot more than 100 bacterial colo-nies 'per ml on a gelatin. plating mediumincubated 3 days at 200 C.

2' Liter developments led to inoculationof samples on duplicate plating media,with one set incubated at 37QC and theother at 200 C.

'a Results were used to develop a ratiobetween the 370 C counts and the _200C counts.

'1.

b Waters having a predominantcount at 37° C were regarded asbeing of probable sanitary signifi-cance, while those giving

,predominant counts at= 20° C wereconsidered to,be of pkobable soilorigin, or natural inhhbitants ofthe water being examined.

8 °

B Groups Tested

There is no such thing as "total" bacterialcount in terms of a laboratory determination.

1 Direct microscopic counts do notdifferentiate between livimiand deadcells:

2 Plate counting methqds enumerate onlythe bacteria which are capable of usingthe culture medium provided, under thetemperature and other gro,7/th conditionsused as a standard procedure. No oneculture medium and set of growthconditiOns can provide, simultaneously,ari acceptable environment:for all theheterogeneous, often conflicting,requirement's orthe total range ofbacteria which may be recovered fromwaters.

C Utilization of Total Counts

1 Total bacterial counts, usingplatingmethods, are useful for:

a Detection 6f cha;iges in the bacterialcoluppeitfon of a water source

b Process control procedures intreatment plant operations

c Determination of sanitary conditionsin plant equipment or distributionalsystems

2 Serious limitations in total bact ialcounts exist because:a No information is given regarding

possible or probable fecal originof bacterial changes. Large numbersof bacteria can sometimes becultivated from waters known to befree of fecal pollution,.

No information of any kind is, givenabout the species of bacteria -cultivated.

c There is no differentiation betweenharmless or potentially dangerousforms.

3 Status of total counts

b

Methodology for the deterinination ofthe Standard Plate Countims beenretained iii the 14th Edition of StandardMethods for the stated reason:

. "total counts may yield useful',information about the quality of

f water And supporting data on thesignificance of conform resultsal o, useful in judging the efficiencyIn operation'of various water treat-.ment processes and may have sig-nificant application as an in-plantcontrol test. It is also valuable forperiodic checking' of finished °distribution water"(abridged for this-inclusion)

3

A


rTechnique for the Standard PlateCount is necessary for theperformance of the Distilled WaterSuitability Test as outlined inStandard Methods and elsewhere withinthis manual;

13, Spore-Forming Bacteria (Clostridiumperfringens, or C. welchil)

1 Distribution.,

This is one of the most widely distributedspecies of bacteria. It is reguhFlypieserit in the intestinal tract of warm-blooded animals. I

.2 Nature of organism

.4'I .C C. perfringens is 'a Gram-poSiTiye,

spore-forming rod. The spores causea distinct swelling of the cell wheniOrmed. The organism is extremelyactive in fermentation of carbohydrates; .

and produces the well-known "stormyfermentation" of milk,

3 Status )

A'. 'The organism; when present indicatesthat pollution has occurred at sometime. However, because of the ex-tremely extended viability of, the spores,it is impossible to obtain even anapproximation of the recency of pollutionbased Only on the presence ofC. perfringens.

The presence of the organism does nt,necessarily indicate an unsafe water:

C Tests for Pathogenic Bacteria of IntestinalOrigin '

1 Groups 'c onsideredinclude Salmonellasp, Shigella sp, Vibrio comma,Mycobacterium sp, Pasteurella sp,Leptospira others.

. '

,2720

ti

4r

2 Merits of direct tests:

Demonstration of any pathogenicspecies would dehionstrate anunsatisfactory water quality, hazardousto persons consuming,or coming into .contact with that_water..

3 Limitations

a There is no aittilable routine pro-cedure for detection'of the fullrange of pathogenic bacteria citedabove.

b Quantitative methods are not avail-able few routine application to anyof the above.oye. '

c The intermittent release of thesepathogens makes it impossible toregard water as safe; even in the.,absence of pathogens.

d 'After detectioR, the public already -would hi* been:expOigd to theorganism; thus, /there is no built-inmargin of safety, as exists with,tests for the conform group.

4 Applications

a In tracing the source of pathogenicbacteria in epidemiological investi-gations

4-b In special research projects

c In eater quality studies concernedwith enforcement actions againstpolltitiOn, increasing attention isbeing given to the demonetratiOn ofenteric Pathogenic bacteria in thepresence of the bacterial indicatorsof Pollution.

D Miscellaneous Indicators

' Wife beyond this discussion to explore thetotal range of microbiological indicatorsof polluMIti that have been piopoied and

,s

7 Bacteriological Indicators of Water Pollution

investigated to some extent. Mention canbe made, however, of consideration of

- :tests for the follow jag.

1 Bacteriophages specific fortany of anumber of kinds of bacteria

2 Tests for Enterovirus

3 Serological procedures for detectionof conforms and other indicators: acertain amount of.recent attention hasbeen given to applications of fluorescentantibodies in such tests

4 'Tests for Klebsiella

5 Tests for PsetTdomonas aeruginosa

6 Tests for Salmonella

4,4( 7 Tests for F'tingi

.8 Tests for Sthylococcus

REFERENCES'

1 Standard Methods for the Examination ofWater and Wastewater, 14th ed.,

: APHA,__AWWA, WPCF. Published Ty ,

American Public health Association,1790 'ft-roadway, New York, N.Y. 1975

.2 Prescott; S. C!, Winslow, C. E. A . , andMcCrady, M. Water Bacteriology. -

John Wiley, & Sons, Inc. '1946. .

3- Parr, L.W. 4oliform Intermediates in}finnan Feces. Jour. Bact. 36:1. . .1938.-

4 Clark, ill F. and Kabler, P.W. The- Pgyaiology of the Coliform Group. .,Prciceedizige of the, Rudolfs ResearchConference on Principles and Appli-cations in Aquatic Microbiology. 1963..

5 Geldreich, E., Bo deer, R.H., Huff,. p and '<abler, P.W.

.Type-Distributioin the Feces of Warm-BlOoded Animals.JWPCF. 34:295-301. 1962.

6 Geldreich et al. The Fecal Coli-AerogenesFlora orSoils from Various GeographicAreas. Journal .of Applied Bacteriology25:87-93. 1962.

7 Geldreich, E. E. , Kenner, B.A. andKabler, P.W. Occurrence ofConforms, Fecal Conforms; andStreptococci on Vegetation andInsectEr;_Applied Microbiology. 12:63-69. 1964.-

8 Kabler, P.W. , Clark, H. F. , andGeldreich, E: E. Sanitary Significanceof Coliform and Fecal ColiformOrganism in Surface Water. PublicHealth Reports. 79:58-60. 1964.

9 plark, H. F. and Kabler, P . W.*Re-evaluation of the Significance of the,

' Conform Bacteria. Journal AWWA.56:931-936. 1964.

10 Kenner, B. S. Clark, 1-i. F. , andKabler, P.W. Obat1 Streptococci.II. Quantification in Feces. Am. J.Public Health., 50:1553-59. 1960.

'11 Litsky, W Mailman, W. L.; and Fifield,C. W. Comparison of MPN ofEscherichia con. and Enterococci inRiver Water. Am. Jour. Public Health-.45:1949. 1955.

12 Medrek, T. F. and Litsky,Comparative Incidence of ColiformBacteria and Enterococci inUndisturbed Soil. Applied Micro-biology. 8:60-63. '1960.

4413 ..31allnian, Litsky, W:Survival of Selectd Enteric Organismsin Various Types of Soil.. Am. J.Public Itealth. 41:38-44. 1950.

14 Mailman, W. L., and Seligmin, E. B., JrA Comparative Study of Media forDetection of Streptococci in Water and

. Sewage. AM. J. Public Health.40:286 -89. 1950._

15: Ministry of Mang (London). TheBacterial Examinationof Water Supplies..Reports on Public Health and MedicalSubjects. 71:34.

38

-

Bacteriolo cal Indicators of Water Pollution

16 Morris, W. and Weaver,Streptococci as Indices of Pollutionin Well Water. Applied glicrobiology,2:282-285. 1954.

. .

17 Windt, J. 0. , Coggin, J. H. , Jr. , andJohnson,Streptococcus

: 'Growth of5 fecalis var. liquefacienson Plants.-' AppliedMicrobiology.10;552-555. 1962.

18 Geldreich, E.-E. Sanitary Significanceof ,Fecal Coliformi in the Envtronment.U. S. Department of the Interior.FWPCA Pul?1. WP-20-3. 1966.

19 Geldreich, E. E. and Kenner, B. 4.Concepts of Fecal Streptococci inStream Pollution. J. WPCF. 41 :R336.1969.

20 Kehler, P.W. Purification and Sanitary.. Control of Water (Potable and Waste)

Ann. Rev. of Microbiol. 16:127. 1962.

21 Lltsity, W., Mailman,' W. L., and Fifield,C. W. CdmparlSon of the Most ProbpbleNumbers of Escheridhia'coli andEnterococci in River Waters. A. J. P. H.45:1049: 1933.

22 Geldreich, E. E. Applying BacteriologicalParameters to Recreational WaterQuality. J. AWWA. 62:113. 1970.

23 Geldreich, E. E. , Best, L. C. , Kenner, B.A.and Van Dotisel, D. J. The Bactgriolog=ical Aspects of Stormwater Pollution.J. WPCF, 40:1860. 1968.

224 FWPCA Report of Water Quality CriteriaCalumet Area - Lower -ke Michigan

,-Chicago, IL. Jan. 19667a

This outline was prepared by H. L-. Jeter,.'Chief, Program SUpport Training Branch,and revised by R. Russomanno, Microbiologist,USEPA, Cincinnati, Ohio 45268.

--miNcor

Descriptors: Coliforms, Escher4chia coli,Fecal Coliforms, _Fecal Streptococci,Indicator BacEbLa, Microbiology, SewageBacteria, -Water Pollutidh

r

d

- Iv

I INTRODUCTION

-

A-

EXAMINATION OF WATER FOR dOLIFORM ANDSFECAL-,.5TREPTOCOCCUS,GROUPS

(Multiple Dilution Tube [MPN] Methods)

The subject matter of this outline is containedin three parts, as follows: =

A Part 1

Fundamental aspects of multiple dilutiontube ("most probable numbers") tests,both from a qualitative and a quantitativeviewpoint.

2 Laboratory ?ench records.

3 Useful techniques in multiple dilution."-'tube niethods.

4 Standard supplies, equipment, andmedia in multiple dilution tube tests.

TABLE

-.B Part 2

Detailed,... day -by -day, procedures in testsfor the coliform group and subgroup];"within the coliform group..,

C Part 3

Detailed, dak-liy-day, procedures in testsfor-members of She fecal streptococci.

D Application of Tests to Routine Examinations

The following considerations (Table 1) apply-to the selection of the Preriumptive Test,the Corifirm?d Test, and the CompletedTest. Termination of testing at the .1*-.

Presumptive-Test level is not practicedby labqratories cithis agency. It mustbe realized that the Presumptive Test alonehas limited use when water quality is toi,be determined.

Examination Terminated at -

Type of ReceivingWater

PresumptiveTest

, Confirmed Test ----Completed Test

.

Sewage Rec iving 41,)

Treatment ]ant - Raw

Applicable

I1

Applicable

m.

Applicable

Applicable )Important where resultsare to be used for contraof raw or finished waterA pp li ratio*, to' a,s tatis-tically valid number ofsamples from theConfirmed Test to establish its validity indetermining'the sanitaryquality.

. .

. .

Chlorinated Not Done. _

Applicable

Bathing-1

Not Done

.,- _

Applicable/.. .Drinking_ . Not Done Applicable A

Other n4..4,,

-..

Applicable in allcases where Pre-sumptive Test aloneis unreliable.

\NOTE: Mention of commercial products tuidmanufacturers does not imply endorsement by the1 Environmental Protection Agency.

W. BA. 3m, 8a 78.

1

0

4

MPN Methods

U BASIS OF MULTIPLE TUBE TESTS

A Qualitative Aspects

1 For purely qualitative aspects of testingfor indicator organisms, it is convenientto consider the tests applied to onesample portion, inoculated into a tubeof culturT medimm, andithe follow-upexaminations and tests on results of theoriginal inoculation. Results of testingprocedures are definite: positive

-(presence of the organism-group isdemonstrated) or,negative (presence ofthe organism-group is not demonstrated. )

2 Test procedures are based on certainfundamental assumptions:

a First, even if only one living cell of-the test organism is present in thesample, it will be able to grow whenintroduced:into the priniary inoculationmedium;

b Second, growth of the test organismin the culture medium will producea result which indicates presence ofthe test organism; and,

"c, Third, extraneous organisms willnot grow, or if they do grow, theywill not limit-groiyth of the test

.organism; nor will they produceePowth effects that will be confusedwith those of the bacterial ii4Oup forwhich the test is designed.

3 Meeting these assumption's usuallymakes it neceilisary.,to conduct the testsin a series of stagei (for example, thePresumptive, Confirmed, and CoinpletedTest, stages, respectively, of standard,tests for the coliform group).

4 Features of a full, multi-stage' test.

a First stage: The culture mediumusually_ serves primarily as anenrichment medium for the grouptroted. A' good first-stage.gftwth

.medium should support growth of allthe living cells of the group tested,and it should include provision forindicating the,Pierence of'the test'

organism being studied. A first-stage.medium.may include somecomponeth which inhibits growthof extraneous bacteria, but thisfeature never should be includedif it also inhibits growth of anycells of the groupfor which thetest is designed. The,PresumptiveTest for the colifortm group is aghod example. 'Thernediumsupports growth, presumably, ofall living cells of the 'conformgroup; the culture container has afermentation vial for demonstrationof gas production resulting fromlactose fermentation by coliformbacteria, if present; and sodiumlauryl sulfate may be included inone of the approved media forsuppression of growth of certain' -noncoliform bacteria. Thisadditive apparently has no adverseeffect on growth of members of thecoliform group in the concentrationused. If the result of the first-stage

)cst is negative, the study of theCulture is terminated, and the resultis-recorded as a negative test. Nofurther study is made of negativetests. If the result of the first-stage test is positive, the culture

may be subjected to further studyto verify the findings of the firststage.' _

b Second stage: A transfer is madefrom positive cultures of the first-stage test,to a second culture medium.This test stage emphasizes provisionto re uce.confusion of results due to

. gr,o h effects of extraneous bacteria,co only achieved by addition ofsel ctive inhibitory agents. (TheCo firmed Test for coliforms meetsth se requirements. Lactose and

) -----ie entation vials rare provided ford monstration of coliforms in themedium. Valliant green dye and .,

4493ile salts are included as inhibitoryagents which ,tend to.supprep growth'of practically all kinds of honcoliformbacteria, but do not suppress growthof coliforrnbacteria when used asdirected).

ti

JP MPN Methods

If result of the second-stange test isnegative, the study of the culture isterminated,- and the result is recordedas a negative test. A negative test heremeans that the positive results of thefirst-atage test were "false positive, "due to one or more kinds df extraneousbacteria. A positive second-stage testis partial confirmatidn of the positiveresults obtained in the first-stage test;the culture may be subjected to finalidentification through application of stillfurther testing,procedures". In routinepractice, most sample examinationsare terminated at the endapf the secondstage, on the assumption that the resultwould be positive if carried to the-third,and final stage. This practice should befollowed only if adequate testing is doneto demonstrate that the assumption isvalid. Some workers recommend contin-uing at least 5% of all sample examina-tions to the third'stage to demonstratethe reliability of the second-stage result!

40

B Quantitative Arects of Tests

1 These methdds for deter,mining bacterial.numbers are based on the assumptionthat the bacteria can be separated fromone another (by shaxing or other means)resulting In a suspension of individualbacterial cells, 'uniformly distributedthrough the original sample when theprimary inoculation is made.

2 Multiple*ailution'tube tests for quantiia-tive determinations apply a Most ProbableNumber (MPN)' technique. In this pro-cedure one or more measured portions**of each of a stipulated series of de-creasing sample volumes is inoculatedinto the first-stage culture medium.Through decreasing the sample incre-ments, eventually a volume is reachedwhere only one cell is introduced into.

h.

some tubes, and no cells are it4rOduce4into other tubes. Each of the several 1`

tubes of sample-inoculated first-stagemedium is tested iittlependehtky,according to the principles previouslydescribed, in the qualitative aspects -

of testing procedures.

3 The combination of positive andnegative results is used in an applicationof probability niathematic8 to securea single MPN value for the sample.

4 To obtain MPN values, the followingconditions must be met:

a The testing procedure must resultin one or more tubes in.which thetest organism is demonstrated tobe present; and

b The testing procedure must resultin one or more tubes in which thetest organism is not demonstratedfo be present.

5 The MPN' Falue for a given sample isobtained through the use of MPN Tables.It is emphasized that the precision ofad individual MPN value is not greatwhen compared with most physical orchemical determinations.

'6 Standard practice in water pollutionsurveys conducted by this organization,is to plaht five tubes in each of a seriesof sample increments, hr samplevolumes decreasing at decimal intervals.For example, testing known pollutedwaters, the initial sample inoculationsmight consist of 5 tubes each in volumesof 0.1, Oti Ql, 0.001, and 0.0001 ml,respectively. , This series'of samplevolumes will yield determinate resultsfrom a low of 200 to a high of 1,600,000organisms per 100 ml.

a

3-3

03

MPN Methods

Ill LABORATORY BENCH REORDS

A Features of a Good Bench Record Sheet

1 PrOvides complete identification of thesample.

2 Provides for full, day-by-day informa-tion abi ote,lA.t1.1 tests performed on thesame

3, Provides easy step-by-step record,applicable to any portion of the sample.

4 Provides for recording of the quantitativeresult which will be transcribed to sub-sequent reports.

5 Minimizes the amount ofriting by theanalyst.

ti Identifies the analyst(s)

B There is no such thing as "standard"bench sheet for Multiple tube tests; thereare many versions of bench sheets. Someare prescribed by administrative suthortity(such as the Office of a State Sanitary

-Engineer); others are devised by'laboratorr:or project personnel to meet specific needs.

It is not-the purpose of this discussion torecommend an "ideal" beech form; however,the form used in this training coursemanual is essentially sundar to thathlaledin certain research laboratories If i isf.organization. , The student enrollecrin.the ,"

course for wp this manual is written '"hi _,should make himself thoroughly familiar .04with the bench sheet and its proper use.See Figure 1.

.:

11./

IV NOTES ABOUT WORKING PROCEDURESIN THE LABORATORY

A Each baciCricaogical examAatiOn of waterby multiple dilution tube methods requiresa considerable amount of manipubition;much is qtiite repetitious. Laboratoryworkers mug' develop and maintain goodroutine working habits, with constantalertfiess to guard against lapses intocareless, slip-shod laboratory proceduresand "short cuts" which only can lead tolowered quality of laboratory. .work.

B Specific attention is brought to the followingby no means exhaustive, critical aspects oflaboratory procedures in multiple dilutiontube tests:

1 Original sample

a Follow prescribed care and handlingprocedures, before testing.

Maintain absolute identification ofsample at all stages in testing.

c Vigorously shake sarnples (andsample dilutions) before plantingin culture Media.

2 Sample measurement into primaryculture medium

a Sample portions must be measured\faccurate)y into the culture mediumfor yeliablequantitative tests to bemafle. Standard Methods prescribesthat calibration errors should notexceed + 2.5 %.

4

43

4

. BACTERIOLOGY BENCH SHEET

Collection Data

Project

Sample Station

7

ilr

p1_

Multiple kilutioti Tube Tests.

*4 7Dates fr,4f Time 1'60 By.Tempe a ure °C pHOther Observations

Analytical Record

Behch Number of Sample jaAnalyst Adz.Test sta ted at /..5c.6. By

ml^-unple

Coliformfr est Fecal1

con-Fecal

Streptococdus*-1

Remhrks.LTB BGLB EMB LSTB Gram form A - D , EVA

24 ,,;48 24 : 48 11 1111 I 48 stain 24 24 48 24 ! 48

: Mi a_ 1 1 0 E 3. --=-M1Ar4 I OM I1M.4 I 6 3 I VWlqSO 4 ra I I -Al I 4:

.../1111101110171%1101111102----'411110111111MMIIP' 11111111

a_ II I(1

,4

I

I .MA a 4 OM al I 0 PMWit Rigri I_M S' 'd. '-- A "41 E '...4 V. :4 Li i ita 1

'-D 1 ,

/0 . : . ....,, ___.

,

,---.i

if

---,. .. .

.

4 _,_ ._, .

4 __ _f_______.

o., .--..}: _____

t -. --,- ,

o

1

i----1 i.. ' 1--

GIP / ,-

: i, .

.

, r

i.

.,

' -.

I-4,--__I

. -

:..,...IF

---,f

.

_.

...

.,,

.

,---

,

,.

. ,......4_ . ,i

I

.,

_I1

. i_,____._

1

-.-

1

1

OOP' -4111110'-" -14W-lop.-_,...0--.11. --.......-

--.

. 4. ..

_AL-7

, 1----'411

, ,°4tgr=0km.

,i. .

.

, .

Con `mlConfirmed:Completed:

Fecal Conform MPI44,`44

Figure 1. SAMPLE BENCH SWET

FecalStreptococcus MPN/100 ml- EVA:

3'5

MPN Methods

A

t

Suggested sample Measuring practicesare as follows: Mohr measuringpipets are recommended. 10 mlsamples are delivered at the top of

. the culture tube, using 10 ml pipets.1.0 ml samples are delivered downinto the culture tube, near the sur-face of the medium, and "touchedoff" at the side of the tube when thedesired amount of sample has beendelivered. 1.0 ml or 2.0 ml pipetsare used for measurement of thisvolume. 0.1 ml samples aredelivered in the same manner as 1.0ml samples, using great care that,the sample actually gets into theculture medium. Only /, 0 ml pipetsare used cor this sample volume.After delivery of all sample incre-ments into the culture tubes, theentire rack of culture tubes may beshaken gently to carry down any ofthe sample adhering to the wall ofthe tube above the medium.

Workers should demonstrate actualtests that the pipets and the techniquein use actually delivers the rate d voltune swithin the prescribed limits of error.

b Volumes as small as 0.1 ml routinelycan be delivered directly from thesample with suitable pipets. Lessersample volumes first should be diluted,with subsequent delivery of suitablevolumes of diluted sample into theculture medium. A diagrammaticsqheme for making dilutions is shownin Figure 2.

3" Reading of culture tubes for gasproduction

a On removal from the incubator,shake culture rack gently, to

,, encourage release of' gas whichmay be supersaturated in the'culturemedium.

b *Gas in any quantityis a posittve test.It is necessary to work in conditionsoftsuital;le lighting fqr easy recog-nition of the extremely small amountsof go. s inside the tops of somefermentation vials.

4 .4eading'cif-liquid culture tubs for ,

krowth as indication of a positive teatrequires good lighting. Growth isshown by any amount of increasedturbidity or opalescence in.-the culture

si\ medium,' with or without deposit ofsediment at the bottom of the tube.

6

5 Transfer of cultures with inoculationloops and needle

a ways sterilize inoculation loopsnd needles to glowing (white hot)

in flame immediately before transferof Culture; do not lay it down or--touch'sit to any non sterile objectbefore making the transfer.

b After sterilization, allow sufficienttime for cooling, in the air, to avoid

. heat-killing bacterial cells which willbe gathereb on the wire.

c to' ops should be at least qram in insidediameter, with a capability of holdinga drop of water or culture.

For routine standard transfersrequiring transfer of 3 loopsful ofculture, (Fecal Steptococci) many

.workers form three. 3 -mm loops ,on thesame length of wire.

As an alternathre to use of standardinoculation loops, the use of ,"applicator sticks" is describett in the14th Edition of Standard Methods.

4 5.

10

5

Figure 2.

Dilution Ratios:

PREPARATION OF DILUTIONS

1:100

Tubes

,!ctual volumef is ample in tube

1 ml

1 ml O.

C.)

1 ml

MPN Methods

1:10000

Petri Dishes or C1ture Tubes

0..1 0. 01 ml 0. 00Tm1

The applicator sticks are dry heatsterilized (autoclave sterilization isnot{a.cceptahle because of possiblerelease of phenols if the wood issteamed) and are used on la 'single-service basis. Thus, for everypositive culture tube transferred, anew applicatorfstick used.

This use of applicator sticks isparticularly attractive in field

' situations where it is inconvenient orinipossible,,to provide a gas burnersuitable for .sterilization of theinoculation loop.. In addition, use ofapplicator sticks is favored inlaboratories where room temperaturesare significantly elevated by use ofgas burners.

Ir

\14,

1ml 40. 1 ml

0. 4001 ml 0. 00001 ml

7 Streaking cultures on agar surfaces,

a All streak-inoculations should bemade without breaking the surfaceof ihe agar. Learn to use a lighttouch with the needle; however,many inoculation needles are sosharp that they are virtually useleakin this respect. When the needle isplatinum or platinum-iridium wire,it sometimes isbeneficial to fusethe working tip into a small sphere.This can be done byinsertion of a well nsulated (againstelectricity) wire into a carbon arc,or same other extremely hot environ-ment. The sphere should not be morethan twice the diameter of.the wirefrom which it is formed, otherwiseit will be entirely too heat-retentiveto be useful.

-r

3-7

air

MPN Methods

t

When the needle is nichromeresistance wire, it cannot be heat-fused; the writer prefers to bendthe terminal 1/16 1/8" of the wireat a slight angle to the overall axisof the needle. The side of theterminal bent portion of the needlethen is used for inoculation of agarsurfaces.

ft When streaking for colony isolation;avoid using too much inoculum. Thestreaking pattern is.somewhatvariable according to individualpreference. The procedure favoredby the writer is shown in theaccompanying figure. Noteparticularly that rhen going fromany one stage..of the streaking to thenext, the inoculation needle is heat-sterilized.

8 Preparatioti of cultures for Gramstain

a The Gram stain always should bemade from a culture grown on anutrient agar surface (nutrientagarslants are used here) or from nutrientbroth.

b The culture should be young, andshould be actively grlowing. Manyworkers doubt the validity of theGram stain made on a culture morethan 24 hours old.

c Prepare a thin smear for the stainingprocedure. Most -beginning workerstend to use too much bacterial sus-pension in preparing the dried smearfor staining. The amount of bacteriashould be so small that the dried filmis barely visible to the naked eye.

t

V EQUIPMENT AND SUPPLIES

Consolidated lists of equipment, supplies, aand culture media required for all multipledilution tube tests described in this outline .are shown'in Table Z. Quantitative infor-mation is not presented; this is variable -according to the extent of the testing pro-cedure, the number of dilutions used, andthe number of replicate tubes per dilution.It is noted that.-requirements for alternate

,procedures are fully listed and choices aremade in aocordance to laboratory preference.

So,

ti

croMPN Methods

1.

1 a Flame-sterilize an inoculation needle and air-cool.

b Dip the Lip of the inoculation needle into the bac-terial culture being studied.

g Streak the inoculation needle.tip lightly back and-forth over half the agar surface, as in (1), avoid-ing scratching or breaking the agar surface.

d Flame-sterilize the inoculation needle and air-cool.-

2 a Turn the Petri dish one-quarter-turn and streak theinoculation needle tip lightly back and forth over one-half the agar surface, working from area (1) into one-half the unstreaked area of the agar. 4,31

b Flame-sterilize the inoculation needle aid air-cool.

3 a Turn the Petri dish one-quarter-turn anti streak theinoculation needle tip6lightly back and forth over one-half the agar surface, working from area (2) intoarea (3), the remaining unstreaked area.

b Fla Me-sterilize the inoculation needle and set it aside.

c Close the culture container and incubate as prescribed.

Figure 3. SUGGESTED 'PROCEDURE FOR COLONY ISOLATION BY ASTREAK-PLATE TECHNIQUE

AREA 1 (14avy inoculum)

AREA 3(1,so fated colonies).

St

AREA A-2

(Moderate growth]

APPEARANCE OF STREAK PLATE

AFTER INCUBATION INTERVAL

3-9

MPN Methods

4

0

-TABLE 2. APPARATUS AND SUPPLIES FOR STANDARDFERMENTATION TUBE TESTS

D scription of Item Total Conform Group Fecal CedUorms

Presumptive I ConfirmedTest ' I Test

Completed-Test

-(EC broth)

Lauryl tryptose broth or Lactosebroth. 20 ml amounts of 1.5 XConcentration medium, In 25 X150 mmculture tubes withinverte0 fermeb-tation vials, suitable caps.

Lauryl tryptose broth or Lactosebroth. 10,ml amounts bt singlestrength medium in 20 X 150 mmculture tubes with inverted fermen-tatioti vials. Gullible caps.

.Brilliant green lactose bile broth. 2%

in 10 ml amounts. single strength,in 20 X 150 mm culture tubes with

. inverted fermentation vials,suitable caps.

Eosin methylene blue agar, pouredin 100 X15 mm Petri dishes

Endo Agar, poured in 100 X15 mm' dishes

.1'1'4i-fent agar' slant, scresZ cap tube.-

EC Broth, 10 ml amounts of single .

strength medium in fermentationtub t s:

Culture tube racks. 10 X5 openings; '

each opening to accept 25 mm dia-meter tubes.

Pipettes, 10 ml, Mohr/ type, sterile,

in suitable cans.

Pipettes, 2 ml (optional), Morh typesterile, in suitable cans

Pipette,. 1 ml, Mohr type. sterilein meta.) suitable cans .

Standard buffered dilution water,sterile, 99-ml amounts in screw-capped bottles.

Gas burner, Bunsen type

Inoculation loop, loop 3mm Ma- ,meter, of nichrome or platinuTn-Wt.:limn wire, 26 B & 9 gauge, insuitable bolder. (or sterile *pr./testae'Wick)

Inoculation needle. nichrorne. orplatiOum-iridium wire, 26 B & Szgauge, in suitable older.

Incubator, adjusted to 35 + 0.50C

Waterbath incubator, adjusted to44.6 + 0.2°C.

.

Glass microscopic slides. _1" x3"

Slide racks (optional) -,Gram -stain solutions, complete set

.=Compound microscope, oil Immo-

,ion lens, Abbe' condenser

B.iiket for discarded cultures..,Container for discaNied pipettes. .","'

,--

,

X

..

4',X

X

X

X

X

X

X

X

It

("A

.

I

I

I et

I1

I

I `

.

.

.t

-

t

X

.

X

X

X

.

X

X

X

X

X

-

II'

,

'

.

t

-

.

0

.

.

X

X

X

X

CN

X

X

X

X

X

X

X

X

X

.

--)

,

...

0

X

0

x

_r--

i X i

X.

..

".X

...

t:

.

X

Sri.

.

.

.

49

A

R

9

i44,4

Y.

Part 2ios

DETAILE ASTING-PROCEDURES FOR-MEMBERS OF-THEk --COLrFORM GROUP SY MULTIPLE DILUTION TUBE 'METHODS

.

r SCOPE

A- Tests Desalbed

1 Presumptive Test,

2 Confirmed Test

3 Completed Test

4 Fecal Test rt

B .Fort of,Presentation :

.The P mptive, Ccnifirmebd, andComple Tests aEe presented as total,independent procedures.. It is'y eco'gnizet

-- that this form of presentation ks somewhat'repetittous, inasmuch as the PreiumptiveTest is preliminary to the ConfirmedTest, and both thit,Prestimptive Tist aridthe Confirmed Test are prelintinary to theCompleted Test for total coliforins.

.II TESTING TO PRESUiVIPTIVeTEST

.STAGE

A First -Day Procedures

f Prepare a laboratory data sheet forthe sample. ,Record the followinginformation: assigned laboratorynumber, source of sample, date andtime of collection, temperatuee of the

` source, name of sample collector,date and time of receipt of simple inthe laboratory. Also show the date

/ and time of starting tests in thelaboratory, name(s) of workei(s) Per-

In using these procedures, the workerlast know at the outset-what is to be thestage ate which the test is, lb be, ended, andthe details of the procedures throughout,in order to prevent the possibility, of

t-discarding gas-positive tubes beforeproper transfer procedures have been 'followed.

il'hus, if the worker knows -thatthe test willbe ended at the Confirmed Test,. he willturn at once totSectIon III, TESTING TOTHE CONFIRMED TEST STAGE, and will/ignore Sections II and IV.,

The Fecal Coliform Test is describedseparately, in Section y, as anadjunct to the Confirmed Test and to theCompleted Test.

forming the laboratory tests, and thesample volumes planted.

24*. .

Lebel the tubes of lauryltryptose brothreqtiired for tie initial planting of the

?,. mple (Table 3). The label shouldb r three identifying marks. Theup riitunber is the identification of

'h worker(d) performing the test(applicable to petsonnel in training

.2 Courses), the number immediately. below is the assigned laboratoryAum-

-ber, corresponding with the laboratoryr'ecorcfsheet. The lower -number is 'thecodeLto designate the sample volumeand which tube of a replicate series isIndicated.

NOTE: Be .sure io use tubes containing,the correct concentrations of culture medium .for-the koCulumitube volumes. (See the;Ichapar on media and solutions for multipledilution tube methods or refer to the current,edition of Standard Methods for WaterlindWastewater).

0

MPN Methods

. .

Table.-3.SUGGESTED-LABELING: SCHEME FOR ORIGIN -L CULTURES-AND---SUBCULTURES IN MULTIPLE DILUTION TUBE ESTS

Tube1

Tube2

Tube3

Tube4

;Tube5

Sample volumerepresented

Bench numberVolume & tube

Bench numberVOlume & tube

Bencjt number_

1Volume & tube

.



'312A,

312 -

a

4312_a.

3122a

312z; B

3.12b

31.2 ,

b

3121.13 ld12b

312C

312c

312c

312

3122c

312D

3I2d

312d

312

3122d

312E

3121,e

312e

312le

3122e

Tubes with 10 mlof sample \.

.

Tubes with I mlof sample

Tubes' with 0.1 mlof sample

Tubes with 0.01 mlof sample

.400orTubes with 0:001 ml

of sample..-

The labeling of cultures can be. reduced by labeling only the first tube ofeach eries of identical sample volumes in the initial planting of the sample.

4 All subcultures from initial plantings should be labeled completely.

3 Place the labeled ture tubei in anorderly arrangement iirkculture tuberack, with the tubss intended for thelargest sample volumes in the frontrow, and those intended for smallervolumes in the succeeding rows.

4 Shake the simple Vigorously, approxi-mately 25 tiMes, in an arc of one footwithin seven seconds and withdraw thesample portion at once.

'NS

-5 Measure the'predeterMined sample.'volumes itito-thelabeled-tubes-of-lauryltryptose troth, using care to avoid'intrbdtiction of any'bacteria into-theculture inediumexcept-those in thesample.

XTypical mpleA

iib. Worker

entification

each Number

Sa\ ple Volume'

ube of Culture ediu n

b When usin= the pipet to wit drawsample por ions, do not dip thepipet more han 1/2 inch int thesample; oth rwise sample ru ingdown the out ide of the pipet illmake measu ements inaccurat

6 After measuringsample into theirmedium, gently sinoculated tubes tof sample with theAvoistaig_orous shamay beshaken intovials and 4t ereby in

all portions of threspkective tubes flake the rack of

insure good mixinculture medium.in , as air bubble

f

a Use a 10 ml pipet for 10 ml sampleportions, and 1 ml pipets for portionsof -2- mror less:. Handle sterile' pipetsonly near the mouthpiece, and protectthe delivery end from external con-tamination. Do not remove the cottonplug in the mouthpiece as this isintended to protect the,user fromingesting any sample. .

3-12

he fermentationlidate the test.

7 . Place the rack of ino, u/ated tubes in theincubator at 350 + 0. C for 24 +2 hours. .

24-hour Procedures

1 Remove the rack of lau tryptosebroth cultures from the i ubator, andshake gengy. If gas Is abut to appearin.the ferffiFift4ti% vials, t e shakingwill speed the procbss.

51

_,f

MPN Methods

2 Examine ach tube carefully. Record;in the c umn "24" under LST on thelatiorat ry data sheet, each tube showing

' gas in e fermentation vial as a positive(+) te= and each tube not shoidng gas

. a n gative (-) test. GA's IN ANY 2

. QUAN TY IS A POSITIVE TEST,

e Dipca d all gas-positive tubes of lauryltrypt s se broth, and return all the gas-.negat ye tubes to thq 35°C,incubator for

, an a itional 24 ± 2 hours,

C 48-hour procedures

1 Remthe ipr

ye the rack of culture tubes fromcubator, read and record -gasction for each tube.

2 Bed re to record all results under the48-h ur LTB column on the data sheet.Disc rd 'all tubes. The PresimiptiveTest is concluded at this point, andPres mptive coliforms'rier 100 ml canbe c mputed according to the methodsdesc ibed elsewhere in this manual.

III TESTING TO CONFIRMED TEST STAGE

Note that-the d scriptiodstarts with thesample inocula ;on and includes thePresumptive-Te stage. The Confirmed

. Test preferred i Laboratories of this agencyis accomplished b nieans of the brilliantgreen lactose bile roth (BGLB) and the

'acceptable alternat tests are mentioned inIII F. In addition, e,Fecal Conform Testisincluded as an optionkl adjunct to the procedure,

.

A First- Day Procedues

1 Prepare a labora ry date sheet for thesample. Record t e following,infor-

,mation: assigned laboratory number,source of sample, date and time of

collectiorr, temperature of the source,name of sample collector, date andtime of receipt of sample in thelaborattili. Also show the "date and

I

time of starting tests in the laboratory.name(s) of worker(s) performing thelaboratory tests, and the samplevolumes planted.

Label the tuber:: of lauryl tryptose brothrequired for the initial planting of thesample. The label should bear threeidentifying marks.: The,upper numberis the identification of the worker(s)

.perforrning the test (applicable topersonnel in training courses), thenumber immediately below is theassigned laboratory number, corres-ponding with the laboratory recordsheet. The lower number is the codeto designate the sample volume andwhich tube of a replicate series is indicated.

NOTE: If 10-m1 samples are beingplanted, it is necessary to use tubescontaining the correct concentrationof culture medium,- This has previouslybeen noted in II A-2.

3 Place tie labeled culture tubes in anorderlArrangerdent in a culture tuberack, with the tuhesintanded for thelargest sample volumes in thgfrontrow, and those intended for smallervolumes in the succeeding rows.

4 Shake the sample vigorously, aproxi-mately 25 times, in an up-and- downmotion.

5 --Measure the predeterrnined samplevolumes into the labeled tubes of lauryltryptose broth, using care to avoidintrodUction of any bacteria into theculture medium except those in the sample.

a Use a 10-ml pipet for 10 n4 sampleportions, and 1-ml pipets ter portionsof 1 ml or less. Handle sterile pipetsonly near the mouthpiece, and protectthe delivery end from external con-tamination. Do' not remove the cottonplug &the mouthpiece as this is intendedto protect the user from ingesting anysample.

3-13

"4.

4e

MPN Methods. -

b When using_the pipet to withdrawsample poitions, do not dip thepipet more than 1/a inch into thesample; otherwise sample runningdown the outside of the pipet willmake measurementsinaccurate.

c When delivering the sample into theculture medium, deliver samplepoi-dons of 1 ml or less down intothe culture tube near the surface ofthe medium. Do not deliver smallsample .volunesat the,top of the tubeand allow them to rundown insidethe tube; too much of the samplewill fail to reach the culture medium.

d Prepaie preliminary dilutions ofsamples for portions of 0.01 ml orless before delivery into the culturemedium. See Table 1 for preparationof dirutions. NOTE: Always deliverdiluted sample portions into theculture medium as soon as possibleafter preparation. The intervalbetween preparationof dilution andintroduction of sample into TheMedium never should be. as muchas 30 Minutes.

6 After measuring all portions of thesample into their respective tubes of;medium, gently shake tfie rack ofinoculated tubes to insure good mixingof sample with the culture medium.Avoid vigorous shaking, as air bubblesmay be shaken into the fermentationvials and ihereby invalipte the test.

7 Place the rack of inoculated tulles inthe incubator at 350 0.50C 24.1-2 hours.'

B' 24-hour Procedures

Remove the rick of lauryl tryptosebroigbultures from the incubatOr, yidshake gently. If gas is about to appearin the fermentation vials, the shakingwill speed the process.

P

3

lo

. ; .2 EXElinine *lack tube carefully. Record,

in the column "24":Under LST orilikiftboratci?y data sheet': each tube 'showinggas in the fermentation vial as apositive (+) test and each tube not 'showing gas as a negative (-) test.GAS IN ANY QUANTITY IS A POSITIVETEST.

3 Retain-all gas-positive tubes of lauryltryptose broth culture in their placein the rack, and proceed.

4 Select the gas-positive t es of lauryltryptose broth culture for ConfirmedTest procedures.f Confi med Testprocedures may not be required for allgas-pbsitive cultures. If, after 24-hours

O of incub_qtion,-ill five replicate culturesare gas-positive for two or more con--secutive sample volumes, then selectthe set alive cultures representingthe smallest volume of sample in whichall tubes were gas-positive. ApplyConfirmed Test procedures to all thesecultures and.to any other gas-pOsitive,

-cultures representing smaller volumesof sample, in which some tubes werekas-pesitive and some were. gas-negative.

5 Label 'one-tube of brilliant green lactosebile broth (BGLB) to correspont witheach tube of lauryl tryptose brothselected'for Confirmed Twit procedures.

6 Gently shake the rack of PresumptiveTest cultures. With a flame-sterilizedinoculation-loop transfer one loopful ofculture from Ikach gas- positive tube tothe corresponkpg tube of BGLB. Made'each newly inoculated culture into B(LBin the position of the/original gas- positivetube.

7 After making the tratuafei.s, the rackshould contain .some 24-hour gas-negative tunes of lauryl tryptese brothand theiriVy inoculated BGLB.

8 If the-fecal Coliforg Test is includedin the testing procedures, consult

"Section V of this part of the outline oftesting procedures.

53

e ft

to

'/

:

,

-;

-r

MPN Methods

9 Incubate the 24-hour gas-negativeBGLB tubes andfany newly-inoculatedtubes of BGLI3 an additional 24 + 2hours-at 35o + 0.-5 C.

.

C 48-hour Procedures

1 Rembve the rack of, culture tubes fromthe incubator, read and record gasproduction for each rube.

2 Some tubes will be lauryl tryptose brothand some will be brilliant green lactosebile broth (GLB). Be sure to recordresults from LTB under the 48-hourLTB column and the BGLB results underthe 24-hour column of the data sheet.

3 Label tubes of BGLB to-correspond withall (if any) 48-hour gas-positiok.`, culturesin lauryl tryptose broth. Transfer one

' loopful of culture from each gas-positiveLTB:culture to the correspondingly-

. labeled tube of BGLB. NOTE: Alltubes of LTB culture which werenegative at 24 hours and becamepositiveat 48 hours are to be transferredThe option described above for 24-hourcultures does not apply at 48 hours.

4 If the Fecal-coliform Test is includedin- the testing procedure, consulfSectionN...of the part of the oUtlineof testing procedures. _

5 Incubate the 24-hour gas-negativBGLB tubes and any newly-bloc tedtubes of BGLB 24 + 2 hours at 35o0. 50 C.

6 Discard all tubes of LTB and all 24 -fourgas-positive BGLB cultures.

D 72-hbur Procedures' ,

1 If any, cultures remain to be examined,all will he BGLB. Some may be 24 .

vi)

hours old and some may be48.hoursold.' Remove such cultures from theincubators examine each tube for gasproduction, and record results on thedata sheet.

2 Be sure to record the results pf-24-hourBGLB cultures in the "24" column underBGLB and the 48-hour results under the1'48" column of the dita sheet.

3. Return any 24-hour gas-negative culturesfor incubation 24 + 2 hours at 35 +0. 50 C. .

4 Discard all gas-positive BGLB culturesand all 48-hour gas-xtegative culturesfrom BGLB.

' S

A5 It Is possible that all cultural work andresults for the Confirmed Test havebeen finished at this point. rf so, codifyresults anti determine Confirmed Testcoliforms per 100 ml as described inthe outline on use of MPN Tables.

E 96-hour Procedures,

At most only a few 48-hour cultures inBGLB may be present. Read and recordgas production of such cultures' in the "48"column .under BGLB on the data sheet.Codify resultaand,determine Gonfirmed,Test coliforms pet 100 ml.

F Streak-plate methods for the ConfirmedTest using eosin methyle u orEndo agar cepted proce esin Standard Methods. The worker whoprefers le.use one of these media inpreference to BGLB (also approved inStandard Methods) is advised to refer tothe current edition of "Standard Methods.for the Examination of Water and Waste-water" for procedures.

a.4'

54A3-15

O

1001

MPN Methods `

IV TESTING TO C9MPLETED TEST STAGE

(Note that this description starts with thesample inoculation and proceeds throngh thePresumptive and the Confirmed Test stages.In addition, the Fecal Coliform Test isreferred to as an optional adjunct to theprocedure. )

A First-Day Procedurest o

1 Prepare a laboratory data sheet for thesample. lie,cord the following information:assigned-laboiatory number, source ofsample, date and time of collection,temperature of the source, name ofsample collector, date and time ofreceipt of sample in the laboratory.Also show the date ancrtime of starting*tests in the laboratory, name(s) ofworker(s) performing the laboratorytests, and the sample volumes planted.

2 Label the tubes of lauryl tryptose brothrequired for the initial planting of thesample. The label should bear threeidentifying marks. The upper numberis the identification of the worker(s).performing the test (applicable'topersonnel in training courses),the number immediately below is the-

assigned laboratory number, airres-ponding with the laboratory record

-sheet. The lower number is the codeto designate the sample volume and s

which tube of a repliCate series isindicated. Guidance on labeling far .

laboratory data number and identificationof individualtubes is described else-where in this outline.

-3-16

(

7

NOTE: If 10-ml samples are being,plated, it is. necessary to use tubescontaining the correct concentrationof .culture medium. This has previouslybeen noted elsewhere in this outlineand referral is made td tables.

3 Place the labeled culture tubes in anorderly arrangement in a culture tuberack, with the tubes intended for thelargest sample volumes in the frontrow, and 'those intended for smallervolumes in the succeeding rows.

4 Shake the sample vigorously, approxi-mately 25 times, in an up-and-downmotion.

5 Measure the predetermined samplevolumes into the labeled tubes of, lauryltryptose broth, using care to avoidintroduction of any bacteria into theculture medium except those in thesample.

a Use a 10-m1 pipet for 10 ml sample .portions, and 1 -ml pipets for portionsof 1 ml or less. Handle- sterilepipets only near the mouthpiece,and protect the delivery end fromexternal contamination. Do not movethe botton plug in the mouthpiece.s this is intended to protect theuser from ingesting any sample.

b When using the pipet to withdrawSample portions; do not dip thepipet more than 1/2 inch into thesample; otherwise sample runningdown the outside of the pipet willmake measurements inaccurate.

c When delivering the sample into theculture medium,. deliver sampleportions of leml or less down into

,

r

O

s

the culture tube i'mar the surface ofthe medium. Do not deliVer smallSample volumes 'at the top of thetube and allow them to run downinside the tube; too much of thesample will fail to.reach the culture'medium.

d Prepare preliminarylklutions ofsamples for portioni of 0.01 ml orless before delivery into the culturemedium. SeTable 2 for preparationof dilutions. NOTE: Always- dtiverdiluted sample portions into theculture medium as soon as possibleafter preparation. The interval _

_between preparation of dilution andintroduction, of sample into themedium never should be as much as30 minutes'.

6 After measuring all portions of thesample- into their respective tubes ofmedium; erl ja shake the rack ofinoculated tubes to insure gOod mixingof sample with the culture medium. ,

. Avoid vigorous shaking, as air bubblesmay be shdken into the fermentationvials and thereby invalidate the test.

7 .Place the rack of inoculated tubes inthe incubator at 350 +_ 0.50C for 24 +2 hours.

B 24-hour Procedures

1 Remove the r ck of lauryl tryptose brothcultures from e incubator, and shakegently. If gas is about to appear in thefermentation vials, the shaking willspeed the prress.

2' lExamine each tube carefully. Record,in the column, "24" under LST on thelaboratory data sheet, each tube showinggas in the fermentation vial asalpositive

6 (+) test and each tube not shoviing gasas a negative (-) test. GAS INTA'NYQVANTITY IS 'A POSITIVE TEST.

?v43 Retain all gas-positive tubes of lattryltryptose broth culture in their place inthe rack, and koceed.

. . . ,

ti

MPN Methods

4 Select the gas-Posithie tubes of lauryltryptose broth culture for the ConfirmedTest ,procedures. Confirmed Testprocedures may not be 'required fbi'all gas-pOsitive cultures. If, after24-hours of incubation, all fivereplicate cultures are gasrpositive fortwo more 'consecutive samplevolumes, then selectthe set of fivecultures representing the smallestvolume of sample in which all tubeswere gas-positive. Apply Confirmed*Test proCedures to all,these culturesand to any other gas-positive culturesrepresenting smaller volumes ofsample, in which some tubes weregas-positive and some were 'as-negative.

Label one tube of brilliant green lactosebile broth (BGLB) to correspond witheach tubei of lauryl tryptose brothselected for Confirmed Test procedures

1

6 Gently shake the rack of PresumptiveTest cultUres. With a flame-sterilizedinoculation leop transfer one loopful ofculture from each gas-positive tube tothe' corresponding tube of BLB. Placeeach newly inoculated culture intoBGLB in the position of the'originalgas-positive tube.

7 If the Fecal Coliform Test is includedin the testing procedure; consultSection V of this Outline for details ofthd testing proce /ure.

8 After making he transfer, the rack13hould contain &me 24-hOur gas-negatiye tubes f lauryl tryptose berthand the newly inoculated BGLB.Incubate the rack of cultures at 350 C+ 0. 50 C for 24 + 2 halt's.

5

C 48-hpur ProceduresN

1 Remove the rack of culture tubes fromt he incubator, read and record gasproduction for each tube.

2 Some tubes will be lauryl'tryptose brothand some will be brilliant green lactose

56

t.

S

.1V

4,

MPN Methods

bilte,broth (BGLB). Bs sure to recordresults from LTB under the 48-hourLTB column and the BGLB resultstinder the 24 -hour column of the datasheet.

3 Label tubes of BGLB to correspond withall (if any) 48-hour gas-positive culturesin lauryl tryptose broth. Transfer oneloopful of culture from each gas-positiveLTB cultu1e to the correspondiligly-

. labeled tube of BGLB. .,,,NOTE: All tubesof LTB culture which were negative at24 hours and became positive at 48 hoursare to be transferred. The Optiondescribed above for 24-hour LTBcultures does not apply at 48 hours.

.

4 Incubate the 24-hour gas-negative BGLBtubes and any newly-inoculated tubes ofBGLB 24 + 2 hours at 350 + 0.50,C.Retain all 24-hour gas-positive culturesin BGLB for further test procedures.

5 Label a Petri' dish preparation of eosinmethylene hlu'd agar (EMB agar) tocorrespond with each gas-positiveculture in BGLB.

6 Prepare a 'streak plate for colonyisolation frOm each gas-positive culturein BGLB on the correbpo'ndingly- labeledEMB agar plate.

Incubate the EMB agar plates 2A + 2hours at 350 + 0.50C.

.

D 72-hour Procedures

1 Remove the culture's from the incubator.Some may be on BGLB; several EMBagar plates also can be expected.

2 Examine and ecord gas productionresults for an cultures in BGLB.

3 Retain any gas-p itive,BGLB culturesand prepare streak late inoculations,for colony isolation EMB'sgar.Incubate the EMB agar plb.tes 24 +2 hours at 35 + 0.50 C. Discardllie'''7.gas-positive BGLB cultures site%transfer.

3-18

9Reincubate-any gas-negative BGLBcultures 24 + 2 hours at 350 + 0.50C.

5 Discard all 48-hour gas-negative BGLBcultures.

es.

6

rExamine the EMB'agar plates for thetype of coloniv developed thereon.Well-isolated colonies- having a darkcenter.(when viewed from the lowerside, held toward a7light) are termed"nucleated or fisheye" colonies, andare regarded as "typical" conform

colonies. A surface sheen may or. maynot'be loresent on "typical" colonies.Colonies which are pink or opaqtke butare not nucleated are regarded as"atypical colonies." Other colonytypes are considered "noncolifoim. "

- Read and record results as + for"typical" (nucleated) colonies for"atypical" (non-nucleated pink oropaque colonies), and - for other typesof colonies which might develop.

7 With plates bearing "typiail" colonies,select at least one well- isolated colonyand transfer it to a correspondingly-labeled tube of lactose broth and to anagar slant. As a second choice, selectat least two "atypical" colonies (iftypical colonies are not present) andtransfey,thim to labeled tubes oflactose Ifro,t and to agar slants. As athird-choice, in the absence of typicalor atypical cbliform-like colonies,select at least two well-isolatedcolonies representative of thoseappearing on the EMB plate, and trans-fer them to lactose broth and to agarslants.

Incnbate* all pultures transfered fromEMB agar plates 24 + 2 hours at 35 +0.50 C.

E 96-hour Procedures

1 Subcultures from the Samples beingstudied may include: 48-hour tubesof BGLB, EMB agar' plates, lactose .

broth tubes, and agar slant cultures.

5

MPN Metha4s

2 If any 48-hour tubes of BGLB arepresent, read and record.gas production"in the "48" column under BGLB. Fromany gas-positive BGLB cultures pre-pare streak plate inoculations for colonyisolation on EMB agar.. Discard alltubes of BGLB, and incubate EMB agarplates 24 -+ 2 hours at 35 + 0.5°C.

3 If any EMB plates are present, examineandrecord results in the "EMB" columnof the data sheet. Make transfers toagar slants and to lactose broth fromall EMB agar plate cultures. Indecreasing order of preference, transferat leant one typical colony, or at leasttwo atypical colonies, or at least twocolonies rem:esentative of those on theplate.

4 Examine and record results from thelactosi brcith cultures.

5 Prepare a Gram-stained smear fromeach of the agar slant cultures, asfollows:

NOTE: Always prepare Gram stainfrom an actively growing culture,preferably about 18 hours old, and

.never more than 24 hours old. ,Failureto observe this precaution often resultsin irregular staining reactions.

a Thoroughly clean a glass slide tofree it of any trace of oily film.

b Place one drop of distilled water onthe slide.

c Use the inoculation needle to suspenda Iftyi amount of growth from thenutrient agar slant culture in thedrop of water.

d Vfix the thin suspension of cells withthe tip of the inoculation needle, and.allow the water evaporate.

e "Fix" the smear by gently warmingthe slide over a flame.

f Stain the smear by flooding it for 1minute with ammonium oxylate-crystalviolet solution.

tg Flush the excess dye splution

off in gently running water.

h Flood the smear with Lugol'siodine for 1 minute.

i Wash the slide in gently runningwater.

Decolorize the smear with acetonealcohol solution with gentle agitationfor10-30 seconds, depending uponextentof removal of crystal violet dye.

k Counterstain for 10 seconds withsafranin solution, then wash in running

-water and gently blot dry with bibulouspaper.

.1 Examine the slide under the microscope,using the oil immersion lens. Coliformbacteria-are Gram-negative (pink to redcolor and nonspore-forming, rod-shapedcells, occurring singly, in pairs, orrarely in. short chains.

m If typical coliform staining reaction, andmorphology are observed, record 4 inin the appropriate space under the "GramStain" column of the data sheet. If typicalmorphology and staining reaction are notobserved, then mark 'it + or -, and makesuitable comment in theThremarks'l columnat the right-hand side of_the-data sheet.

n If spore-formidg bacteria are observed, itwill be necessary to repurify the culturefrom which the observations were made.Consult the instructor, or refer to StandardMethods, for procedures.

At this pqint, it is possible that all culturalwork for the Completed Test, has been finished.If. so, codify results and determine CompletedTest coliforms per -100 ml.

'56

3-19

r'

ti

t

MPN Methods

F 120-hour Procedures and following:

1 Any procedures to be undertaken fromthis point are "straggler" cultures onmedia already described, and requiringstep-by-step methodology already giveniii detail. Such cultures may be on:EMB plates, agar, slants,, or lactosebroth, The same time-and-temperatureof incubation required for earlier studiesapplies to the "stragglers" as do theobservations, staining reactibns, andinterpretation of results. On con-clusion of all cultural procedures,codify, results and determine CompletedTest coliforms per 100 ml.

V FECAL COLIFORM TEST

A General Information L1 The procedure described is an elevated

temperature test for fecal conform`bacteria.

2 Equipment required for the tests arL.those'required for the PresumptiveTest of Standard Methods, a water-bathincubator, and the appropriate culturemedia.

B Fecal Coliform Test with EQ Broth

1 Sample: The test is applied to gas-positive tubes from the StandardMethods Presumptive Test (lauryltryptose broth), in parallel withConfirmed Test procedures.

2 24-hour Operations. Initial proceduresare the planting procedures describedfor the Standard Methods PresumptiveColiform test.

a After reading and recording gas-production on lauryl tryptose broth,temporarily retain all gas-positivetithes.

b Label a tube of EC broth to corre-spond with each gas-positive tubeof lauryl tryptose broth. The optionregarding transfer of only a limited

3-20

number.of tubes to the ConfirmedTest sometimes can be applied here.However, the worker is urge&40avoid exercise of this option untilhe has assured the applicability ofthe option by preliminary testa onthe sample source.

c Transfer one loopful of culture fromeach gas-positive culture in lauryltryptose broth to the correspondinglylabeled tube of EC broth.

d Incubate EC broth tubes 24 + 2 houi-sat 44.5 .2. 0.2°C in a waterbathwith water depth sufficient to comeup at least as high as the top of thecultu?e medium in the tubes. Placein waterbath as soon as possibleafter inoculation and always within 430 minutes after inoculation.

3 48-hour operations

a Remove the rack of EC culturesfrom the waterbath, shake gently,and record gas production for eachtube. Gas:in any quantity is apositive test.

b As soon as results are recorded,discard all tubed. (This is a 24-hour tesefor EC broth inoculafionsand not a 48 -hour test. )

c Transfer any additional 48-hourgas-positive tubes of laup'l tryptosebroth to correspondingly labeledtubes of EC. broth. Incubate 24 +2 hours at 44.5.1 0.2 °C.

4 72-hour operations

a Read and record gas production foreach tube. Discard all cultures.

b Codify results and determine fecalconform co'unt per 100 ml of sm.-Lyle,

59

.sr

Examination of Water for Co Worth and Fecal Streptococcus Groups

TESTS FOR COLIFORM GROUP

ISAMPLE I r

LACTOSE OR LAUREL ERMINE MTNFERMENTATION TUBES (SERIErt DILUTION)

GAS POSITIVE124 id 3 2 NI.)

1

1

1

OAS NEGATIVE

I

OAS POSITIVE

;:.

OAS NEGATIVECOLIFORM GROUP

ABSENT

CONFIRMATORY POEN11111LUANT GREEN LACTOSE TILE

EMB PLATESOR ENDOAGAR PLATES

ALTERNATECONFIRMED

OAS POSITIVE,. 1 COLIFORM GROUP

1 CONFIRMED

OAS NEGATIVE

COLIFORM GROUPNOT CONFIRMED

LACTOSE IL LAGIVEL TRYPTOSE

SA0111 ARE INTERCHANGEABLE MEDIA

AND ARE INCUBATED AT 35 DEG C30 5 DEG C

OAS POSITIVES TUBES (ANY AMOUNTOF GAS) CONSTITUTE A POSITIVE

RESUMPTIVE tiff

TOTAL INCUBATION TIME FOR LACTOSE011Eli IS 41 NRS 3.3 NOS

INCUBATE SOLI TUBES FOR 41 Nrs3 NRS AT 35 DEG C3 0 5 DEO C

INCUBATE EMS oit ENDO AGAR

PLATES FOR 24 NU 2 2 FIRS AT35 DEG. C3 0 5 DEG C

NUTRIENT AGAR SLANT LACTOSE &ROTH TUBE/ N \ORAN AND GRAM NEGATIVE GAS POSITIVE OAS NEGATIVESODS AND/Olt RODS NO SPORES

. '"

COLIFORM GROUPSORE/ORMERS ABSENT

Is COLIFORM GROUP PRESENTCOMPLETED TEST

IF ALSO IF ALSOGAS POS ME OAS NEGATIVE

IN LACTOSE IN LACTOSE

TRANSFER 10 EMS PLATEAND REPEAT PROCESS

COUFORM GROUP ABSENT

Nos

Part 3LABORATORY METHODS FOR-FECAL STREPTOCOCCUS

(Day-By-Day Procedures)

I GENERAL INFORMATION

A The same sampling and holding proceduresapply as for the coliform test.

B The number of fecal streptococdfin watergenerally is lower than the number ofcoliform bacteria.- It is good practicein multiple dilution tube tests to start thesample planting series with one sampleincrement_largertban for the coliform _test. For example: If a sample plantingseries of 1.0, 0.1, 0.01, and 0.001 mlis planned for the coliform test, it issuggested that a series of 10, 1.0, 0.1,and.0.01.rill be planted ,for the fecalstreptococcus test..

C. Equipment requir4d for the test is the sameas required for the Standard MethodsPresumptive and Confirmed,Tests, exceptfor the differences in culture media.

II STANDARD METHODS (Tentative)PROCEDURES

A First-Day Operations

41,

1 Prepare the sample data sheet andlabeled 'tubes of azide dextrose brothin the same rnamier, as fossthePresumptive Test. NOTE; If 10-mlsamples are included in the series, besure to use a Special concentration

_(ordinarily doublestrength)of azide.dextrose broth for these sample,portions.

2 Shake the sample vigorously, approxi-mately 25 times,, in an up-and-downmotion.

=3 Measure the predetermined samplevolumes into the labeled tubes of azidedextrose broth, using the samplemeasurement and delivery techniquesused for the Presumptive Test.

4 Shake the rack of tubes of inoculatedculture media, to insure good mixingof sample- With medium.

5 Place the rack of inoculated tubes inthe incubator at 350 + 0.50 C for 24 +2 hours.

B 24-hour Operations

.61 ,

1. Remove the- rack of tubes from the ..

incubator. Read and record the resultsfrom etch tube. Growth is a positivetest with this test. Evidence of growthqnsists either of turbidity of theedium, .a "button" of sediment at the

bottom of'the culture tube, or both..Label a tube of ethyl violet azide brothto correspond with each positive culture' '-of azide dextrose broth. It may If tpermissible to use the same confirmatory

. transfer option as described for thecoliform Confirmed Test, in this outline.

3 Shake the rack of cultures gently, toresuspend cells which have settled ..,out to the bottom of the culture tubes.

2

4 Transfer three loopfuls or use awood applicator to transfer 'culturefrom each growth-positive tube ofazide dextrose broth to the correspond-ingly labeled tube of ethyl violet azidebroth.

5 As transfers are m ade, place the newlyinoculated tubes of ethyl violet azide_broth in a separate rack while returningtheAp. tubes to their former positionsin the rack.

6 Return the rack, all azide dextrosebroth tubes and newly-inodulatedInbesof ethyl-violitlizidetroth, to-the in-cubator. Incubate 24 + 2 hours at 35°+ 0. 5°C.

,

3-23 4

a

MPN Methbds

C 4'8-hoUr Operations

1 ,Remove the raciOf tubes. from thtincubator. Read-and report results.Growth, either in azide dextrose brothor j.n.,ethyl violet azide broth, is ap(isitive test. Be, sure to report the ,;results of the azide dextrose brothmedium under the "48" column for ahatmedium and the results of the ethyl-violet azide broth cultures under the"24" column for that medium.

2 Any 48-hour growth-positive culturesof azide dextrose broth are to betransferred (as before) to ethylvalet azide broth.' Discard all 48-hour'growth-negatiA tubes of azide dextrosebroth and all 24-hbur growth-positivetubes of ethyl violet azide broth.

3 Re-incubate the 24.-hour growth-negativeEthyl Violet azide tubes after again re-inoculating with their respective positiveAzide Dextra`se tubes and the Awly-inoculated tubes of ethyl violet azide .broth 24 + Aours at 35° + 0.5°C.

--

D 72-hour Oiier.ations

1 Read and report growth results of alltubes of ethyl violet azide broth.

2 Discard all growth-positive culturesand all 48-hour growth-negativecultures.

3 Reincubate any 24 -hour growth.:negativecultures in ethyl violet azide broth afterreinoculating with their respectivepositive azide dextrose! tubes- for anadditional 24 + 2thours at 35° + 0.5°C.

E 96-hour Operations

1 Read and report growth results of anytemaining tubei of ethyl violet azide

,broth.t

3-24.

2 Codify results and determine fecalstreptocotbi per 100 ml.

REFERENCES

1 Standard Methods for the Exarni,nati"o"n ofWatei and Wastewater (14th Ed).-Prepared and published jointly byAmerican Public Health Association,American Water Works Association,and Water Pollution Control'Federation. 1975.

2 Geldreich, E. E. , Clark, H. F. , Kabler,P. W. , Huff;. Q.B. and Bordner, R.H.The Colifortin Group. IL ,Reactionsin EC Medium at 450C. Appl.Microbiol. 8:347-348. 1958.

3 Geldreich, E. E. , Bordner, R. H. , Huff,CAI., Clark, H.F. and Kabler, P.W.Type Distribution of Conform Bacteriain the Feces of Warm-Blooded Animals.J. Water Pollution Control Federation.34:295-301. 1962.

This outline was prepared by H. L.Jeter, *id. Program SupportTraining Branch, USEPA, Cincinnati,

Ohio 45268

Descriptors: Coliforms, Fecal ColiformsFecal Streptococci, Indicator Bacteria,Laboratory Equipment, LaboratoryTests, Microbiology, Most ProbableNqmber, MPN, Sewage Bacteria,Water Analysis

624

MEDIA AND SOLUTIONS FOR MULTIPLE DILUTION TUBE METHODS

I INTRODUCT ION

A This chapter is intended to present, detailedinformation on preparation and managementof media and solutions needed*with the testsand observations described elsewhere inthis course manual.

B The preparation and management ofsupplies of culture media and solutionsis one hf the most critical aspects of abacteriological water quality testingprogram.

1 In the same manner that the chemist.relies on correctly prepared'andstandardized reagents .for- his analyticalwork: the bacteriologist must depend kl

on satisfactory culture media for thetype of analysis with which he is con-.cerned.

. ,2 In many -laboratories Preparation,of

media-is entrusted to subprofdasionalperSonnel. Most such personnel,properly trained and guided, 4'e ableto perform the required tasks efficientlyand reliably.

3 The professional supervisor shouldmaintain close attention to all details,however, to guard against gradualintroduction of bad habits, in preparingand preserving media and.othersupplies.

II GENERAL INFORMATION'

A Use of Commercially AvailableDehydrated Media

A1 The preparation of all, media deperibed

in this chapter is given in termis-of the

J

11

individual components, and preparationof the finished medium. This is done,even through commercially availableathydrated media-are widely used, toacquaint the worker with the compo-sition of the media and to indicate therequired specifications of each medium*.

2 The use of commercially availabledehydrated media, requiring onlycareful weighing and dissolving of thepowder in the proper quantity andequality of distilled water, is stronglyrecommended. Such media-are muchmore likely to have uniformity at anatceptably high level of quality thanare media compounded in the laboratoryfrom the individual constituents.

3 It is recommended that the worker,when using commercially prepareddehydrated media, keep a carefulrecord of the lot numberi'of Mediabeing used. With first use of each .

new lot number of a given medium, itis suggested that the medium be checkedfor stability, pH after sterilization,arid to, see that performance is satis-facter'y. 'While rare, an occasionallot of medium will have some unforeseenfault which reduces or destroys itseffectiveness. Maintenance of lotnumber records on medium givesopportunity for communication with ,

the manufacturer to determine whethersimilar,problems are being encounteredin other laboratories.

.B Quality of General Materials

1 Distilled water

Distilled water, or demineralized water,Wrequired. It must be free from

NOTE: Mention of commercial procUfe and manufacturer's is for illustration and does not implyendorsement by the Environmental Protection Agency.

W. BA. met. 19e, a, 78 63 4-1

F

Media a,nd Solutioni for Multiple Dilution Tube Methods

Q.

dissolved_me.tals or chlorine. Freedomfrom bactericidal constituents or growth,promoting substances Should;:e dem-onstrated through laboratory Tests .A procedure for this test is describedelsewhere in-this course manual.

2 Beef extract

Any brand of beef extract is acceptable,provided that it is knoWn to give resultsacceptable fo the-user. Meat'infusionis not acceptable..

3 Peptones

Peptones are sold under a wide varietyof trade.names. Any peptone shownsatfsfactbry.by comp-a,rative tests withanacceptible peptone, maybe accepted.

4 Sugars4

All sugars must-be cherhically pure,'and suitable foi; bacteriological medi

5 Agar:

. Any form of bacteriologic- grade of- .

= Agar can beused.

6 'General chemicals- must be reagentgrade or A-CS if used in culture media.Chemicals used in the distilled waterquality-test must be of the highest purityavailable.

7 Dyes

AU dyes used in culture media must becertified by the Biologica.l'Stain

-=Commission- they will be so labeledon the container:"-.^

C Quality of Equipment and Supplies Usedfor: Preparation of Media _

1 glassware ,t a.. .. ,,

..itis recommended that all glassware.

beof borosilicate glass. Such glassis not subject to release of solubleproducts into the culture medium, aswith -aome of the so-called "soft glass. "

O

(

2 Balance

A balance with sensitivity of ± 2 grams}with a load of 150 grams is the minimumacceptable standard fors weighing ofculture, media in dehydrated form.

3 pH meter

An electrometric meter is recommended.While a comparator block with pHindicator solutions is useful for suchmedia as lauryl tryptosebroth, itcannot be used satisfactorily with dye-contain' g media such as : brilliant:green la tole bile broth. Thereforeit is silk ested that all pH controlwork on baMeriological media be donewith an electrometric type of pH meter.Accuracy of the-meter should be estab-lished thrbugh calibration against astandard buffer.

4 Autoclave

The autoclave shOuld be of sufficientsize to permit loose packing of tubedmedia when normal load is beingsterilized. This is to permit freeaccess of steam to'a.lisurfaces.

Operation should be such that sterilizingtemperature is reached in not more than30 minutes. s.

A pressure gauge should be present.More important, the autoclave shouldbe equipped with at least 1 thermometer,which should be located properly in theexhaust line.

Pressee regulation should permitoperation up to and-including 1210C.

-When Medie, containing carbohydratesare present, sterilization should becontinued e12. - 15-minutes; in rriedia,not containing carbohydrates, normalsterilization time should be a standard15 minittes.Me .

After sterilization, media, should be,removed from, the autoclave as soonas possible. In no case should anautoclave simply be turned off after

644

Media anc

X

Soltitions for Multiple Dilution Tube Methods

the usual exposure'to.steam tinderpressure, and allowed to stand until thefollowing morning betbre .removing media.

Ei

5 Utensils for mixing and prepai^ing media

Borosilicate glass 54suggested, butother materials, such its stainlesssteel, :porCelain (unchipped) containers,or other containers free of solublebactericidal or bacteriostatic materials,are acceptable. In any case, the con-tainers must be thoroughly clean.

III CONCENTRATION OF MEDIA

A Basic formulas of all media described inSection IV are presented as single-strength

' media. Most media are used in the single-strength. concentration.

B The concentration of primary, inoculationmedia (media into which the measuredportions of the original sample. aredelivered) 'requires special consideration.

1 When the amount of medium is 10 ml orgreater, and the volume of sample orsample dilution is 1 ml or less, thensingle- strength medium is satisfactory..

2 When the sample volume introducedinto the primary inoculation mediumis greater than 1 ml, then.it is necessaryto aompensatefor the diluting effectof the sample on the culture medium.In such cases, it itt necessary toincrease -the initial concentration ofthe medium so that after sampleinoculation the concentration of nutrientsiyn mearumLpius-sample is equivalent'to the concentration of nutrients in thesingle strength medium.

IV PREPARATION OF MEDIA AND SOLUTIONS

A Lauryl Tryptose Broth (Lauryl Sulfate Broth)

1 Use: Primary inoculttion medium inPresumptive Test

65

2 Composition:

Tryptose (or Trypticaseor equivalent)

Lactose

420. 0 g

5.0 gDibasic Potassium 2.75 gPhosphate (K2HPO4)Monobasic Potassium 2.75 gPhosphate (KH2PO4)Sodium- Chloride 5M gSodium Lauryl Sulfate (1.1 g(Total Dry Constituents ' `35 60 g)Distilled-Water .1.000 ml

Sterilization.: 12 - 15 minutes at 1210 CReaction after sterilization: pH 6.8approximately)

3 Compensation for diluting effect Ofsamples

No, mlmediumin tube

M1 ofsample ordilution

Nominalconcentra-tion beforeinoculation

No. gramsdehydratedmedium perliter

10 0.1 - 1.0 lx 35.610 10 ' 2x 71. 220 10 1. 5x 53.435 100 4x 137.3

B Brilliant Green Lactose Bile Broth

1- -Ise: Confirmed Test.

2 Composition

Peptone (Bacto or equivalent)LaCtose0411 (dehydrated)Brilliant Green'(Total weight dry constituents

Distilled.Water

10.010.0 g20.0 g0.0133 g

40. 0133 g)

1000 ml

Sterilization: 12 - 15 minutes at 1210 CReaction after sterilization: pH 7.1 to 7.4

\

A

A

Media and Solutions for Multiple Diluhon Tube Methods

C Eosin Methylene -Blue A r (Levine-4Modification)

1 COnfirmed Test

Use: 'Isolation of colitorm-likecolonies as a preliminary.toCompleted Test procedures t

.'2' Coniposition ,

Peptone (Bacto or equivalent) 10 gLactose 10 gDipotassium Phosphate (K2HPO4)Agar 20 gEoSin Y 'gMethylene'Blue O:65 g

s.ec(Tcital weight dry constituents 43 :.05 g)

Distilled Water . 1000 ml

Sterilization: `2 -15 minutes at. 1210C

. ,

3 Special suggestions on preparation:

a' This mediuin can be prepared anddispensed into bottles or flasks inportions of lop ml or 200 ml each.The sterile medium .may be storedfor extended,periods in cool placesout of the light.

b When ready_for,use of such medium,the medium. should be melted byimmersion 'ofthe bottle of preparedmedium in a boiling water bath,after which it is dispensed into.sterile Petri dishes in portions ofapproximately 15 ml. After coolingand solidifying in the Petridish, th6medium is ready for, use. It shduldbused preferably on the day it ispoured into Petri dishes, but can bestored foxl a day or two in therefif gerator.

c) An alternate method of preparingthis medium reciuires preparationthe agar hasemedium whichincludes ali =thg constituentsof the mediumxcept the dyes.When ready to use such a prpparation,,the agar babe medium is melted ina water bath, and to each 100 ml ofthe melted agar base medium, 2 mlof 2% of-aqueous solution of eosin Yand 1.3 ml of 0. 5% tiiethylene blue

S.

44.

solution is delivered with a pipet.The medium islmixed.thoroughlY,poured into Petri dishes, and Used4s. previpusly described.

D Agar Slants

1 Use: This medium is used in theCompleted-Test, to cultivate pure

_ cultures of strains of babteria beingcultivated in preparation of a Gram-

. stained,smear.

2 Composition: The medium is nutrient,agar

Pe oneBe f extractAg r(Total weight dryconstituents

5.0 'g3.0 g

15.0 g23.0 g)

Distilled Water 1000 ml

Sterilization: 15 minutes at 1210 C

Reaction after sterilization: pH 6.8'approximately

3 Special instructions: Dissolve the'con-stituents, using heat as neede'd; dispensein amounts of approximately 8 ml pertube. Screw-capped tubes extend shelflife of the medium. After sterilization,remove the melted'medium from the

.autoclave and place in a slantingposition until the Medium hasebecomeSolidified. A routine procedure shouldbe established so that a uniforni volumeof medium and a uniform surface ofslanted medium be pregent in each tube.While this has no particular bearing onStandard Methods procedures, certainother laboratory procedures doyequireuniform exposed surface area of theslanted mediiim.

ti

E Plate Count A gars, .

1 Use: This medium is used in thedistilled wateritest. It is npt used inother Standard Method8 proceduresdescribed in this course manual.

66

.

Media and Solutions for Multi le- ution Tube Methods

2 Composition: (Tryptone GlucoseYeast Agar)

,Peptone-,tryptone or eqUivalent) 5.0 gYeast extract 2.5 gGlucose '(dextrose)Agar( Total weight--,dryconstituents


Sterilization: 15 minutes at 1210C

1.0 g15.0823.5 g

Reaction after sterilization: pH 7,. 0 +-0.1

3 Special instructions in preparation:Use heat as needed to dissolve andmelt the constituents. Dispense t4erLmediuM in flasks or bottles in portalsof 100 or 200 ml each and sterilize. Inthis state it can be preserved for many'months, provided that it is protectedfrom e,vapbration of the water.

atWhen ready to use", melt the mediumby;heating, and cool to 4500. At thistemperature the medium still should f:It -melted, and will be satisfacotry forpreparation of pour plates fdr platecounts, '

.4

s4 r,

.4P I

.3 This medium is dispensed into culture

tubes with invertedlermentation vials,and suitable caps.

G Azide Dexinse Broth

'1 Use: Primary'inoculation medium forfecal streptococCal presumptive test.

22Composition:

Beef extract -N

' Tryptone or PolyPeptoneGlucoieSodium chlorideSodium a zide(Total dry constituents-

4.5 g15. g7.5 g7.5 g0.2 g

34.7 g)


Sterilization: 12 - 15 minutes at 1210 CtiJ

Reaction after sterilization: about pH 7.2.3 FerMentation vials are not used with

azide dextrose broth.P °

H Ethyl Violet Azide Broth

'4; Use: Confirmed test for, fecal.streptococci

4

F EC Broth: .A t. ,..,-

, ,1 Ude: Test for lecal_coliform bacte"riC. ;. , .. ,.. , . ,.. .

' 2 omdosition . ;-e- i t . . .

.,ATryptone (Be ctO or' 19,uivatit)' -.4. 20.0 F

. .

Lactose' 5.0 g..- Bile Salts (Bacto' #3 oyeliui ent)v 1.5 g

t DiAtsssium Phosphate iK2H 0) 4.0 g. MoriOpotasdium giOdpliatel 04 m

), 1 5 ges i t

Sodiuinchlori,d'e f 4 -r . 5'. Q g:.1(Totariteight7dry Constifdents : .^.37.0;g)...:: .' 1 k' :1

.1% Distilled Water't,

1000 ml.

-Sterilizatioh: 12 - 15 minutes at 1210C. -"'.. .

2 ,CompositiOn:

Tr.y.ptone oF'BiosateGlucose-Sodium chloride

20 g5 .g5 g

lbotassiem.phosphatg, 2.7 gdiabasic (K2HPO4),

Potassium'phosphate, 2.7 gZ- in,..onobasip°(KH....P0 )

Sodium .azide,. Ethyl violet (certified 'dye

if 'available)(TOtal dry constituents

* .Reaction after:sterilization:0'pH 6..9*

1.

bistilled Water

sterilization: 1 -e

0.4- g. 00083 g

35..84040.

100Q ml

15 m inutes at 1210 C

Reactiori after sterilization: about pH 74

of

Media and Solutions for Multiple Dilution Tube Methods.

.

3 Fermentation vials are riot used withethyl,violet az ide broth.

I Buffered Dilution Water

1 Use: Preparation of samp le dilutionspreliminary to primary inoculation, inmembrane filter work, and in platecourts.

2 COmposition

a Stock phosphate buffer solution

Monobasic PotassiumPhosphate (KH2PO4)

Distilled Water

34.0 g

500 ml

1N NaOl solution (about 175 ml)to give pH . 2

Distilledwffter sufficient to bring finalvolume to 1000 ml

b Working solution of phosphate buffered. distilled' water 41

.-Stock phosphate buffei;solutail

Distilled water 1000 ml

13 Preparation acid handling:

a StOck solution: After preparation thestock solution should be stored in therefrigerator until dse. Hat any' Uniteevidence of mold'or other =tam-imam appears, the stock solutionshould be discarded and a freshsolution prepared.

r t

Weyking Dispense therequired amount into,distilled water,and deliver into screw-capped bottlesfor 'dilution water: The amount addedshould be .such that, aftei= sterilization,the bottles will contain 99 ± 2 ml ofthe dilution water.. Ordinarily thisrequires initialaddition of approxiI,,- -mately 1p2 ml f the solutionpriorto sterilizati .

.

o'

c Sterilization is 20 minutes,at1210C.

d Tightly stoppered bottles of thedilution water, protected againstevaporation, in suitable containers,appear to last indefinitely.

J Solutions for Gram Stain4, .

1* Ammonium oxalate crystal violet 'solution:,

a Dissolve 2 g crystal violet(approxiinately 85% dye .eontent) in20 ml of 95% ethyl alcohdl.

b

c

d

Dissolve 0.8 grams ammoniumoxalate in 80 ml distilled water.

ft

Mix solutions a and b.

Filter through cheesecloth or coars4.filter paper.

e Problemawith th- e gram stain. technique. frequently are traceable

, to the ammonium oxalate -crystalviolet solution. In the event thatdecolorization does not seerh satis-lactOry, the amount `of crystal violetin Vie solution can be reduced ,to aslittle as 10% of the ecommendedamount.

2 Lugol's iodine: Dissolve 1 g iodinecrystals and 2 g potassium iodide inthe least amount (usually about 5 ml)of distilled water in which they are -soluble. After all crystals are insolution, add sufficient distilled waterto bring the 'final. solution'to a volumeof 300 ml.

,

Counterstain: Disiolve 2.5'n-urns of. safranin in 100 mil of 95y. ethyl alcohol.For ke working solution of. counter stain,add NO ml of this solution of safranin to

0 ,

6V

100 ml of'distilled water.

N.

Am.

o

Media and Solutions for Multiple Dilution TubeMethods

.

r/ .

This outline was prepared )Dy He, L. Jeter,Chief, Program Support Training Branch,

1 -USEPA, Cincinnati, Ohio 45268 ,1 St.4.nda.rd Methods for the Examination of

. t Water and Wastewater. 14th ed. 1975.1

0

J

REFERENCES

t

Descriptors; Cultures, Bacsteria,Enteric Bacteria,' MPN, .MultipleDilution Method, Most Probable Number,Coliform, Fecal C9liforrh

AO,

n4.

-4

p

USE OF TABLES OF MOST PROBABLE NUMBERS

Part 1

I INTRODUCTION

A Using probability mathematics it ispossible to estimate the npfnber of bacteriaprociticing the observed result for any com-bination of positive and negative resultsin' dilution,tube tests. Because thecomputes ion8 are so repetitious and time -consuming, it is common lboratorypractice-to use Tables of Most ProbableNumbers. The'se tables are orderlyarrangements of the possible culturalresults obtainable from inoculating varioussample increments in differential culture,media. Each possible combination' ofpositive and negative tube results isaccompanied by the result (MPN) of thecalculated estimate and the 9`5.% confidencelimits of the MPN.

B The Tables.of Most Probable Numbersused in the current (14)tdition of StandardMethods for the examination of Water andWastewater were developed by Swaroop. if)Previous editions of Standard Methods haveused the tables prepared by Hoskins. s. (2)

.

1 Most of the tables are based on using3 sample volumes in decreasing decimalincrements. Thus, the systems arebased on using volumes of 10 ml, 1.0 inand 0.1 ml, etc. Other quantityrelationshipscan be used, such as

fir' 50 ml, 10 ml, and 1.0 ml in a table.Tables of Most Probable Numbers cap.,be ptepared for any desired series of 'sample increments.

2 In addition,4 tables can be devisetLfor'different.numbers of replicateinoculations of individual samplevolumes. For example, the MPN fTable most commonly used in the

' laboratories of this agency is basedon five replicate 10 ml -portions, five1.0 ml portions, and five 0.1 ml portions.A separate table is required for anoffiercombination,of sample volumes, con-sisting of five replicate 1'0 rnal.portions,

W. BA. 42g. 1.78

one 1.0 ml portion, and one 0.1 mlportion. This is popular in bacteri-ologicarpotabilitetts on water.MFN Tables can be prepared for anydesired combinations of replicates ofthe sample increments used in a,dilution tube svies.

3 An approximation of the MPN valuesshown in the Tables can -be obtainedby a simple calculation, developed byThomas. (3) The formula andapplication of this calculation is shownon a later page of this chapter.

C The method of using a Table of MostProbable Numbers is, described here,based on the table for five 10 ml portions,five 1.0 portions, and five 0.1 portions.The principles apply equally to the othertables presented -in the current edition ofStandard Methods for the Examination ofWater and Wastewater.

II DETERMININ8.THE MOST,PROIMBLENUMBER ,

A Codifying Results of the Dilution TubeSeries

If five 1.0.m1 'portions, five 1. 0 ml portions,and five 0.1 ml portions are inoculate'dinitially, and positive results are securedfrom fiv'e of the-10 ml portions, three ofthe 1.0 ml portions and none of the 0.1 mlportions, then the codecl result of the testis.5-3-0. The code can be looked up inthe MPN Table, and the MPN per 100 mlis recorded `directly. If more than theabove three sample volumes are to beconsidered, then the determination of thecoded result may be more complex. Theexamples described in Table 1 are usefulguides for selection of the significant seriesof three sample volumes,,

5-14

Use of Tables of Most Probable Numbers ti

Table 1. EXAMPLES OF CODED RESULTS

No. ml sample per tube -- 100No. tubes per sample vol. -- 5

'_10°5

1.05

0.15

0.015

0.0015

Code See Below,

No, tubes in sample givingpositive results in test

.. -

'.

.

5

.' 5

5

5

4

5

t5

0

0

1

4

4

1

4

5

5

0

'1

0

1

0

0

1.

5

5

00

0

0

0

1

4

5

0

0.0

0

0

0

5-4-1

5-4-04 -1 -0

5-4-2

5-5-4

5-5-50-0-0

0-1-0

1-0-0

/

-

(1)

(2)

(3)

'(4)

(5)

(6)

,(7)

(8)

Discussion of 'examples:

1 When all the inoculated,tubes of morethan one of the decimal series givepositive results, then it is customaryto select the smallest sample volume(here', 10 ml) in which all tubes gavepositive results. The results of thisvolume and the next lesser volumes 'are used to determine'the coded result.

2 When none of the sample volumes givepositive results in all increments ofthe series, then the results obtainedare used to designate the code. -Notethat it is not permissible-to-a-Ssumethat if the next larger increment hadbeen Inoculated, all tubes probablywould have giVen positive results andtherefoye, assign a 5-4-1 Code to theresults.,

3 Here the results are spread throughfour of the sample volumes. In suchcases, the number of positive`tubes inthe smallest sample volume.is addedto the number of tubes in the thirdsample volume (counting down from thesmallest sample volume in which alltubes gave positive results).

4 Here it is necessary to use the 5-5-4code, becauseinoc'ulations were not .

made of 0.001 ml sample volumes;and it is not permissible to assumethatoif such sample volumes had beeninoculated, they would havegivennegative results, or any other arbitrarily:designated result.

This is an-indeterminate result. Ma.' nyMPN,tables do not give a value for sucha result. If the table used does nothave the code, then look up the resultfor code 5-5-4, and report the result"greater than" the value shown for the5-5-4 code. The first number of the5-5-4 code is based on the 1.0 mlsample volume.

6 Like (5), this is an indeterminateresult. If the code dcies not appear in .

' the table being used, then lookup theresult for code 1 -0 -0, and report theMPN as "less than" the value shownfor the 1-0-0 code.

7 The current edition of StandardMethods stipulates this type of codedesignatiori, when unusual resultssudh as that occur.

.,

Use of Tables of Most Probable Numbers

8 Note the difference from (7) above. _Inoculations of 100 ml portions werenot made, and it cannot be assumedthat the result would have 'calltd forcode 0 -1- 0.

.

B Computing and Recording the MPN

When the dilution tube results have been' codified, they are read and recorded from

theappropriate MPN Table.

1 If, as in the first four of the examplesshown under (A) the first numberin the coded result represents a 10 mlsample volume, then the MPN per 100 rnis read and recorded directly from theappropriate column in thetable.

2 On the other hand, if the first numberin the coded result represents a samplevotarrie other than 10 ml, then acalculatiOn is required to give the

lbacorrected MPN. For example (4) under (A)""-above, the first "5" of the 5-5-4 code

represents a sample volume of 1. 0 ml.Look up the 5-5-4 code as if the 1. 0 mlvolume actually were 10 ml, as if the0.4 ml volume actually were 1.9 mland as if the 0.01 m.1 volume actuallywere 0.1 ml. The MPN obtained (1600)then is-multiplied by_a factor of 10 togive the corrected value. A simpleformula for this type of correction isst}own on a later page of this chapter.

III PRECISION OF THE MPN VALUE

A The .current edition of Standard Methodtshows for each MPN value, the 95%confidence limits for value. Thisdraws attention to the fact that a givenMPNevalue is not a precise measurement,but an estimate. The 95% confidencelimits means that the pbserver will becorrect 95% of the time when he considersthat the actual number of cells producingthe observed combination of positive andnega. 1 e tubes was somewhere betweenthe stat d upper limit and thestatedlower imit.

B The greater the number of rdplicates ofeach sample volume in a dilution series,the greater the precision (in other words,the narrower the limits of the 95%confidence cange) of the test. Theprecision of results, based on numbersof tubes inoculated per sample volume,is shown in Table 2.

C Woodward (4) and other workers havestudied the precision of the MPN indetail. Such reports should be studiedby those desiring further informationregarding the precision of the MPN test.

Table 2, Approximate Confidence Limits for Bacterial Densities asPer Cent of MPN as Determined from Various Numbers of Tubes

in 'Three Decimal Dilutions*

rtt

Number of tubesin each dilution

50%Lower Upper

7'5%Lower Upper

80%Lower Upper

90% 95%Lower Upper Lower Upper

. 1, 33 186 18 340 15 402 10 637 6. 5 9552 47 .160 31 246 '27 276 20 383 15 5113 53 150 38 215 . 34 2'37 26 311 21: 3955 64 139 49 182 46 196 37 41

4...289

44.9 76. 127. 63 152 ° 60 160 52 184 446' .?08

*The interpretation of these-figures is as follows: When MPN estimates aremade on the -basis of dilution tests using one tube in each of three decimaldilutions, you will be right 50% of the time if you sayqhat the true bacterialdensity is between 33% and 1E16% of the MPN. If you had used 5 tubes in eachdilution you could reduce this intervalto fi-rom 64% to 139% of tlie.MPN and stillbe right 50% of the time If a greater certainty were desired, sdy 95%, youweulal have to widen this interval to from 31% to 289%;

72 _3 A

4

Use of Tables of Most Probable Numbers

IV OCCURRENCE OF IMPROBABLE TUBERESULTS

A Many of the theoretically 'possible tuberesults are omitted from the MPN Table.For example, codes 0-0-3, 0-0-4, and0-0-5 are not includedas well as manyothers. These are omitted, because, inthe opinion of the authors of the tables,the probability of occurrence of suchresults is so low as to exclude them frompractical consideration.

B The frequency of occurrence of variouscode results is shown in the Table 2 bothon a theoretical basis and on the basis ofactual laboratory experience.

ti

4

FIVE- TIT

INCLUD

C From the MPN tables, it can be inferredthat the codes omitted from the MPN

6 Table can be expected to occur up to 1%of the time. If, in reviewing laboratorydata, the theoretically unlikely codesoccur appreciably more than 1% of thetime, ther'e4 isan inditation fold inquiryinto the causes. Such results / /can occur (1)as a consequence-of faulty laboratoryprocedures, or (2) as a result ofextraneous influences in the samples.

D The current edition of Standard Methodsdoes not include MPN values for manyrare combinations listed in previouseditions. By pruning out those codes listedas Group IV in Table 3, the table has beenconsiderably 'condensed. Table 4 suggestsmaxiinum permissible numbers of samplesfor various numbers of samples tested.

Table 3E AND THREE-TUBE CODESI'HAT

99 PER CENT OF ALL RESULTS

GroupTheore ally Ex-petted P rcentage

of Re Its

Theoretically Ex-petted Cumulative

PercentageObserved Percentage

of 360 Samples

Five -Tube-Test.. .

Class 1 codes550, 551, 552, 553, 67.5 67: 5-'''- - -68.0554, 500, 510, 520,530, 540, 100, 200,300, 400.

Class 2 codes ' \511, 521, 531, 541, 23.6 91. 1 23. 1542, 110, 210, 310, . .

410, 420. ,[

1rClass 3 codes501, 010, 532, 320,

,7.9

.99.0 75

522, 220, 543, 430,120, 533, 330, 502,,020, 544, 440, 301,401, 431, 201, 411,101, 311, 421, 211,001.

Improbable codes 1.0 100.0 1.4

e Three-Tube Test

4Class 1 codes .

380, 331, 332, 300, 81.5 81.5 81.7310, 320, 100, 200.

. k

Class 2 codes321, 311, 301, 210,110, 010.

14.9, 96.4,

14.1

Class icodei i*:.322, 220, 201, 101 2.7 ` 99.1 ' 3.7312, 120. ..Improbable codes 0.9 10'O.0 0.6

N

Table 4,,

MAXIMUM PERMISSIBLE NUMBERS9F IMPROBABLE CODES FOR VARIOUSNUMBERS OF SAMPLES TESTED

..pumber of Maximum NumberSamples ° of'Improbable Codes

Use-of Tables-of-Most-Probable-Numbers

Vpample: From a sample of water, 5 outa five 0. 01 - ml portions, 2 out of five0.001 - ml portions, and 0 out of five0. 0001 - ml portions, gave positivereactions.

. From the code 5-2-0 In the MPN table,the MPN index is 4'9

1 - 15

16 - 4546 - 83

1

2

3

49 10= 00049,(from table) #0.01

'84 - 130 4 MPN Index = 49, 000131 - 180 5

181 - 233 6 A simple approximation of the most234 - 290 7 probable numbed may be obtained from291;_350 8 the following fo/mula (after Thomas):351 - 413 9'414 - 477 10 MPN/ 100 ml =478 - 543 _ 11

F.

F

Table 5 is from International standardsfor Drinking-Water, published by the WorldHealth Organization, Geneva (1958). Thelast three values, not shown in the WHOpublication, are from Woodward, "HowProbable is the Most Probable Number. "(4)

Several theoretically possible combinationsof positive tube results are omitted inTable 5. These combinations are omittedbecause the statistical probability ofoccurrence of any of the missing resultsis less than 1%. If such theoreticallyunlikely tube combinations occur in morethan 1% of samples, there is,need for -review of the laboratory procedures andof the nature of the samples being tested.

\ - :\-- When the series of decil dilutions is

\\'' other than 10, 1.0 andO. 1 1, use the\ MPN in Table 5, according t the following

\formula:

MPRX

10`(from table) Largest quantity tested

.

, No. of Positive Tubes X 100

`4 No. of ml in negative tubes) X (No. of ml .

in a.11 tubes)

Example: From a sample of water, 5 outof five JO - ml portions, 2 out of five1.0 ml portions, and 0 out of five 0.1 mlportions gave positive results.

MPN/ 100 ml II X 100 50.22(3.5)X(55.5)

MP] i/ 100 .50'.

Note that the MPN obtained from the tableon the preceding pages with these tuberesults is 49'. ,"Most probable numberscomputed by the aboye formula deviatefrom values given by the usual methods1411 amounts which ordinarily areinsignificant. The formula is notrestricted as to the number of. tubesand dilutions used --" (Thomas) -

Use of-Ta. ViaOf Most Probable Numbers

Table 5. MPN INDEX AND 95% CONFIDENCE LIMITS FOR VARIOUSa COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS WHEN FIVE

10-ML PORTIONS,, FIVE 1-ML PORTIONS AND FIVE 0.1 ML PORTIONSARE USED

a

No. of Tubes GivingPositive Reaction out of MPN

Indexper

100 ml

95% Con-fidence Limits

No. of Tubes GivingPositive Reaction out of IVIPN

95% Con-fidence Limits

5 of 10ml Each

5 of 1ml Each

5 of 0.1ml Each

Lower Upper 5 of 10ml Each

5 of 1ml Each

5 of 0.1ml Each

Indexper

100 mlLower Upper

00

0

0

.0 <22 <0.5 7 2 1 26 '9 78

0 1 0 2 <0.5 7 4 3 0 27 9 80

0 2 0 4 <0.5 11 4 3 1 33 11 934 4 0 34 12 93

1 0 0 2 <0.5 7

1 0 1 4 <0.5 11 5 0 0 23 7 70

1 1 0 4 <0.5 11 5 0 1 31 11 89

1-1

1

21

0

'6

6

<0.5<0.5

1515

555

0

1

1

20

1

433346

151116

110 .93

120

2 0 0 5 <0.5 13 5 1 2 63 21 150

2 0 1 7 1 17

' 2 1 0 1 17 5 2 0 '49 17 130

2 1 9 2 21 5 2 1 70' 23 170

2 2 9 2 21, 5 2 2 94 28 220

2 3 0 12 3 28 55

3

30

1

79 25_31

190250A 0 8 1 19 5 3 2 140 37 340

3 ., 0 1 2 25 5 3 . 3 180 , 44 500

3,3

1

1

0

1

1114

24

2534

55

44

0

1

130170

35..43 °

300490

3 2 o, ., 14 4 34 5 4 2 220 57 700

3 2 1 17 5 46 5 4 3 280 90 850

3 3- 0 17 5 46 5 4 350 120 1, tioo,4 "0 0 13 3 31 5 5 0 240 68 750

4 0 1 17 5 46 5 5 1 350 120 1,0004 1 0 17 5 46 5 5 2 540 180 1,4004 1 1 21 7 63 5 5 3 920 300 3,2004 1 2 26 9 78 5 5 4 1600 640 5,8004 2 0 22 7 67 5 5 5 52400

-5-6

\.

Use of Tables of Most Probable Nuinbers

Table 6. MPN AND 95% CONFIDENCE LIMITS FOR-VARIOUSCOMBINATIONS OF POSITIVE RESULTS IN A PLANTING

SERIES OF FIVE 10-m1 PORTIONS OF SAMPLE

No. of Positive Tubes Out of:

Five 10-m1 Tubes

MPN per

100 mlr"

Limits of MPN

Lower ,Upper

0 ' 2.2 0 6.0

1 2.2 0,1 12.6

2 5.1 9. 5 19.2

3 9.2*)..

1.6 29.4

:4' 16.0 . 3.3' 52.9

- 5 > 16 8.0

44,

Table 7. MPN AND 95% CONFIDENCE LIMITS FOR VARIOUSCOMBINATIONS OF POSITIVE RESULTS IN A PLANTING SERIES OF

. .FIVE 1.0-ml, ONE 1-ml, AND ONE 0.1-ml PORTIONS OF SAMPLE c

.No. of Positive Tubes Out

,of: -

MPNper

100 ml

Limits of MPN

Five 10-mlTubes

One 1-mlTube

One 0. 1 -mlTube Lower Upper,

0 0 0 <2 5.90 1 0 . 2 0.050 131 0 0 2.2 0.050 13

'1. 1 0 4.4 0.52 142 0 0 5 0.54 49

2 1 0 7.6 . 1.5 193 0 0 8.8 1.6 293 1 0 . 12 3.1 304 0 0 15 3.3 464 0 1 20 . '5.9 48

4 ;fir 1 0 21 6.0 535 0 0 38 . 6.4 3305-. 0 1 96 , 12, 3705 1 -

1

01,

240>240

1288

3700

.

" 765 -7

Useof Tables of Most Probable Numbers

.

IV TABLES OF MOST PROBABLENUMBERS

is"These tables' consist of the MPN indices and95% confidence limits, within which theactual number of organisms can lie,' forvarious combinations of positive andnegative tubes. Thre MPN tables arepresented. -Table K i based on five I0 mlfive 1.0 ml and five 0, ml sample portions.Table 6 is based-on five la ml sample por-tions; and Table 7 is based on five 10 ml,one 1 ml' ana one 0.1 ml sample, portion.

REFERENCES

1 Swaroop, S. Numerical Estimation ofB. coli by Dilution Method. Indian J.Med. Research. 264.'353. 1938.

Hoskins, J.K. Most Probable Numbersfor Evaluation of Coli-Aerogen Testsby Fermentation Tube Method.. PublicHealth Reports. 49:393: 1934.

.1

3 Thomas, H. Al, Jr. Bacterial. Densities from Fermentation Tubes.

J.A.W.W.A. 34:592. 1942.

4 Woodward, R. L. How Probble is theMost Probable Number? J.A.W.W.A.49:1060. 1958.

5 Standard Methods for the Examinationof Water and Wastewater. 14th,Edition. Prepared and publishedJointly by Americah Public Association.American Water Works Association,and Water Pollution Control Federation.

This outline was prepared by H. L. Jeter,-tor, National Training Center,

Water Programs Operations, EnvironmentalProtection Agency, Cincinnati, OH 45268.

5-.8

.

1

.

I DERIVATION OF THE MPN

.A Assumptions

The validity of the MPN procedure is basedupon two principal assumptions.

I. In statistical language, the firIeigkthatthe organisms are distributed rajeomlythroughout the liquid. This means' thatan.organism is equally likely to befound in any part of the liquid, and that cathere is no tendency for pairs or groups 2of organisms either to cluster together 04

or to repel one another.

MPN THEORY

Pari 2

2 The second assumption is that eachsample from the liquid, when incubatedin the culture medium, is certain toexhibit growth whenever the samplecontains one or more organisms.

B The Probability Equation

Babe d-upon-these-assumptionsi-an-equation--for the probability of the observed com-bination of positive and negative tubes canbe derived as a function of the true density.6. By solving this equation for differentvalues of 6a curve can be plotted as -shownin Figure 1..

Curves of this type always have a singlemaximum,or peak. The value of 6, 'say d,which corresponds to the peak of the curveis called the most probable number,commonly designated as MPN,

The MPN is "most probAb, :in the sensethat it is the number-Which maximizes theprobability of the observed results. It isinteresting to note that althbUgh theoriginal derivation 'of the MPN predatesmodern statistical MPNprocedure corresponds to the rrentlyaccepted estimation procedure knownasthe "method of maximum likelihood." .

;ON: fdkk%

0 I

-d(MPN)BACTERIA PER 100 ML

FIGURE 1

C Indeterminant Solutions

The_MPAT provides a meaningful estimateof 6 only if there are, both positive andnegaive tubes in at least one dilution.If all tubes are negative, the maximumof the probability, curve occurs when 8 is \set equal to zero (see Figure 2) and thusthe MPN is zero. If all tubes are positive,the maximum of the probability curveoccurs when 6 is set equal to.infinity,(see Figure 3) and thus,the MPN is infinity.

e

- MPN Theory

'ALL TUBES NEGATIVE1:00

MAXIMUM AT 8=o

1.00

BACTERIA PER 100 ML

FIGURE 2

ALL TUBES POSITIVE

0

MAXIMUM AT 8=0

,BACTERIA PER 100 ML

FIGUREA

4 DISTRIBUTION OF MPN VALUES

*,A Skewed Distribution-

If a.very large number'of independentMPN determinations were made on thesame water sample, the,distribution.ofthe MPN values would be such that veryhigh values relative4O the median value

. would occur more frequently than verylow values. Thus the distribution of MPNvalues-is skewed to the,,right as shown inFagure ,

. .

MPN

FIGURE 4

B Logarithmically-Normal Distribution

Since it is mathematically inconvenientto work with data distributed asymmetri-cally, it is desirable to transform theskewed data in such a way that the trans-formed values have a symmetric distri-bution resembling the normal. In thecase of MPN values The logarithms of,the MPN's are approximately normallydistributes' as shown in Figure 5.

SYMMETRIC(NORMAL)

IOg(MPN)

FIGURE 5

79 S

sr'

ti

C Precision of MPN Estimates0 0.

The lick of_precision of MPN estimatesof bacterial densities is generally recog.t.'nized. A measure of the precision isgiven by the confidence limits on theestimate which can be computed on the-basj.s ofthe.normal distribution of thelogaritInns.of the .MRU.,...valties . It hasbeen verified that three-tube and five-tube MPN estimates are appr4ximatefylogarithmically normal and the Standarddeviation of the logarithms of the MPN'sis given by the formula:

0.58a =log. n

where a the standard deviation ofthe.logarilffms of the MPN estimates andn is the number of tubes in each dilution.'

The upper and lower 95% confidence limitsof an MPN estimate are given by theformulas: 1.

. UCL = antilog (log MPN + 1.96a log).

opoi MpN k,.

LCL = antilog (log MPN 1..960 log)

= k,

where k = antilog (1.96a log).

a=

ti

4

MPN Theory

Notice that the confidence limits are notsymmetric about the MPisT estimate.

The 'precision of the MOT estiriiate canbe increased byinpreasing thenumber,Of tithes per dilution. Figure 6 show thewidth of/the 95% confidenceintervalexpressed' as a percentagel-of the MPNestimate for various values of n.. Noticethat the width of the confidence intervaldecreases as 'n increases°.

Ill PLANNTG A DILUTION SEfi,IES

4 The Rationale

450

400 ->

`61 3130z2i -z _0u 209

1_

100-

6'

It was entionea that-the MPN procedure ,

pro es a. reasonable estimate of the truedensity only,,if there are both positive andnegative tubes in at least one dilution.It follows'that in a series of dilutions theexpected number of organisms in thehighest sample volume (lowest dilution)v should beat least one, otherwise alltuloies,may be negative and the result willbe anindeterminant value.

80

0

111111.1111 1 11111111,13 5 10' 12 .....2.0

NUMBER OF TUBES PER DILUTION ,

J FIGURE 6

5-11.

$

$

MPN Theory

Similarly, the expected number ororganisms in the lowest sample volume(highest dilutiOn),vL should not exceedone, to'avoid the risk that all tubes willbe positive.

B The Mile

4406

The above line of reasoning leads'to,therule that a dilution series is capable ofestimating any density between 1/v and1/v,T . In.practice, we use the ruleIoy .

first guessing two limits4 and (5T betweenwhich we are fairly certain that te aetual

4

density ffes. The sample volumes Areten chosen to satisfy the rules

1vH ' vL

Lf

,1.4

Table 1 di4lays the -range of densitiescovered by various decimal dilutionseries.

a

ct

4t,

4`

(r.

SAMPLE VOLUME1ML)

101

10°.X0-1

' 210-

:- 10-3

.._ `10 -4

Al 0.- 5.

::.0-6*01

a'' ° 10-7

TABLE 1

. (COLIFORMS/100RANGE

10 1

COVEREDML)

10 3

16 4

10 5

106

10 7108

910

jj2

41 310

.. 104

-. 106

106,7

10

5-12

This outline was prepared by J. H. Parker,former Statistician, Analytical QualityControl, Bureau of Water Hygiene, EPA,Cincinnati, OH.

.

Descriators: Bacter,is,, Coliforms, MPN',Most Probable Nbimber, MeasureMent,

----Tricrobiology, Laboratory Tests

THE MEMBRANE FILTER IN WATER BACTERIOLOGY. e

4.0

I HISTORICAL BACKGROUND

. '\There is sometimes a tendency to look uponMembrane filters and their bacteriologicalapplications as new developments. Both thefilters and many of their present basterio:-logical applications are derived from earlierwork in Europe.

A Some European developments prior to4.7 1947 are as follows:

.

1 Fick is credited with application ofcollodion membranes in biologicalinvestigations in 1855.

2 Sanarelli, in 1891, reported develop-ment of membrane filters impermeableto bacteria but' permeable to theirtoxins. .;

'4

3 Bephhold, in the early 1900's madea systematic study of the physico-chemical properties of a number ofvarieties 'of these ffiembranes. After1911 numerous investigations weremade in several countries with respect.to-the properties of collodion membranes.

4 Zsigmondy and Bachmann, 19Y6-1918,developed improved production methodswhichere applicable on a commercial

_ scale. Membrane filters have beenproduced for many years at theMembranfiltergesellschaft, SartoriouS-Verke, in Goettingen, Germany. In1919 Zsigniondy-appliedior a U.S.patent on his productipn methods; it-was gi-arit-ed in. 1922.

5 In the 1930's, W.' J. Elford in England,and P. Graben .in. France, made newcontributions iq developing and teachipgmethods for making collodion- membraneswith controlled pore' size.

determination of bacterial counts,coliform determinations, and isolationof pathogenic bacteria from water andother fluids. Most early interest indeveloping these techniques seems tohave been in Germany and in Russia.During World War II Dr: G. Muellerapplied Membrane filter techniquesto the bacteriological examination ofwater6 following bomb destruction ofmanyof the laboratories.

4B Developments in the United Stateg

1 In 1947,, Dr. A. Goetz reported on amission to Germany as a scientificconsultant to the Technical IndustrialIntelligence Branch, U. S. Departmentof Commerce. He obtained detailedinformation about the nature, methodof preparation, and specific bacterio-

mlogical applications of the Zsigmondy-Membranfilter,being manufacturedby the Membranfiltergesellschaft inGoettingen.

6 Beforp World War II filtration .

procedures using the Zsigmondy -menibrancliad been s4ge4ted,,for the

2,, After his return to this country,-Dr, Goett developed methods forpreparing and improved type'ofmembrane filter from domesticmaterials. Ori a small scale hemanufactured filters under agovernment contract; afterwardmembrane filter manufacture wascontinued by a commercial orfanization.

3 In, 1950,° bacteriologists Of the PublicHealth4Service began intensive studyof the applicalions't)f mernbragie filtersin bacteriological examinations of -:water. Their-firskreport,waspublished in 1951, and' wai Mowed,by numerous reports Of other similarinvestigations. Such studies have

bekeriltidely:expanded,:as indicated in,

tr*references shown elsewhere in this

, manual.

-*s '-

-!------ -0,,,

. . -- -- '.Nalt: Mention+n , ()I lamereifil products and manufacturers does not imply encioa ..'emert by tilt.

oWP, F.aii, iron mtaniti- Brut ection Agency. 1.

. : .82 ,

ttr..BA!".1,1i4m. ,-0}1,. 8;77 , 6-1

t'

The M mbrane Filter ,iii Water Bacteriology

: 0

4-1In 1955 the310th Edition of "StandardMethods for the Examination of 'Water,Sewage, and Industrial Wasted" included.a tentative method for ceilifornis by mem-'vane filtpr method. In;the-llih and 12theditions, the membrane filter methodfor conforms has become official. In

Addition,' methods for epterocoOcus(fecal streptococor) areancluded as

The 13th editiontad given tne feoat Itrept600ccus test`a standard designaticin and the tentativeMethod status reserved f8r:the "pourplate" technique of quantitation

e

5 The membrane, filter is an officialmethod for examination of potable wafersin interstate commerce. The PublicHealth Service Drinking Water,Standards11962) state "Organisms of the colifockugroup.... All tile details of technique

shaltbe in accordance with SdndardMethods for Elcardination of Water andWaStewater, ra,:ff edifiop... " Thusacceptance byes ndard Methods asofficial automatically validates a method*/for use with interstate 'Waters. °

. .

-

I(' /ROPERTIEJS .44,M.EMBRP,NE. FILTERS

Membrane filters used in water bacteriologyare flat, hiely.pOrous, flexible pla4tio discsabout's% i5 millimeters in thickness apd usually

1 47150millimeters in dial ter. r"

1.

3 The emulsion is cast on plates and dried-in an environnient rigidly controlled as tohumidity and temperature. The dropletsof insoluble fluid retain their size andidentity in the dried film, eventuallybecoming the gores of the finishedmembrane.

4 The dried porous filinis cut into filterdiscs of-the-desired-size. Representa-tive discs are subjected to control testsfor accueatadetetmine.tiOn I the poresize obtained.

5 ,Particle retention by membrane filtersks at or very near the filter surfade, bya mechankial, sieve-like action. (Thisapplies 3jhydros °is,. pOt to aerosols.)Throup manufacturine,control it ispossible to make membrane filters withcontrolled pore size, within narrow limitg....

B Some Important Characteristics ofMembrane Filters

'4 1

It A Principle of-Manufacture e< IP

,.... :1

-The procedures described below are from-- FIAT Report 1312: Whilelhdrnathods in-

dicated by goetzdOot necessarily describethe current nianufacturing processes, it is '-assumed thatlaimilar principles of manu-facture still apply. .

-..

T One or more cellulose esters, such las,cellulose nitrate, is dissolild in asuitable solvent.

2, Water, or some other liqrrid insoluble ,*in the cellulose.solutioriis added and. .

inbred, to form an emulsion having greatuniformity inn size and distribution ofdroplets of the insoluble liquid.

6r2 1.7

AI

1 The membrane filters used in micro-.biology, should be Slat, circular, gridded,. .of uniform thickness and porosity, non-toxic to microorganisms, wettqle,able to withstand commonly employedsterilizing conditions, andunaffected bythe fluid to b'eiltered. '

2 Withoutrefegence to specific manufacturers;some particulars of their products haveincluded:

Average pore diameter rangingom 5 minimicrons to 10 microns.

' Thicknesses. ranging from 70 to150 micrOns. Can be sterilized'by au:toddling at 12 ecefs 10.0minutes.

..mean flow pore size ranging from.7.5 mininii.crons to 5 microns.The pore si;e liked in waterbacteriology having a standarddiameter, has tiwater. floAri rate

''of.70bcimilifcm ,and must pass100',m1 of ba' 11i:0.e-free igaterwithin 9.1ecorlds: o

ker ,

9 4

1

8a...

- of

.k

I

Auk

4

,a

O

I

currently produced in morethan twenty distinct pore sizesfrom 14 microns to 10 Milli-microns in discs ranging from13 mm.to 293 nun in diameter.

. The total range of pore sizedistribution of the type used inwater microbiology of 0.45 microns

. is plus or minus 0.02 micron.,

d ... membranes are offered ingraduated pore sizes ranging,fibm 12 microns to ,5 milli-micronA. The types used inwater bact,griology have a dis-tilled wa-:ow rate of .65ml /min /cm at 700 mm Hgdifferential pressure or an airflow rate of 0.4 liters/min/cmat a differential pressure of500mm water. _

The embrane Filter in Water,Bacteriolo

1

4 Membrane filtf s are wettable. Thus,...after sample f ltration, when a filteris placed on m t culture mediumthemedium diffuses through the pores andis available, to organisms collected onthe opposite surface:

5 Mgmbnane filters are free of solublechemical substances inhibitory to bac-terial growth. Water soluble plasticizersare included in one commercially produced filter (glycerol, 2.V/0), Thecellulose esters themselves have someabsorbing tendency illustrated by somedyes and heavy metals. Total ash isvery low, less than 0.0001%, '

6 Membrane filters haire a uniform index 4of refraction. -With membrane filters.:this index is No' 1 5 When wetted'with a liquid havihg refractive indexwithin this range_ihe filters becofnetransparent. This "uszty permitsdirect microscopic examination ofparticulate" matter collected on thefilter surface. 4,

:

.

7 Temperiature.,resistatice 'depends on'plas4cS used in the filter. The nitro--pllulose membrane filter is stubledry

/

up to 125°C in air. Membranescellulose triacetate are aavertised t

withstand dry heat to 266°C, In generalhowever, membranes in current usemust be sterilized cautiously. Con-

4 suit tip laboratory equipment discussionfor details. Overheating of all, typesntprferes with filtration by bi.dcking

pores. D.

C Nomenclature

Membrane, filters used in bacteriologicaltests on water are known under severalnames. Though the names are different,the triflers are similar in fdrm, properties,anc of use. Names commonly en-countered are:

1 Membrane clilter. This is the general*name forofilters made according to'the generalprinciples and having theproperties discussed above. The term.."membrane filter" is most used intechnical reports on filters of this type.

2 Molecular filter. This name used byGoetz for the improyed type of filterthat, he and his associates developedafter study of the manufacturing Methodsat the Me mbranfilte rges ellsc haft inGoettinge n, Germany.

3 Millipore filter is a trade name formembrane filters made by the MillipoieFilter Corporation.

4 Bac -T-Flex filter-is a trade nameapplied to certain membrane` filtersmade by Carl SchleicherAnd SchuellCompany,

5 Okoid filter is a trade.name applietttofiltel's made by ,Oxo, Idondon,England.

81'4

The Membrane Filter in Water Bacteriology

6 Micropore, 'Polypore and Metricel 2) A verified membrane filterform test ..an be used whenas a supplement to the directbrane filter test. Pure ctilures

coli-needed

mem-have been trade names used by theGe(Irrian Instruinent-Coppany.

III APPLICATIONS7IN WATER BACTER OGY

A The basic cultural procedures f bacterio-logical tests on membrane fil s are:'

A sample isJiltered-tfitough a membrane,

Tilte r1.

*2 The filter is placed in aculture con-tainer, on an agar medium or a paperpad impregnated with moist culturemedium:

3 The inoculated filter is incubated underprescribed conditions of time, tempera-ture, and humidity.

4 After incubation, the resulting cultureis examined and necessary iqterpretartions and for additional tests are made:

B With variations in such factors as culturemedia, incubation time, and combinationswith other cultural and biochemical tests,several diffetent kinds of tests areavailable. ' .

1 Totarbacterial counts are made bycultivation of bacteria on membranefilters using an enriched all-purposeculture medium.

2 Tests for bacterial indicators of

sad Conform tests

pollution.,

'

' .6 -4

1) The direct membrane filter tests; .fqr coliforms is one in which,

kt-er sample filtration,, the mem-' brage.fi4eir is incubated in Con-

tact Iv' ne or more specialmedi *At least one of the mediais a s ective, diffecential(rned-taminc ludin conikbpentgmbich13mit cat rrn.bactfrialfo developcol of)ies asily reoogpizab14. 13"'y

lor, sheen,, or other'chatacteristics:

Aff

ale .obtained fr9m individualcolonies differentiated on themembrane filter and subje rtedto further cultural, bioche ical,and staining tests to establishthe identity of the colonies being.studied:

3) The delayed membrane filt rcoliform test was develope toovercome babterial changes fre-quently occurring when there isa delay of one to several daysbetween sample collection andthe-initiation of laboratory tests.The test consists of sample' fil-tration at or shortly after the timeof sample collection. The inocu -lated filter is placed 'on a preser-vative medium and taken or sentto a laboratory, where it is trans -ferred to a growth medium forlhe,differentiation of coliform Colonies.

"111-tcr incubation the culture is ex-amined and the resu 'Its are evalu-ated asiror the direct membranefilter coliform test. -

4) A medium and t chnique.fordetecting and c unting fecalcoliform bact= is has beendeveloped an is called MrFICBroth. .Thi medium currentlyis beiqg u d increasingly iqwater po ution studies.

b. Selective, differential, culture mediahave been developed for direct cul-tural tests for members of the enter-Ococcus group of bacteria.

3 Tests for pathogenic bacteria

a 'Workers.are currently testingmedia forthe differentiation omembers of the Salmonella-Shgroup .of enteric pathogens. Aable information indicates potusefulness of a scrviiing med

1

85

ne'w

ellsail -ntialum

The Membrane Filter in Water-Bacteriology .

for differentiation of nonlactose-fermenting, non urease-producing

.kacteria.,

b One medium has bele usedfor screening tests in detection ofSalmonella typhosa.

c Further confirmatory cultural, bio-_ s - chemical, and serological tests _are

necessary to establish the identity, ofbacteria differentialed with thesescreening, media.

C Menibrane filter techniques can be appliedboth in the labdratory and under field con-ditions. Several varieties of portable mem-brane filter field units have been developedon a commercial basis.

IV ADVANTAGES AND LIMITATIONS a

This evaluation is limited to tests for the con-form group. Similar, but separate evaluationswould have to be made for any other bacterio-

1 Results are ebtainei in approximately24 hours. compared with 48-96 hoursrequired thestandard fermentationtube method.1

.2 Much larger, and hence more 'represen-

tative samples of water can be sampledroutinely with membrane filters:

3, Numerical results from membranefilters have much greater precision(reproducibility) than is expected with.the fermentation tube method.

4 The equipment and suppliesrequired arenot hujj,../ A great many samples can be''examined with minimum requirements'for laboratory space, equipment, andsupplies..

B Limitationso

,Endo type culture, media sometimesgive difficulty. In such cases a highratio of these noncoliform bacteria tocoliforms results in poor sheen pro-duction, or 6.reti suppression, of thecoliform organisms.

2' In samples having low conform counts'and relapvely great amounts of sus-pended solids; bacterial growth some-

_ "timesdevelops in a continuous film onthe' membrane surface. In such casesthe typical coliform sheen sometimesfang to develop.

3 Some samples containing as much as1 milligram per liter of copper or zinc,or both,' show irregular coliform bac-terial results.

*4 Occasional strains of-bacteria growingon membrane filters producing sheencolonies prove; on subsequent testing,to be acid but not gas-producers fromlactose. Where this occurs it may

4 give, a falsely-high indication of coliformdensity.

Such limitation's as these are not frequent,but they do occurotten enough to requireconsideratiOn. In samples where these dif-ficulties often occur, the beq course ofaction often is to av,oid.use of membranefilter methods and us^e the multiple fer-mentation tube procedures.

1 '-Samples .haying high numbers of non-` conform bacteria capable of growing on

g. 4

V SUMMARY t.

The 'development of membrane filters andtheii bacterial applications-has been discussbriefly, from their European origin to theircurrent status in this,country. Membrane

.filters currently available here have beendescribed, and their properties have been.considered. Applications of membrane, filtersiti water bacteriology are indicated in generalterms. Some of the advantages and limitationsof 'membrahe- filter methods are presented. forcolifOrm teats.

86

4

The,Menibrane Filter in Water acteriolo

-.11 Goetz, Alexander. Materials, Tec iques,

and Testing Methods for the Sa ation(Bacterial Decontamination) of mall -Scale Water S upplies in the Field Usedin Germany During and After the War.Technical Industrial Intelligence Bratich.U.S. Department of Commerce. FIATFinal Report 1312. December 8, 1947.

2 Clark, 'Harold F., Geldreich,Jeter, H. L., and Kabler, P. W. TheMembrane Filter in Sanitary Bacteri-ology. ,Public Health Reports. 66:951-77. July 1951.

.Goetz, A. and Tsuneishi, N. Applicationof Molecular Filter Membranes to thet3acteriological Analysis of Water.Journal American Water Works Associ-.ation. 43:943-69. December 1951.

^ v

0

4 Clark, H. F. , 'Kabler,0 P. W., andGeldreich, E. Advantages andLimitations of theXembrane Filter .

Procedure. Water and Sewage Works.104; 385-387: 195x7...

i Public Health Service Publication 956.'Public Health Selivice Drinking WatStaidards. 1942.

6 Wiridle-Tay-16r, E.;/[aridThe Application of 1VIembrane FiltratioTechniques to the Bacteriological Ex-amination of Water. Journal AppliedBacteriology, 27:294-303. 1964.

r

This outline was 'prepared by H. L.Jeter, Chief, Program SupportTraining Branch, USEPA, Cincinnati,Ohio 45268

1

AtrY

Descriptors: Filters, Membrane,Bacteria, Microorganisms, LaboratoryTests

0

tpi

\:`

MEMBRANE FILTER EQ IWTENT AND ITSI PREPARATION FOR LABORATORY USE

A

I tome equipmdnt and supplies used in thebacteriological examination of water withmembrane filters are specific for the meth6d.

, ;Other items are standard in most well-equipped bacteriological laboratories_ andare readily adapted to membrane filter work.This chapter describes needed equipment andmethods far its preparation for laboratoryuse. Where more than one -kind of item isavailable or acceptable for a given function,

'sufficient descriptive information is pro-vided to aid the worker in selecting the onebest suited to his own needs.

II EQUIPMEA' FOR SAMPLE FILTRATION* AND INCUBATION

A Filter Hoping Unit

1 The filter holding unit is a device forsupporting the 'memtlrane filter and for

. holding the sample until it passesthrough the filter. During filtration

.the sample passes through a circulararea, usually about 35, mm in diameter,in the center of the filter. The outer ,

part of the filter disk is clampedbetween tlie'two 'essential Componentsof the filter holding unit. -(See Plate 1)

a The lower, element, called the filterbase, or receptacle, supports themembra,ne filter on a plate about '450 mm in diameter.. The centralpart of this plate is a porous diskto allow free passage of liquids.The outer part of the plate is asmooth nonporous surface. Thelower eier ant includes fittings fdrmounting!..ne unit in a suction flaskor other container suitable forfiltration with vacuum.

b The upper.ele ment, usually called'the funnel, holds The sample untilit is drawn through.the:filter. Its

lower portion is a flat ring that restson the outer part of the membranefilter disk, directly over the non-_porous part of the filter supportplate.

c The asseinbled filter holding unit isjoined by a locking ring or by oneor more clamps:

2 Characteristics of filter holding unitsshould include:.

a The design of filter holding unitsshould provide,fbr filtratiOn withvacuum.

(A) ASSEMBLED FILTERHOLDING UNIT

(B) UPPER' ELEMENT,

LOCKING RING

(C) LOWER ELEMENT

PLATE 1

NOTE. Mention (Jr commertle oroductsand manufacturers does not imply ericiorsement by the'-41.1.vr-Iirre-fhe:--Environmental,Proteetion_Agency._

W. BA. nkent. 601i.: a. 77. -ti

88 7:1 -

Meml?rane Filter' Equipment and its Preparation for Laboratory Use

b Filter holding units may be madeof glass, porcelain, plastic, non-corrosive' metal, or other imperviousmaterial.

c Filter holding units should be madeof bacteriologically inert materials.

d All surfaces of the filter holdingassembly in contact with the ,water

P-ri-c-ir-tc3- ifs -pa-Ts .-t hro Ughthe membrane filter should beuniformly smooth and free fromcorrugations, seams, or other sur:.face irregularities that could becomelodging places for bacteria.

. .

e Filter holding units should' be easilysterilized by routine methods.

The filter holding unit should beeasily an quickly assembled anddisassem ed. in routine operationaluse.

f

t4

g Filter holding units ishciuld be' durableand inexpensive. Maintenance shouldbe simple.

3 Several forms 31' filter holding unitshave been develOped for use withaqueous suspe4ions. '

a SS 47 Membrane Filter Holder(Plate 2, Figure 1)

Conical- shape, funnel with a 500 mlcapacity. The' base section includesg wirescreen membrane support. -Funnel and base section are evenly

..----....e-"-- joined by a locking;ring mechanism.1-This assembly is designed to holaa47 mm diameter membrane firmly in

1(place owing an effective filterareaof appr xirdately 9:6 square centi-meters. The entire filter unit ismade of §tainless steel with thefunr!q, interior having a mirror-likefinish:- , .

.b

7 -2

The Millipore Pyrex Filter.Holder'(Plate 2,* Figure 2)

The unit is made of pyrex glass withcciarst grade fitted support_in base .

q

for filter. The upper element ofearly models of glass filter holdershad a capacity of 1 liter. Currentlyavailable units are supplied withupper elements having 300 mlcapacity. The assembled filterholder is joined with a springclamp which engages on flat sur-faces encircling the upper andlower elements.

Millipore Standard Hydrosol FilterHolder (Plate 2,. Figure

Mos t, components of this unit aremade of stainless metal. -Theporous membrane support p/ate.isfine-mesh stainlesssteel screening.The upper eleinent is a itfaight...:

sided cylinder .4. to 5'inches in ,fliarrieter, constricted to a narrow-eylinder at the bottom, to fit theloiter element. Capacity of the

'funnel element is about 1 liter.assembled filter holding unit is

joined by a bayonet joint and lockingring. Accpsories may be obtained'for collection of small amounts offiltrate and for anhydrous sterilizationof the filter holding assembly.

d' Gelman "Parabella Vacuum Funnel"(Plate'2, Figure 5),

89

The unit is mi.de of spun stainlesssteel. The locking ring is a bayonet-type fitting, and is spring-loaded.The funnel element has a 1- literscapacity.

e Th Sabro Membrane Filter Holder(P to 2, Figure 4)

The unit is mostly of -stainless steelconstruction. The lower element isa combination vacuum chamber,filtrate'receiver, and filter support-ing elemeht. It consists of a-stainlesssteel cup' with a metal, cover. The'cover is fitted within rubber gasket'permitting airtight fit of the coverinto the top. of the cup., A porousWintered stainless steel membranesupport disk is mounted in iliacenter of the cover. At the side

"Irak

;

Membrane Filter Equipment and its Preparation for I.,aboratoiy Use

.

of the beaker is a valve, to which a -pumping device can be fitted. 'theupper element is a stainless 'steel

. tunnekwith about 500 rhl Capacity..4.4fie assembled filter holding unit isfned'by a locking ring at the base

". of the upper element. This engageson three spring clamps on the coveringplate of the lower element.

"Sterifilu filter, unit(Plate 2, Figure 6)

A tunnel and flask unit of poly-carbOhate with filter. base andsupport of'pol ropylene. Manu-facturers tabl should be referredto,regarding c emicals which maybe Present in the sample and theireffect on the holder and flaskelements. This unIt can be safely

o sterilized under steam pressure.

FILTER HOLDING UNITS. ,KOR AQUEOUS SUSPENSIONS

FIG. 4

FIG. 6

FIG. 2

4 Care and maintenance of filter holdingunits

I

a Filter holding units should be keptclean and free of accumulatedforeign deposits.

b Metal filter holding units should beprotected from scratches or otherPhysical damage which could result-in-formation of surface irregularities.The surfaces in contact with mem-brane filters shotild receiveparticular care to avoid formationof shreds of metal or otherirregularities which could cause--physical damage to the extremelydelicate filters.

c Some filter hOlding units haverubber components, The rubberparts may in time become worn,hardened, or cracked, necessitatingreplacement of the rubber part ,

involved.

d The locking rings used in somekinds of,filter hiilders have two ormore small wheels or rollerswhich engage on parts of the filterholding assembly. Occasional

, adjustment or cleaning is necessaryto insure that the wheels turd freely

- and function prOperly. On someunits, the wheels are plastic, andare not intended to turn. Whenworn flat,. they should be loosened,turned a partial turn, and tightenedagair.

B Membrane Filters and AbsorbentPads

1

FIG. 5

, PLATE 2'

The dekred propertres of membranefilters have been discussed elaewhere.Typical examples, commerciallyavailable include:

a Millipore Filters, Type HA, whtie,grid-markedi 47 mm in diameter

b. S & S Type B-9, white, black-gridmark, 47 mm diameter

3

Membrane Filter. Equipment and its Preparation for Laboratory Use

C

;

c Oxoid cellulose acetate membranefilters, 4.7 cm, grid-marked

Art absorbent pad for nutrient is apaper filter disk, us y, th; same

-diameter as the membra filter.Absorbent ilia-smust be, free ofsoluble chemical substances whichcould interfere with bacterialgrowth. They should bd of such

= thickness that Ay-will-retain- 1.82 ml of liquid culture medium.

During incubation of cultures on .

membrane filters an absorbent padsaturated with liquid culture mediumis the substrate for each filter.Absorbent pads are supplied withthe purchase of membrane filters.Addjtional absorbent pad may be-purchased separately. Sterilization,in_anakoclave_ is recommended forabsorbent pads.

Vacuum

Almost any, form of culture containeris acceptable it it is made of imperviouSbacteriologically inert material. Thpculture container should, or course, belarge enough to, permit the membranefilters to lie perfectly flat. The followingare widely used

4 Glass petri dishes

Conventional borosilicate_glass-culture -dishes are widely used in laboratory

,applications of,membrane filters. Forroutine work, 60 mm X15 mm petridishes are recommended. The common100 nm X 15 mm petri dishes are -acceptable, but are subject to difficulties.

t Water can be filtered through a membranefilter-by gravity alone, but the-filtrationrate would be too slow to be practical.For routine laboratory practice, twoconvenient iethods are available forobtaining vadimm to hasten sample filtration.

1 Artflectric vacuum pump may be usedconnected to a filtration apparatusmounted in a suction flask. .The pump-need not be a high-efficiency type. Forpro4ection of the pump, a water trapshould be'incltided in the system,between the filtration apparatus andthe vacuum pump.

2' A water pump, the so- called "aspirator"gives a satisfactory vacuum, providedthere is reasonably high water pressure.

3 in emergency, a rubber suction bulb, ahand pump, or a syringe, may-ge usedfor vadtuitn. It will be necessary toinclude some form of valve system toprevent return flow of sir.

D Culture Containerk(Plate 3).Most membrane filter cultures are .

incubated in individual containers.

4

HYDRATOR

ALCOHOL JARWITH

FORCEPS

'st

-4

--)

SUITABLE TYPES OF FORCEPS

GLASS PLASTIC .

TYPES OF CULTURE CONTAINERS

PLATE 3

t

7

a

4

"1.

. .MembraneFilter Equipment and its Preparation for Laboratory Use

.

2 PlastiZ petri dishes ,'

Plastic' containers have been deve ldped 14,

for use with membrane filtercultures.Their cost is fairly low, andisingle-service use feasible. They cannot beheat-sterilized, but are suppliedsterile. They must be frde fromsoluble...toxic-substances. They caneither be loose-fitting or of a tightto base friction fit.

4

Er Other Equipment and Supplies Associated' with Sample'Filtration

1

1 Sudtioa flask (Plate 4)/

a 'Most types of filter holding apparatusare fitted in a conventional suctionflask for sample filtration. Whileother sizes may be used, the 1 -liter

,size is. most satisfactory.

b The suction flasitwan be connected .

to the vacuum facility with thick-"walled rubber tubing. Latex rubber,tubing:3/16H inside diameter, withwall thickness 3/32", is suggested,. .

This tubingdoes:not collapse upioervacUlipl,yet it is readily closed with

'.aipinch clamp.

c A pinch clamp'on the rubber tubingfs a convenient means og cutting off

the vacuum.fRem the suction flaskduring intervals when samples arenot actually being filtered.. It is

4 mos convenient to ha_ye the vacuumfacty in continuous operation during

17 sample filtration work.'

d ,In laboratories conducting a high -

voAime of filtration work, thesuction flask may be dispensed.Filter-holding manifolds are availableto receive up to three filtration,units.The filtrate water is collected inatrap (in series with the vacuumsource)''which is periodically, emptied.-

e Another arrangenlent can be madefor dispensing with the suction flask.%-thie case the receptaci&element

of the filtration 'unit is mounted in

the tench top. Instead of using asuction flask, the lower element ofthe filter: holding unit hasa dualconnection with the vacuum sourceand with the laboratory drain. A

solenoid-operated valve is used todetermine whether the vacuum systemor 'the drain line is in series withthe filtration unit. :

Ring stand with split ring (Optional)(See Plate 4)

SUCTION FLASK RING STAND WITH SPLIT RING

-PLATE 4

't/Wten the filter holding unitdisassembled after -sample filtration,the worker's 'hands.must be free tomanipulate the membrane filter.,Upon dilassemblg of the filter holdingunit, many workers place the funnelelement, inverted, on the laboratory ,bench. Some workers, 'to preventbacterial contamination, prefer a rackor a support to keep iAe funnel elementfrom any possible source of contam-ination. A split ring on a ring stand 1sra convenient rack for this pux.peas.

)

Jp 3 Graduated cylinders

In laboratory practice, .100 ml graduatedborosilicate glass cylinders aresatisfactoryfor measurement ofsamples greater than 20 ml.

.54,

Membrane Filter Equipment and its Prepiration for taboratOry Use

4 Pipettes and tans.

a _graduated Mohr pipettes areneededTor many procedures, such agmeasurement of small samples, andfor preparing and dispensing culture

Pipettes ghoulthbe,availablein 1 ml and 10 ml sizes. .1

b Holding tang may be round or'squarebut must not be ma-de-of -copper.

. Aluminum or stainleSs steel areacceptable:

Aicciriol jar with forceps(Plare 3)

a 'All manipulation of 'membrane filtersis with sterile forceps. For steri-lization, forceps are kept with theirtips immersed in ethanol or methanoWhen forceps are to be used, theyare removed from the container aridthe alcohol is burned off.

b Forceps may be straight or curved.They should be designedto permit'easy handling of filters withoutdamage.' Some forceps havqaporru-gations on tlieir gripping tips. It isrecommended that such corrugations

_ be filed off fOf membrane fiitei; work

6 A gas burner or alcohol burner is neededto ignite the alcohol prior to use offorceps.

Dilution water

The buffered distilled water described,in "Standard Methods for the Examinationof ater and Wastewater': for haCterio-

gical examination of water is usedmenibrane filter methods. Dilution

water is conveniently used in 99 + 2 mlamountsstored in standard dilution,. -

115-dttleST7Stsme-workei.:s-prefer-to-use9.0 + 0.2 ml dilution blanks.

8 Culture mediumAI"

Bacteriological, culture media used withmembrane filter techniques ate dis,cussed t length in another part of thismanual.

*'

F Incubation Facilities"f

ti

Requirements

a Temperature

For cultivation of, atgiven kind ofbacteria, the same temperairerequireMents apply with membranefilter methods as with any othermethod-for cultivating the bacteriain question. nil.' example,bation temperature for coliform,.tests on membrane filters shouldbe 350C + 0.50C.

b Humidity

11,

0

Membrane filter cultu-res must beincubated in an atmosphere main-tainedaVor very'neat to 100yorelative humidity. Failure to -maintain high humidity duringincubation results in growth failure,or at best insmall or poorlydifferentiated colonies,

2 The temperature and humidity r.7.4"...quire-.

ments can be satisfied in any ofseveral types of equipment..

a A conventional incubator may be. used. With large walk -in

incubators, it is extremely difficultto maintain satisfactory humidity.With most conventional incubators,membrane filter cultures can be

,-incubated in tightly closed con-tanners, such as plastic petriIn such containers, required

,hurhidity conditions are- established.with evaporatibn of some of theculture medium. ecanse thevolume of air n-&-tightly closedcontainer is smi.11,_thii result's innegligible changein the 'ctilturetmeheri.- If glassiyatri dishes orotherdoosely fitting eoittaiKers areused, the containers shotrld beplaced in a tightly closAcontaire,with wetPaper-or cloth inside4to

fobtaimthe requir;d'hurrij.dityst on°-dftions. A vegetabld crisper, suchas used in most home refrigerators,'-

O- 1

, Membrane Filter Equipment and its Preparation for Laboratory Use ..

is useful for the purpose. (SeePlate 31

r.

b A covered water bathmaintaining`44.5oC,+0.2o C is neeessary`forthe-fecal coliarm test and thiswilrnecessitate.the use of'a water,-bath having forced-:'circulation of water.

III STERILIZATION OF MEMBRANE FILTEREOUIP.M gNT A_NaSDP.P LEES

A Filter Holding Unit

1 When is sterilization necessary?

aN The filter holding unit should besterile at the beginning of eachfiltration series. A filtration seriesis gopsicterea to be intrruptedthere t,s An interval of 30 minutes

lotiger betw'een sample filtrations.After such interruption any further!-

» sample filtration is treated as anew filtration series and 'requiresa*.sterile filter, holding unlit.-t -

b It is not necessary to sterilize thefilter holding unit between successive:filtrations, or between successivesamples, -.of a filtration series.

After etch 'filtration the funnel wallsare flushed:with sterile water to-free them of bacterial contamination.If properly done, the flushing pro-sedure will remove bacteria. 'remaining on the funnel walls andprevent contamination of latersamples. -,. ..t2

"4".

2 Methods-for st erilifz'ation of filter -,hording unit*

7a Sterilization in the autoclave ispreferred. Wrap the funnel and

receptacle separately in Kraft paperand Sterilize hi the autoclave 15mintftes at 121 0 C. At the end ofthe 15,minutes holding period-in the

4.

autoclave, release the steampressure rapidly, to elcouragedrying of the filter ho ng unit.'

b ,The unit may be sterilized byholding it 30 minutes in a flowingsteam sterilizer.

s--c 'The unit may be immersed 2 to 10

minutes in-boiling-water. This-method is recommended for 'emergency or field use.

d Some units (Millipore Stainless Unit)are available with aeeessories-per-,- --mitting anhydrous sterilization with .formaldehyde. The method consistsof introduction of methanol into awick or porous plate in the sterili-zation accessory, assembly of thefilter holdilg unit for 'formaldehydesterilization, ignition of themethanol, and closure of the unit.The methanol is incompletelyoxidized in the closed container;resulting in the generation, of 'formaldehyde, which is bactericidal.The filter holding unit is kept closedfor at least 15 minutes before use.

e,Ultraviolet lamp sterilizers areconvenient to uset. A device now

). commercially available 'for ultra-.violet sterilization of membranefilter,funnel units.

Sterilization of Membrane, Filters, andAbsorbent Pads

1 Membrane filters '0'1" - ,

, a Membranes a, supplied in units4 of 10 in kraft velopes,, .or in

: ackages of 00 ndnibtanes. Theymay be sterilized C8nveniently in'the packets of 10, bilt shoulder ** .,- --repackaged-if supplied in units-of.- ,no. Large packages of'filters car'be distributed in standard 100 mmXII_ mm petri dishes, or they can

5 be wrapped in kraft paper packets

1

for sterilization. $

94

e

)

7-7

Membrane Filter Equipment and its Preparation for Laboratory Use

b Sterilization in the autoclave ispreferred. Ten minutes at ..1210 Cor, preferably, at 1160C isrecommended. After bferilizationthe steamIsKessure is released as -rapidly as possible, and the filters'are removed from the autoclave and-dri.e._d at room temperature. Avoid,excessive exposure to steam.

c In emergency; membrane filters may.be sterilized by immersiOn'in boilingdistilled water.for 10 minutes. Thefilters should first she separated fromabsorbent pads and paper separatorswhich usually are included in the .package.' The boiling water methodis-not recommended for generalpractice, as the membranes tend toadhere to each other'and must beseparated from one another with,forceps. " °

.2 Absor8ent.pads' for nutrient

a Unsterile absorbent pads canwrapped in kraft_paper or*stacloosely in petri dishes; and autclaved with membrane. filters (t

eed

nminutes or longer at 1-210C or.1.160.C).

. -

b After starilizationTastorbent padsfOr nutrient should be dried befOre

.-'use.

C Glasswa.Aa

, ../ ' ; ..- *#'.. 1 Sterilization at 1700C for.not lesstithap,e "* 1hour is prIfetreildettOsteglassware, (p?p ette s:. g raiduate d- cylinders ' g, 'as s . -,'

. 4 -petri,dishes4, Pi# tesican be slerilized t,

in aluminum or sta eas steel ?Anil, or ,individu i21the ma be wi. ed

exhaust the steam pressure rapidly,and vent the containers momentarily. .This allows the vapor to leave the canand prevents wet pipetteS.

4 -

D -Culture:Containers

1 Glass petri dishes

a Pet*rf dishes may be sterilized inaluminum or stainlessOsteel caps,or wrapped in kraft paper or metalfoil. They can be wrappedindividually,or, more conveniently,in rolls of up to 10 dishes.

b Preferably, sterilize glass petridishes at 1706 C for at least 1 hour.

Alternately, they may be sterilized. the autoclave, 15 minutes at

Lilo C. After sterilization steampressure should be released, rapidlyto facilitate -drying 'a the dishes. ,

Other suggested methods for .sterilization of plastic dishes.include exposure to ethylene widevapor (0:5 i,prethylene oxide per,.liter. of container yolurne), or' ,

Vieposure to ultraviolet light.Ethylqne oxide is adangerouschemical being both toxic andexplosiye, and.it shottld beused

:only when more' convenient-andsafer, methodt are notomillable.

4,

. 2 ePla.stie culture container>s

40 . t.y y pt ypaper. The opening of ghiduated 41(cylinders shohld tiecovered with Paperor metal fop prior to sterilizatiop

4 #,Glassware wftherubber fittitts niuSAiot

. be sterilized ht 1706C,'_as the rubberwill be $lamaied.° # M

.

2 Sterilization ih the autoclave, 15 .es.at 1/11:1C1, is satisfactory,

and referred by Hang workers. Whensterilizing pipettes it is important to

7-8

"' a 'Becal.as.of the thermo-labile_ charaiteristics of the plastic,.

these Ziontdiners cannebp hqat .., .... sterilized. Manufacturers supply::

.these in.a..4terile lognchtion. A . le ,C

P ' ..4r., . 3 4'..,./i .,,". 4. - ,i4;0, ,''b For practjpal-purposes, tolat,,ic ' 11/:* st».

1 ,, .1,

hes, maj bemstegfifized y. .-''' 2$- .. -.0 ; ts' P. e . . dr- St.*, 4meersiffn in 70/0.-iolutrory3f. 0,,, i .,,

s

Ys

.4 -.tii,thanol in *ate , ,i'brgtr leaf 40.. nIrInutes goDishoTinust, be allowed ' .

.1 'to drAntanci diii before.. use, as

sk -ethavol,rvall.influenee the perform-:' Ariceof tuiture media. .

e.

This Otliqe,was prepared by' H. L. Jeteri,Chief, Program Support training Branch,tsEpik' Cincinnati, Ohio .45268 -.c

on....-

bestiptors _Laboratory Facilities, '40-.Bacteriology, Microbiology; Enteric . , .0Bacteria, Filters, Menibrane.

r

a

orf

I INTRODUCTION

a

0

MEMBRANE FILTER' EQUIPMENT FOR FIELD USE.399e Z /

One of the-most troublesome problems in,bacterial water analysis is-the occurrenceof changes in the bacterial flora of water

- samples between the -time-Of-sample collection.and Ifie time the actual bacterial analysis isstarted. Numerous studies have4bPPn madeon this problem. FrozOhese have come such

ommendations (Standard Methods, 10th ed)as holding the sample at 0 -10°C and starting'laboratory:tests as'asoon as possple after .collection of the sample. Recommendations ofStandard Methods, 12th Edition, wts to holdthe saMpIR,as close aspodeible to. the 'tempature of th.rsodrte andto start the labdiattests preferably within 1 hour and always within-a maximum of 30 hours after ,collection.Changes the *3th edition of Standard Meth-

s,od (1 1) willagain call for the icingsa,mples, and further, that.eimples ofrntireonmental waters be held for not more than .8`hours total elapsed time beforele-niples'afeOatedor used for mitrobj.ological testing.

44 3g hour maximum elapsed sarriple hgailingo 4

.tifnevtill, still be rettainecjOr potablewatec.-,aa .n e .

.7:p, . .-;

w.N,

t

. 1

r' A -E-.4y.y vwuiti be used in certain routinewater quality'control Oerationg.,Examplesinclude such places as on_.board ships;some airlines, partitulazny in overseas

-operations. and some national parks. Ineae41 example, it is:seen that there is anobvious difficultyin getting water samples,

, ,,'r. .4. 14;, ;.'

:*,to the examining laboratory in time foraarly examination.'

. *NB In addition, such units would be invaluable

in emergencies when existing laboratoriesare overburdened or inoperative. Portablekits already have proven extremely helpfulin testing many small. ater supplies in a

°short period of time. Further there is apredictable need for' such equipment in the

'event of-a vie.rtime, civil defense'disaster.'Experience of the -Germans in'the vicinityof lisamburg during World War II lendssupport to this concept.

''

!IS

The purpose`of this-discussion is tointrodpee some of the portable equipmentwhich has been developed and to poilit outnoteworthy feataresot)f each. Adttiaiipractiee and experience with these UnitsI _

reveal strong point and Weaknesses in *.each type.

*..The membrane filter'method has been'accepted by the Federal Government forthe bacteriological examination of waterunder its jurisdiction. This acceptancewas based on inethpds develbped andprocedures applied in fixed laboratories. rWhile the use elekita is. not excluded;

. no specialkonc ssion haA been maderegarding the st ndards of perforAlanceof marobrane filter field,kits. 9 Thus; in .planning to use a membrane filter fieldkit for the bacterlologiCal examination of

.water, 'it is thd reiponsibility of the' .individual. Yaboratory.to,establish beyond a.reasonab doubt, by., compairison witlfStarklardMIdthOde ferthentatiOn tube testapr established labofat6ry.4rikerphratzrefilter riieth'oda, the.valte Of uta of the.membrane filter fief kit in dd rm>j ngthe sanitary quality-of -w,ater.eupplies ,

-,v examined: ,:

t TYPES. CbMIVIERCIAI:LY 2WVILIALEMEMBR FitTER EQUIP ENT PORI,FIELD USE .

A Sabro Water Laboratory

This unit represents a fixed Meinbrgnefilter laboratory in miniature, ,

'adaptations for special situations to beencountered in the field; Notable features:,

0.1 The funnel unit supplied on oldv,units

, is- glass. A newer model has nenreleased with anall-metal funnel unit:

.2111'9e vacuum source is a hand pump(modified bicycle pump) oiILstiopally,an all-metal syringe., -

h :

3 The manufacturer sells. preparedampouled,medium, in a liquld,state.;

- The medium should be kepi. at a co61.,M1 ,

>'"-*-r --., .04 NOTV Vfention of 'commercial Products and mcantifanturers does not imply endorsement by the

Federal.Water. PollutiOn Cnntrol Administration.W .-.11A.. men. 63g. 8. 77

. , 4 <

.

,`-Mci.mbrane Filter Equipment for Field Use

1,

4

"'

temperature, out of the light. Itsuseful shelf life is uncertain, butlimited tests by this agency indicatethathe inediurirperforms accept-ably-with storage up to one year.

Inedbation of the cultures is in anincubator drawer having1/4 capacityof 18 1-ounce culture Containers, or ,

36 plastic containers, and operateselectrically at 110V, aricl-With suit-

, able converters, at, 6V, 12V. Abattery also can be used.with thisunit.

5 Sterilization of the funnel unitcarried out by a "light flamingtechnique" or, Optiahally, byimmersion of the funnel unit inhot or boiling water.

6 Useful accessories provided includethermometer, .alcohOl lamp, measur-

.1....ing cup, anfoi2sreTi.e.

B Millipore Field Monitor Units V

These units differ radically from anyother field equipment that has, appeared...Significant reductiofts in bulk"of equip-ment have been brought about through .major changes in function and des-ign_pf*-.-the usual_equipment. Although not rel-commended for valid data due todifferences in quantitation wheri comparedto standard tests procedures, the Unit isuseful for rapid field testing to establish,ball park" figures for latetesti4 with

approved test procedures. Notable features:.. 1

1 The fur/gel unit has been eliminated inits usual form. This has been done-by..development of a carefully fitted, single-use combination filtration unit and cylturecontainer. This feature eliminates most 'of the handling and use of accessoryequipment.. -

4

container. Alternately,. the manufacturermakeil available a delayed-incubationmedium in the ampoules. Other cultUremedia can be used at the discretion of theuser, but some difficulty can be anticipatedin introducing the medium without specialequipment.

Incubation of the cultures is provided in thefield through use of an associated portableincubator and equipment carrying. kit. ThiEg,incubator has room for about 25 cultures.It' is electrically operated, and throughselection of available switching positions, .operates at 6V, -12V, 110V, or at 220V.

'5 Sterilization of components in th'e field isunnecessary. The culture containers andplastic tubes are single-use units suppliedin a sterile condition. Samples do not come

a in contact with the syringe until aftbrhave passed through the filter.

C Millipore Field Unit for Military Use

1 A modified Millipore field unit based on thecase and incubator described in B, 4 above,twiefleenldopted by the U. S. Departmentof Defense. This unit includes 'a miniaturizedstainless metal funnel unit instead of theMonitors.

111r

.2 'The vacubm source is ahlall metal syringe

with e. Mtn providing fpr direct cohnec-tion to the culttfre container.

3 The culture medium provided by the manu-facturer -includes M-Endo Broth, MF, ready -to -use, in g/a4 ampoules. These ampoulesare so designed,as to permit easy introducL.tion of the Clilture medium into the culture

8-2

2 The vacuum source is an all-rhetal syringe.3 Sterilization of funhel unit is by formaldehyde

generated through incomplete combustion.ofmethyl alcohol.

COMMON DIFFICULTIES ENCOUNTERED INCOMMERCIAT,LY AVAILABLE FIELDEQUI PM EN

A, 'line most conspicuous problem arising IA ith fielduse of most units is their ultimate reliance on afixed laboratory for essential supplies.

B These portable laboratories will permit simulta-neous incubation of up to 30 membrane filters.

C 14or protracted field work, a fairly large amountof reserve supplies and equipment will be,neces-san. Such a reserve would include culture media.membrane filters, °ult.-Live container's, fuel, andother expendable supplies required in the field,organized in a supplementary carrying case.

No currently available.field unit provides illumi-nation or optical assistance for interpretationif results.

D

E Some of the terilization methodsrecommen e by manufacturers areunacceptable. If field sterilization in:boiling water is needed, then there mustbe a heat source and a metal can or beaker.Such equipment could be carried in theease suggested in C alDcWe.

V IMPROVISED FIELD EQUIPMENT-

The initial cost, of most of the commerciallymanufactured units has met some objection-.This factor, coupled with need for additionalaccessory supplies and equipment,' hasaroused interest in improvised units. Sucha unit could consist largely of equipmehtnormally used in a fixeklaboratory, packagecin one or two fiberboard cases.

A The funnel unit could be one of thefamiliar stainless steel units used inmany laboratories; or it could be speciallydesigned, smaller than ordinarily used;permitting use of up to a dozes or moreupper filter holding, elements in the field.

B The vacuum source could be the modifiedbicycle pump (leathers reversed), andprovided with a by-pass valve.. The suctionflask could be the standard side-arm glassflask, or ametal unit could be devised.

C M-Endo Broth or LES Endo agar aresuitable medisa. Both are available as.dehydrated medium which must bereconstituted and boiled in the field.M-Endo Broth MF is now available inliquid form, sterile,.in sealed ampules.A shelf life of approximately one year i.:stated when stbred under moderatetemperatures in the dark.,

LES MF Holding Medium.- Coliformrequires merely dissolving in distilledwater. No heating is necessary. Such

-medium`would be arr advantage whereapplir

4.

Membrane Filter Equipment for Field Use

ly

98

oi 5, _gracylinders, media, etc., would be throughImmersion in boiling water for 2 minutesor longer, as indicated for the materialbeing sterilized. Provision for bgi3.inewater is easy through use of a srfiall campstove or other simple burner.

' ua

Improvised equipment, such as discussedabove, would have great usefulness inemergencies, where commercially availablemembrane filter field units are not on hand.

,

V In a separate.outline are detaileddescriptions of procedures for use of r.commercially available membrane filterfield equiptnent. In some cases thesuggested methods are different, fromthose recommended by the manufacturers.In each case such departures are basedon a series of experimPntal studies modeby this agency, which suggested needfor modification ofsexisting recommend-ations.

4

REFERENCE

1 Laubusch, E. J. What You Should KnowAbout the Membrane Filter, 'PublicWorks, 89: 106-la, 162-68, 1958.

This outlinewas,prepared by H. L.Jeter, Chief, Program SupportTraining Branch, USEPA, Cincinnati,Ohio 45268

4

,FieldOn-Site Laboratories,'

Field Investigations, Filters,membrane, Bacteria, Microorganisms,Labdratory Tests

I.

8-3

41

/

PRINCIPLES OF CULTURE MEDIA FOR USE WITW MEMBRANE FILTERS

I INTRODUCTION_

A Marcy kinds of Membrane filter mediahave been described for use in bacterio-Ngical tests on water. This noteworthyip view of the relatively few-years-the-filters have been widely available in this'cur try. This discussion is to considerseveral of these media in terms of theirpurposes, composition, an the ways inwhich they are used.

B Basic Considerations

1 Filtration of water sample through amembrane. filter results in depositionof.bacteria and particles of suspendedmatter on the filter surface. Thebacteria can be cultivated in place ifsuitable culture medium is made avail-able for their growth.

2 The bacteria are cultivated by placingthe membrane, on a pad of absorbentpaper saturated with liquid culturAmedium, or on an agar medium. Theculture medium diffuses through thepores of tnekPilter, and is available tothe bacteria on the opposite surface.Proper time, temperature, and humidityof incubation results indeyelopment ofbacterialcolonies. In principle, eachbacterial cell multiplies to become a,.single bacterial colony.

_3 Some culture media, satisfaatory fortribe cultures or agar plate cultures,

- do not perform well' when used withmembrane filters.due to a seJ.eetiveadsorptive prpperty of the filter itself.

sIn the process of diffusion through thefores some component's of the culturemedium may be removed, completely,

. or reduced in concentration. Thus, thecomposition of a given culture ineditmlat the filter surface where it is avail-able fqr bacterial groNthmay be dif-fe rent from its composition beneath theAnernbrane filter.

There is evidence that improved cul-tural results sometimes are obtainedwith increased concentration of certainnutritive constituents of membrane filterculture media.

Pare cultures may be recoveredfrom membrane filters and subject-ed to supplernentary biochemical,cultural, and serological proceduresfor identification studies or for veri-fication of interpretations based ondirect observation of membranefilter cultures.

The same use can be made of agar plat-ingmedia; however theimembrane filteroffers advantages due to the ability toconcentrate organisms from'a largevolume of sample in which the organ-isms are present in low density.

C Applications of Membrane FilterMedia

The composition of bacteriological culturemedia designed for tube or plate culturesshould be subjected to critical studyibe=fore they are applied to membrane filterprocedures.- Media based op well-knownbacteriological media have been modifiedfor use with membrane liners for the

' following purposes in testing water.

1 Bacterial plate counts

2 Media for bacterial indicators ofpollution

Coliform organisms

b Fecal strentoc. oceus group

c Clostridium perfringens

4 3 Salmonella and other enteric bacterial' pathogens

NOTE: Mention of commercial products and manufacturers does not imply endorsement byChe FWPCA and.fhe U.S. Department M therlate

W. BA. meth.. 681; I.78 s(9 9, 9-1

Principles 'of Culture Media

---+

.

P Constituents of Membrane Filter CultureMedia

Membrane filter media for the differentia-tion and counting of special groups ofbacteria are based on the same principlesused in differential agar plate media. Thus.the components of a differential medium:"or membrane filter cultures include:

Substances favoring growth of theorganisms for which the medium is,designed. Inclusion of special peptones,fermentable carbohydrates, yeast ormeat extracts, water and chemicals t6adjust pH to a desired level are common,methods of favoring growth of desiredorganisms.- .

.111q, 2 Differential indicator system. Thepurpose of the indicator system is toproduce characteristic colonies of thedesired bacterial groups for easyrecognition when present in a mixturewith extraneous types of colonies. Thisis done through inclusion of (a), a com-ponent which is chemically changed bythe organisms to be differentiated, and(b), indictor substances, which giyevisible Of idence of an intermediate orend product resulting from a chemicalchange of substance (a).

SelectiVe inhibitors. Some bacterialgAciups to be tested may be over-whelMingly outnumbered by extraneoustypes -of bacteria. In such cases, it isnecessary that substances-be-included itthe medium which (a), prevent growthof a maximum number of kinds of e±-traneous baCteria, And (b), havemum aterse affict on growth of thekind of lateria for which the mediumis designed.

E Variety of Methods of Using Media Avail-able- with-Membrane Fine r:Methoda

1 Single-stage tests

After sample filtration, the membranefilter is placed on a designated' culturemedium, and left there throughout theincubation period. lThe Allure resultsaTh=eexaririned-and-inter etecpirectly.

9-2 ,

.

2 Multi-stage tests

A membrane filter can be transferred'from one culture medium to anotherwithout disturbance of bacteria orcolonies on the filte'r. This is uniquewith membrane filter methods, andlends itself to a variety of cultural andtesting procedures. rta The membrane filter, a iteir am.pl

filtration, can be incubated orspecified time on one mediuk, thcntransfered to a second medium. Themethod permits initiation of growthon enriel)ment medium, after whirl,the membrane filter can be transferred to a less productive medium.With grOwth already begun, somedifferential culture media give betterquantitative production titan wouldbe the case without:preliminaryincubation.

1

b After incubation on one or more -media, colonies on the membranefilter can be subjected to bio-chemical tests with reagent's-tootoxic to include in the culturmedium. Such reagents may beflooded over the groWth on thefilter, or the filter May be plaCedon an absorbent pad saturated withthe reagent, in order to mare suchtests.

c A third type of multi-stage Cal" is,one in which the membrane filter,after sample filtration, isplacedtemporarily on a medium containinga bacteriostatie agent. in thepresence,of Such a substance,bacterial growth isinhibited orslowed greatly, but the organiamaare not killed. During a limited,period, the 'Membrane filters Maybe 'transported or stored- at'ambienttemperatures. Tli filter 'can be ,'transferred later to a s'uitable

4 medium and incubated for develbp-ment of coiohieti

9

)*-

1

II CULTURE MEDIA FOR TOTAL BA:CTE-RIAL COUNTS ON MEMBRANE FILTERS

°A Concepts

I Strictly, a "total" bacterial \coun4medium is nonexistent. No qinglemedium and incubation procedure canprovide simultaneously the fug rangeof oxygen requirements, needs forspecial growth substances, pH require-meats, --eltc -4:4 all the kinds of bacteriafound in Wat'er.,

61.tually, "total" Itacte'rial (*bunts arecounts of the bacteria developing visiblecolonies on a defined Vulture mediumat a known pit after incubation for aset time and temperature under aerobicconditions.-

3 Within the foregoing liMitations, thefollowing criteria offer a useful basisfor selection of membrane filter mediato be used for estimates of the bacterialdensity in watcr.

a The medium and its method of useshould produce a maximum numberorcolonies from all types of water.The colony yield shOtild.comparefavorably with the bacterial counts'determined as described in "Stand-ard .Methods for the Examination ofWater, Sewage and Industrial ° °Wastes." 13th Ed. (1971).

b The colonies should develop rapidlyto a sufficient size to be countedafter a minimum incubation period.At present, best results with mem:-'brane filter MetWaaare-obtainedafter about hours incubation.

c The medium should be one which isreproducible and routinely avail4b ein laboratories.

B CompOsition of Total Count Media forMembrane Filters

Almost any rich,' gene nal growth h promotingculture medium is acceptable for total bac-teria). counts on membrane filters. Several

. Principles of Culture Media

such media have beeq suggested especiallyfor membrane filter methods. These differonly in minor aspects, and can be discuss-ed as a group. For details of compositionand specific applications of each, see themedia formulations elsewhere in this

Growth_prornoting substances: All thesubstances inelnded in these media are

.included to eAcourage growth of a maxi-mum numbor of kinds of bacteria. Mostworkers agree that the peptone shouldused ih twice the concentration usuallyfoundiin conventional tube or agarplat-ing nr dia.

2 Indicator substances'are unnecessat.y,witr total count media.

3 Sabatances fort * selective. inhibitionoaf certain bacterial groUps are not in-

.,,eltlded in total count media.

Problems Enountered with Ttal CountAtedia

Bacterial colonial growth habits on mem-

.rano filters are similar toitheir surface

growth habits on simifar-agar-plate-media--

1 As with agar plate media, some speciesj1 ' of bacteria grow continuously, spreau-

ing over the surface of a membraneI filter, tepding to obscure nonspreading

'colonies which'otherwise*could be,tounted.

,2 Some samples contain an appreciableamount of particulate matter. In sam-ple filtration, this is depbsited on the

ace ortheinembrene-filter 4ith the..bacteria. When the culture medium --diffuses through the filter; a caiillaryfilm of liqtfid culture medium.aWimti-lates around the particles of egtraneoqsMatter. Bacteria not ordinarlry Conc.sidered "spreaders" sometimes developconfluent colonies due to the film of. .

liquid medium aCchiritilating-around-such--__.particles..

9-3

?

4

Principles of Culture Media,

D What is the best total.cOunt medium fouse with membrane filters?

Because of the relative ease of,preparation,most workers prefer the commerciallyprepared dehydrated media. Difco M-Enrichment Broth (B 408) or BaltimoreBiological Ltboratories' M-Enrichment.Broth (No. 331) are used interchangeably.TotaLcolotly productivity of these mediais eqUivalent to that of media preparedfrom the individual component's.

III CULTURE MEDIA FOR TOTAL COLIFOR.1"TESTS ON MEMBRANE FILTERS

A Concepts' r"

4

The nature of membrane filter culture 'methods imposes a different definition ofcoliform bacteria than the Standard Meth lids'definition.

1 Standard Methods fermentation tube _

t meth d. "The coliform group includesO `all of the aerobic and facultative'anaero-

bic Gram-negative nonsporeformingrod-G sha e bacteria which fermentlactose g formation within 48hours at C, "

2, Membrane Filter Methods: "In themembrane filter procedure, all organ-isms that produce a colony witha metallic 'sheen in.22-24 hours areconsidered members of the coliform

.group. 'The sheen may appear awe.small central focus or Cover theentire colony." The guiding prin-ciple is that any amount of shAnis considered positive.

3 The Standard Methods definition ofconforms requires demonstration ofthe ability of organiSms to produce gasthrough the ferLnentation of lactose.The membrane tiller method does notlend itself to the demonstration of.gas,production. It relies instead on the /development Of a particular type ofcolony on an Endo-type of culturemedium. , The' culture 'medium is onein which lactose, basic fuchsin, andsodium sulfite comprise an indicatorsystem to cause differentiation of

.0-4 1

conform colonies. While the bacterialgroups measured by membrane filtermethods are not identical with the groupmeasured by Standard Methods pro- '

cediires, they are believed to be es-sentially the same, and to have equalsanitary significance.

B Composition of Coliform Media for,Membrane Filters

Several different media have been suggested,.for coliform tests on membrane filters.The components of these media can beclassed'into three convenient groups forgeneral considerations.

1 Growth-promoting substances. Growthof`bacteria on all the media is favored bythe inclusion of such components aspeptones '(asNeopeptone, Thiotone Cab"tone, Trypticase, and other proprietarypeptOnes), yeast extract, dipotassiumphosphate (for adjustment of reaction ofthe medium), and distilled water. Lac-tose is included.in all these media. Itserves doubly, to favor growth of coli-form bacteria, and as an essential compo-nent of the systems for differentiatingcolifol-rn colonies.

2 ,Two kinds of differential indicatorsystems are available for demonstrationof lactose fermentation on membranefilters.

a Lacto;e-basie fuchsin-odium sulfite .

system (Endo type media).

1) Mcdil..using this sy stem includelactose and a suitable concentra-tion of basic fuchsin which,has beenpartially decolorized with sodium

, sulfite.

2) The basic fuchsin-sodiuni'sultitecomplex requires Very carefulstandardization. An excess of

'either component results in anunsatisfactory culture medium.

'?-

3) The indicator system demonstrateslactose .fe ementation as follows:

402.

I

Principles of culture Media

a) The coliform bacteria producealdehyde as an intermediateproduct of the .fermentation oflactose.

b) The aldehyde is "Complexed"by the sodium sulfite-basicfuchsin indicStor. In this pro-fess a reaction occurs in whichred color is restored to thebasic fuchsin. Colonies ofbacteria fermenting lactoseassume the color of the re-stored fuchsin. As the restoreddye accumulates, it apparentlyprecipitates on the colony,giving the colony a character-istic green-gold surface sheen.The reaction occurs best in analkaline medium. The culturemedium is adjusted to pH 7. 5:

4) Endo type media require verycareful standardization for suc-cessful use in the laboratory.Most workers prefer to use acommercially prepared and stan-dardized medium. M-Endo BrothMF is the recommended coliformmedium for use with membranefilters.

b pii indicator-system

1) Media using this system rely ondeteftion of pH change due to theaccumtilationof organic acids,end products of lactosefermentation.

2) Bromcresol purple, for example,is a pH indicator, 'approachingyellow at More acid pH. Colon-ies fermenting lactose and ac-cupulating organic acids there.forc turn yellow. ,

3) Studies in England with membraLlefilters for boliform tests havebeen based on a modification ofMacConkey's Medium; using thisprinciple of q6lony.differentiationin coliform tests.

I

t

Inhibitory substances in membranefilter coliform media

nfusing and erroneous results incol orm detection can ht. rnlised'by

1) overgrowtn of the membranefilter by extraneous nonlactosefermenting bacteria, preventingcoliform colonies from developingthe .characteristic color' andsheen; and

2)' the development of sheencolonies of lactose fermentingbacteria which produce acid butnot gas in the fermentation of rsLactose.

(

b -These, difficulties can lie r, ,

t 'hrough incorporating of substancesharmless to coliform bacteria butwhich have inhibitory effect on growthof 'extraneous forms. Attention mustbe given to the concentration of suchsubstances, as excessive amounts-also will reduce the productivity ofthe medium for; coliform colonies.The following components of variousculture media have proven useful insuppressing growth of noncoliformbacteria on membrane filters,.

1) Basic fuchsin-sodium sulfite. Al-though these compounds are includedin Endo-type media for their rolein differentiating coliforms fromother types of colonies, they areeffective in preventing the growthof many of the noneoliform bacteria,occurring in water samples.

2) Ethanol (95%... NOT denatured) isincluded ip Ail-grin-troth MF.,, Inthe concentration used,' eIhanol .

suppregses growth of,Some kindsof noncollform bacteria, and tendsto Unlit 'the colony size of others.In addition, theethahol seems to .increasethe solubility of some ofthe- other components of the media.

-O

Principles of Culture Media

q

3) Sodium desoxycholate or bile salts .' are used in such media as M-Endo

Broth MF, and In ihemodifiedMacConkey's Medium for membrane

-filters used in Britishatudies. Theyaie included primarily fdr their in-hibitory effect against Gram-positiVecocci and spore formers.

C 119ds Avaklable for UsingMedia with Membrane Filters

a

1 Single-stage coliform tests

A, a -After sample filtration, the mem-brane filter is incubated for thedesired time on a selective coliforrrdifferentiating medium.

The conform colonies are countedwithout further tests.

M-Endo Broth MF and LES EndoAgar Media are alternate standardsingle-gtage coliform Media.

2 Two-stage coliform tests

a Immediate coliform test

1), After sample filtration, the men,brave filter, is incubated 11 2

'':.hottes on the efiriehment medium-oflauryl try2Loi_te broth.

2) The memlicane is*then transfernto a new absorbent pad Eiaturatedwith the standard differential

r medium for colifOrm bacteria,and incubated tor 20-22 hours at35 +. 0.5C.

-

3) The coliform colonies. arecotinted .

, without further tests.

4) This test procedure, based onEHC Endo Medimina was described 4..

in the, 10th edition of Standard .Methods. With the 12th edition,an official two-stage coliform testhas been adopted, based on LES

.r.P.pto Agar Medium.ti

j

1) Delayed inkthation colifOrm tes

1) After sample filtration, the mem-brane filter is placed on an ad-sorbent pad saturated with benzo

rated Endo Mediuth or with LESHolding Medium. The filter ma),'be preserved up to 72 hours at'ambient temperatures. During 'this time it can be transported orstored. Growth is stopped orgreatly reduced.

2) The membrane filter can be.transferred to a fresh absorbentpad, saturated with such a mediu.nas M-Endo Broth MF, or to LESEndo Agar and incubated up to 24ho,drs.

3) The differentiated coliformcolonies are counted as withother membrane filter coliformmedia.

4) This test procedure makes itpossible to filter samples in thefield,e place the filters on4pre-'servative meaium, then mail or

St transport them to the laboratoryfor completion of the bacteriologi -cal examination. The procedureis designed to eliminate the needfor maintaining sample tempera-

--lure in the interval befweensample collection and.initiationof the bacteriological exrninatiOn. ,In addition, the method ghouldproduce results more nearlyreflecting the quality of the sourceWater than is available with othermethods of collecting and testingsamples.

.3 Verified menibrane filter coliformtest d

a This is used to verify the interpreta-. tion of-differentiatedcolonies on any

type of membrane filter cqliforibmedium. The test is suggested for:self-training of laboratory workers,for evaluation Of new or experimental

f.

t a

Princi les of Culture Media

media, and in any water examinatio!in which the interpretation, of resultsis in doubt or likely to be involved inlegal controversy.

b The test consists of obtaining purecultures from differentiated coliformlike colonies on membrane filters;and subjected therit to further culturaland biochemical tests to establishtheir identity as Gram-negatiVe non-sporeforming bacilli which fermentlattose with gas production. Thetechnical procedures are described .elsewhere in this manual.

. IV MEDIUM FOR THE FEdAL COLIFORMTEST

A' Concepts 0"

':

4* THe selective effect of elevated temperatur. . .has bee/I.-the most important development

in fe,calfoliform tests .since 1904. In that,

year, Eijltman discovered that coliformbacteria from the gut of warm-bloodedanimals produced gas from glucose' at46oC,_while the majority of coliformbacteria from other sources did not.Media variations were of only 'secondary importance.mportance.

. ,

Much medium variation ha.s.resulted frtomattempts to select for Esehericlua coil. )only, as the fecal coliform. While E*. coli

is usually the predominant coliform inhuman (and animal) feces, other typesare present, including the alleged soiland plant coliform, Aerobacteraerogenes in very large numbers.All coliforriis demonstrated by isola- _

tion to have arisen in feces are called herefecarcoliforzrns and are measured empir -idally by theifecal coliform tube test.Membrane filter tests reflect divergence"of attitude on indicators of fecal origin.Delaney et al. (1962) have published:,Measurement of E. con Type I by theMembrane Filter. Geldreiap et-al. (1965)have presented: Fecal-Coliform-prganismMedium .for the Membrane Filter Technique

Temperatures are the same but media aredifferent. Because the fecal coliform testappears more convenient, it will beemphasized.

B Composition of Fecal. Coliform MediumMFC

1 MFC medium is a rich growth mediumcontaining lactose, proteose peptoneno. 3, tryptone and yeast extract. Alevel of .3% sodium chloride producesfavorable osmotic balance-. Vigorousgrowth results. A practical result isshortening of test time to 24 hours.eaThe growth constituents are similar tothose of the tube test for fecal coliform.Both have` 0.15% bile salts to select forcoliforms but elevated temperature isthe more important selective factor.

2 The. indicator s tem of aniline blueresults in blue fe al coktermeolonies.Nonfecal colif m colahles, generallyfew, are gray to cream-colored.

C Special Problems with MFC Broth Medium

1 Temperature control must be accurate.CUrrent recommendations call for44.5 + 0.2°C and the temperature4o

'be maintained in a water incubatorof forced circulation.

2 'ierbperature equilibration must berapid. Nonfecal coliforms may initiatp-growth at lower temperatures and sub-sequently give false poSitive bluecolonies when incubated at 44.50,C.No more than 20 minutes lapse of timeis recommended from filtration toincubation. Submergence in waterproofplastic bags tees attudl temperatureequilibration t 10 n 12 minutes.

3 Rosolit acid presents some problemsin preparation. It is practicallyinsoluble in water and of limitedstability in alkaline soltition. A 1%solution in 0.2 N NaOH should beprepared and this added td the mediumas recommended by the manufacturer.

Principles Of Culture Media

V MEDIA FOR-FECAL STREPTOCOCCUSTESTS

A Introduction

1 The development of rnembrane filterculture metia for the fecal streptococcire cts the continuing interest in thisgroup bacterial indicatbrs of pollution.The pro uctivity of enter+orococcus mediarecently as been greatly. increased,

2 Standards of performance of a goodfecal streptococcus medium correspondwith those of a good colform mediumon membrane filters. Thus, therequirements of productivit, specificity .ease of use, and reproducibility of themedium, are equally applicable tomedium for the detection and enumera-tion of the fecal streptococci.

B `KF Agar

1 This streptococcus medium wasdeveloped at SEC by Kenner, et al.and designated KF agar,

While KF medium is productive fordetection and" enumeration of theFecal Streptococcus Group,, its useis hampered by nonspecificity. Studieshave demonstrated that the medium

. supports growth of S. bovis., and otherforms common in animals, but nOtnumerous in the fecal.acreta of humans.

'2 Composition of KF Agar

a Nutritive requirements of the fecalstreptococci are supplied by peptone,yeast extract, sodium klycerophos-phate, maltose, Ia:ctose, and dis-tilled water.

The'indicatorsystem is phenol,red0 and 2, 3, 5 triphenyl tetrazogum

chloride. OtrKF Agar, used with.membrane filters,: the-fecal' strepto-coccus colbnies develop as smallcolOnies, up to- 2 mm in diameter,colored vaFious shades. from apink to ,a dark wine:color.2

r'.

9-8

he selective component of KF Agaris dium azide, used in 0.04%concentration.

REVERENCES

1 APHA. AWWA, FSIWA.L Standard Methodsfor the Examination of Water and Waste-water. 12th Edition.

2

3

Clark, H. F. . Teter, H. L.and Kabler, P. W. The Men4rane Filterin Sargtary Bactertology- Public HealthReports.. 66;951-77. 1851.

-Fifield, C.W. aed Schaufus, C. P. ImprovedMembrane Filter Medium for the Detection ofColiform Organisms. J. Amer, Water WorksAssn. 50:2!193-6. 1958.

4 Geldreich, E. E., F., Huff, C. B.and- Best, L. C. Fecal-Coliform-OrganismMedium for the Membrane Filter Technique.J. Amer...Water Works Assn. 57231209-14.1956.

.5 Kabler, P. W. and Clark, H. F. Use of..Differential Media mitif the Membrane Filter.Am. Jour. Pub. Health. 42:390-92. 1952.

6 Kenner, Bernard A. , Clark,- H. F.' andKabler; P. W. Fecal Streptoc.occi.1. Cultivation and Enumeration ofStreptococci in Surface Waters.Applied Microbiolody. 9:15-20. 1961.'

06-

I

1

a

Principles of Culture Media

7 McCarthy, J.A., Delaney, J. E. andGrasso, R. J. Measurihg Coliformsin Water. Water and Sewage Woiks108 :238. 1961.

8 Rose,' R. E. and Litsky, W. EnrichmentPrbcedure for Use with,the Membraneniter hfor the Isolation d Enumeratib"of Fecal Streptococci WatersMicrobiol. 13:1:106-9 1965.

9 Slanetz, L. W. , Bentof the Membrane FilEnumerate EnterocoPublic Health Repor

nd Bartley.tor Technique to

ci in Water.s. 70:67. 1955.

10 Slanetz, L.W., °Bartley, C.,H. and Ray,A. Further Studies on Membrane

Filter Procedures for the Determinationof Numbers of Enterococci isn Water andSewage. Proc. Soc. Am. Bacteriologists.56th General Meeting. 1956.

°

This outline was prepared by H. L. Jeter,Chief, Program Support Training Branch,and R. Rossomanno, Microbiologist, USEPA,Cincinnati, Ohio 45268.

Elescriptort: Cultures, Bacteria EntericBictdria, Coliforms, Fecal Coliforms,Streptococcus, Membrane Filters.

II 5

1

X

4

SELECTION OF SAMPLE FILPtiR MEMBRANE FILT

3

4 .I INTRODUCTION

A Wide Range of Filtration Volumes

It The membrane filter permits testingI a wide range of sample volumes, from,

several hundred milliliters to as littleas 0.0001 ml, or even less. Suitable'dilution of sample volumes smaller,than 1.0 ml may be required foraccuracy orsample measurement.

2 While the method lends itself to a wide.it arange of sample volumes, the filter haslimitations yn the number of isojoited(or countable) difrenlioed colonieswhich can develop on the available sur-face areit. Figure 1 illustrates a tom:

, Mon pattern of colony counts over-awide range of sample filtration volumes.

TIO VOLUMESR METHODS

or both the total. coloni6 and the__co iforni2tOlonies there-is a pro-poi-tional relationship between colonycount and sample volume iSkr muchof the range of sample volumes.With increasing colony counts, thereAre some levels abdve Which the pro-portional relationship fails, both fortotal and for conform colonies.

. ,

c in the straight tines in Figure 1,whe're there is proportionality be-tween colony counts and filtrationvolumys, it is possible to computedensity of acteria in the Sample,

d op e equation:

organiSms = 100 X No. colonies countedper 100 ml. 4' No, ml of sample

filtered-

,/'a/tf° a S

, Total Colonies 1

r

-61 °2Coliform Colonies

' I t ' '4

Sample VolumeFigure 1 3

1,

" 4a 'The graph is based on conform' detprminations using M Endo BrpthMF. The,line.slesignated 'TotalColonies" includes both coliform andnoncoliform colonieth: The "ColiformColonies" line refers only*differ-

. entiated Colonies hayingthe typicalcolor and sheen of Conform colonieson the medium.

p

W . BA. mem. 75f. 8.77

The equation is ,not quantitativelyre liable\ in the curved portions ofthe lines.

I .

e of this, Presentation

o explain limitations on quantitatively- -r liable colony counts on membrane

'Si ters. *4`tTolpresent.numbers of Foldiiieabl for quantitative tuts witme ia.

a.

.To emonstrate the different quantitativebac erial density ranges determine y__

sin e-volume filtrations, with availablemed:a.

I -

aCrceptl.avaitablf

4 To demonstrate quantitative rangecovered by a series of filtration volurr't

^ with currently-used differential media.

I

,1.08ipst,/

.x

Lloo.

:Selection of Sample Filtrati54Volumes

- ,

5 To prbvide guidance for selection ofSample filtration vdlufnes under the,.following practical conditions:

a When there is need to determinecomplian6e with established La c-t eiiologicalwater quality standards.

-is- When there is need to determine

Clensity of a specified bacterial group.

1) In the absence of prior bacterio-logical data, and

2) When prior bacteriological dataare available.

U LIMITATIONS ON COLONY COUNTSUSED FOR QUANTITATIVE WORK

A Bacterial Density

1 The minimum sample volume shouldresult in production of at least 20colonies of the bacteria being counted.SaMpte,,volurnes yielding lesser numbersof rolorkieS,are subject to unacceptablylarge.raAdot variations in the computedbacterial density, determined as above

z

. The maximum acceptable colony density,for quIntitalive determinations, is vari-able. IXith the bacterial group tested andthe medium ,used,' Factors influencingmaximum acceptable ,colony densityinclude:

a' Size-of colonies.. In principle, each,colony should represent one.bacteriilcell deposited on the filter, or, con-versely, ea.ch, bacterial cep deposit-ed on the filter should result in, pro-duction of a recognizable 'colony.

edia'producing relatively large"Icolonies (as in the fecal coliformtest) "will spOort smaller numbersof colonies on the filter than mediaproducing smaller colonie (such asfecal streptococci). If t nysize is large andthe num' 1* of tee-

. ° teria deposited on the filter is great,

10-2

some colonies will repre lent two ormore cells initially deposited on thdfilter, and the quantitative reliabilityof the test is impaired,

1

Sle

r's

,b Selectivity of medium. Highlyselective media permit growth ofrelatively few colonies of extraneous,unlvanted, bacteria. The availablearea of the filter is occupied pri- .

marily by colonies of the group ttested. Thus, with a highly selectivemedium such as that used for fecal-streptococci, it is reasonable toexpect good. quantitative results withrelatively high colony counts. Con-versety, media having limitedselectivity (such as Endo-type mediafor co orms) supports growth ofconside ble numbers of extraneousbacteri colonies, and it is necessaryto place arbitrary,limitations on theAmber of colonies per membrane inquantitative studies.

c ,iochemical interference betweenneighboring colonies. Associatedwith the-physical crowding effects'noted ins(b) above, sheen productionof conform colonies maybe inhibitedby OVercro"Vicling of cdlohies. This.reinfOrces the need for restrictionof colony density on the filter. ',

B Suspended Matter

1 Particulate matter in the sample canbe a limitation in application of mem-brane filters, especially when theamount Of susnended matter is relativelygreat,and the bacteriaillenslty is low.

2 Difficulties fronksuspeuded matter inthe sample maylie-apparent in severalways./

109

a The pores °fele filter may beoccluded, limiting the volume*ofsample that cak be filtered. Thisnroble has been noted in watersrich i clays and ip waters containinglarge opulations of certn diatoms 1

or other algae:

b Fibrous matter can be- troublesome,due to the ,tendency for a capillaryfilrh of liquid culture _medium to formaround the fibers. Colonies in contact::with such fibeis tend to grow along

1.

*.,^s

4

the path of the fibers, assuminghighly irregular forms. Sometimesthese, colonies cover abnormal),large areas of the filter surface.

c A more, o less continuous mat ofparticles may be collected fromsome samples, with each particlesoon surrounded by a film ofliquidculture medium. On such cilters,distinct colonies usually fail todevelop as discrete entities, butgrow in a more or less continuous

. 4 film over the entire surface of thefilter:

-3 Problems due to particulate matteroften can be reduced by filtration.ofthe selected,volume of water in two ormore smaller increments, through

1E;parate filters. In effect, this is ameans of enlarging the availablesur-face area or t4 fitter.

Prefiltration of the mple through acoarse filter for prelithinary removalof extraneous particulate matter is notrecommended in quantitative work.Prefiltration invariably results in re-moval of unpredictably large numbers ofbacterial cells.

In some cases the problem of particu-lates cannot besolved, and it mustthen be conceded that the membranefilter method is not acceptable for suchsamples.. It then becomes necessary toresort to other procedurei, such as thedilution tube method or. agar platingmethods.

/1.I LIMITS ON NUM(3ER OF'COLONIES ONFILTERS WITH VARIOUS MEDIA

Referring to Figure 1, a specific number ofcolonies is not shown for acceptable propon.--tionality between colony numberand filtration

_volume. Fixed limits cannot be stated for all..:test,,:satuations, for thede limits are somewhat1,1N-fable from one culture medium to anotherand from one sample source to another.

Selection of Sample Filtration Volumes

'The recommended limits shown in 'Table 1are empirical values ba'sed on -re-search experience. It is believed that quan-titative determinations of acceptable statisticalreliability ca be obtained- if the determidationsare based on olony counts within the limita-tions shown.

IV RANGE OF BACTERIAL DENSITIESCOVERED BY SINGLE-VOLUMEFILTRATIONS

A The equation .used in Section I of this out-line can be used with any sample filtrationvolume to determine the bacterial densityrange over which acceptable counts can bemade. '

For example, assume that a sample of.10 ml is used for a quantitative det'ermi-nation of total coliforms. Based on Table1, quantitative determintitions should bebased on a filtration volume yielding 2080 coli%orm colonies. Computethe coliforms per 100 ml based on 20colonies and on 80 colonies per filter. Thiswill be the bacterial density range covered . -

by a 10 ml filtration volume, thus:

'for 20 colonies:

No. eoliforms per 100 ml = 100X '110°

= 200and for 80 colonies,

:'No. conforms per 100 ml n 100 X'8010

806

Thus, a 10 ml sample portion is apprb-priate for determinatiouoi total coliforms -.in the range 200 - 80b per 100 ml.

B Table 2 illust ates the ranges c red forseveral filfr on volumes, tivi h ccilonycounts in th anges 20 - 60, 20 - 80, and2Q - 100 pe Jijfination volume.

110.48

10-3

'Selection of Sample Filtration Volum esr*

Table 1., RECOMMENDED COLONY COUNT RANGES'FORQUANTITATIVE DETERMINATIONS WITH

MEMBRANE FILTER TESTS

Tist ...Nci. colonies Medium Remarks-

Minimum Maximum

Total Coliform 20 80 M EndO Broth MF, Not more than 200; LES Endo Medium colonies of all types

Fecal Colifo rm -ie

. . 20 60, M FC Broth .

.

Fecal Streptococci 20. 100. M Enterococcus .

Agar, fCF Agar*

TotaLCounts ' 20 200 M Enrichment Spreaders may4 . Broth require, adjustment

Table 2. RANGES COVERED BY REPRESENTATIVEFILTRATION OLUMES

M1 samplefiltered

Bacterial co20 colOnies 60 cold

nt er 100 ml 1:;..sed on80 c lonies 100 colonies,

20. 200

20020,000 -

200, 000

60600

600060, 000

600,000

. 80800

80008d, 000

800, 000

C Application of a Series ofVolumes

1 Examination of Mike 2 shows that forquantitative work on membrane filters,to e?ctend the range of any test, it isnecessary to filter two or more differentsaniplevolumps: The worker uses theone sample volume yielding a quantita-tively acceptable number of colonies tocompute,the bacterial count per 100 ml.

2 Further, ificatOe seen That varying th,filtration Volunies by decimal-incrementswill be ,inappropriate; there areva:lueswithin the total range covered in whichthe colony number would fall outside thecritical countingrange for the test beingmade:

3 In order to- giveomaximum assurancethat a series of varying filtration-e:."!umes will yield at least one membrane

4. ing bx a factor of 5, or less.

1001000

10, 000U-1,100, 000

U-1 000 000

with an acceptable number of colonies,the range of filtration volumes shouldbe along these lines:

a Total coliform counts should bebased on filtration volumes varyingby p. factor of 4, orless.

b 'Fecal coliform counts should bebasedt,on filtration volumes varyingby a factor of 3, or less. _

.4

Fecal treptoCoecus counts shouldbe b d on filtration volumes vary-

V SELECTING FILTRATION VOLUMES. FOR MEMBRANE'FILTER TESTS -

A Total' Conform Counts

1 Determination of compliance with*exist-ing bacterial quality standards.

-47-

s.

a For all tests to determine Whet lywater meets PHS Drinking Waterquality standards, minimum samplesizes are prescribed as 50 ml, with100 ml sample volumes suggested.

b. With tests in whin assumedthat coliforms are present in somenumbers, and the test is to determinewhether some limiting standard (as1000 per 100 ml in natural bathingwaters, prescribed by-someagencies), another approach issuggested. Hdre, select the samplefiltration volume which would bequantitatively most acceptable tocount coliforms at the limiting value.For eXample, with a limiting valueof 1000 per 100 ml:

. organisms No, colonies counteer 100 ail =--100 X 56.iiif(sr sample i 1t '4' 4

1 his preNiously given equation can be rearrange":

s:mple filtration100!umcs in ml

Ind from this

Sample filtration volumein ml

-No, colonies courfte4

organismsNo. 100

10Q X50

1000'O

= 5(The value 50 is'the idrange numberof colonies for an,ac eptable colonycount of 20 - 804or omputing con-

: irms per 100;m3),

;`' 2''''Wquantitative work, to eterminetiti.ipber of conforms per 100 ml theworker may or inay not h ve prior

. information or standards o use asguidance imselecting filtr tion volumes.

a In absence' of prior bact riologicaldata v

. .. .

1) ,Unpolluted raw surface water, 1,4, 15, and 60 ml samples willcover a count range of 83 - 8000.per ,100,m1;

. .Selection of Sample Filtration Volumes

2) Polluted raw surface water, 0.020.08, 0. 15, and 0; 5 ml sampleswill cover a count range of 4000to 4,00, 000 per.100 ml.

3) Sewage and dilute sewage, withfiltration volumes of 0. 0003,0. 001, 0.413, and 0,01, willcover a count range of 200,000to 27, 000; 000 Der 100 nkl.

, b If prior conform data are available

Use the equation:

3asic filtration'olume in ml

50= roo x Average coliform

Example: Assume that prior dataindicate average coliform count of35, 000-per 100 ml. Using theequation:

,3asic filtration volume in ml = 100 X35,000

k,

=0.143 ml

50

B

Round off the filtratiOn Voluine to0.15 ml. .

To assure a reasonable count-rande,filter increments of 0.04 and+0. 60 mlin addition. This will provide for-acceptable conform cotints in therange of 3300 to 200;000 per100 ml

Fecal Conform Counts,4

1. Currently, no drinking water s andardsare based on4ecal conform or nisms. _-

Many states have environmental waterquality standards which are based Onfecal conform organisms.

2 Determination of fecal conforms. In theabsence of prior data.

a..13npollutedsraw surface water:, IFilter 1, 3, 10, al3d 30 mI sample-portions. These volumes will.cover a fecaPeoliform range of67 - 6000 per 100 ml.

0

Cr

Selection of Sample Filtration ValuMes,% A

t'b Polluted raw surface water: "sifter

portions of O. 1, 0.3, 1, Q, and 3.0nil.' This will cover a fecal coliformcount range of 670 to 60,000 lief 100nil.

c Sewage and dilute sewage: Filtersample. pcirttona:of 0.0003, 0.001and 0. 003 ml. This will provide for

. counts of 670, 000 to 20, 900, 000 per100 ml.

Determination oftfecal coliforms inpresence of prior data

a When previous fecal coliform countsare available:

40Filtration voluthe = 100 X .

Av, fecal coliformin ml count per 100 ml

Example: Prior data show 8000 fecalcoliforms per 100 ml.

Basic filtration volume in ml = 100 X 408000-1

9.5Filter volumes of 0. 15, 0.5 and 1: 5ml. This will, be suitable for fecalcoliform counts over the range 1300

-,to 40, 000.

b -When previous total coliform data .

are available,but no fecal coliformdata are available, use the totalcoliform value as above, but filter3x and 9x the computed basic volurfie.

Example (from above):basic value = 0.5 mlFilter voluiries of 0.5,ml.

Computed

1. 5 and 5.0

C Fecal Streptococbus Determinations

b Polluted surface water: Filtersample portions of 0. 1, 0.5, and2.0 ml. This will provide for fecalstreptococcus counts in the range1000 to 100,000 per 100 ml. Pro-vision for rather high counts offecal streptococci is made becauseof possible situations in which pol-lution of the water originates from

domestic or wild animals. In theevent that such pollution is highly'improbable, a filtration series of0.2, 1.0, and 5.0 ml (covering acount range of 400 to 50, 000,,per100 ml) viouldat)e more appropriate.

2 When pr pr data are available

a If coliform, bit not fecal strepto-coccus data are available, computea basic filtration volume as in A, 2above, but use the average coliformcount as 'la point of reference. Ifsignificant pollution from doniesticor wild animals is believed, ent,filter 0.2X, 1X and 5X the basicfiltration volumes. If the pollutionlevels are believed due primarily tohuman urces, use IX, 5X aild 25Xthe ba c filtration volumes. '.

b If pricyr streptococcus data are avail-able--; use the equation

60Basic filtration, = 100 )"volume its ml Av. Streptococcus

count per 100 ml

and filter 0.2X, 1X, and 5X the basicfiltration volume for streptococci.

1 In absence of prior data

a Unpolluted raw surface water: Filtersample portions of 1, 5, 25 and /00-4'ml. This will provide for fecalstreptococcus counts in tfie range 20.ft,tci,10,000 per,100 ml.

4

0,6113

ttEFERENCES

1 The Federal RegiAter, October 23, 1956,pp 8110-11; and March 1, 957. p 1271;

2 Clark, H. F., Kabler, P. W., andGeldreich, E.E. Advantages andLimitations of the Membrane Filter

"Procedure. Water and Sewage Works.Spritember 1957.

This outline Was prepared by H. L. Jeter,Chief, Program Suppbrt Training Branch,USEPA, Cincinnati, 'Ohio 45268

Descriptors: Bacterfa,-Mnple TestingProcedures, Pilfer, Membrane

I-

C

Iwo

DETAIlliD MEMBRANE FILTER METHODS

r BASIC PROCEDURES

A Introcluction

Successful application of membrane filter .-methods requires development of goodroutine operational practices. Thedetailed basic procedures described inthis Section are applicable to all mem--brane filter methods in water bacteriologyfor filtration, incubation, Vony-counting; -and reporting of results. In addition,equipment and supplies used in membranefilter procedures described here are notrepeated elsewhere in this text in such,detail.

Workers using membrane filter methodsfor the first time are urged to becomethOroughl,y familiar, with these basicprocedures and precautions.

B G,eneral Supplies and Equipment List

Table 1 is a check list of materials.

7" C "Sterilizing" Media

Set tubes of freshfyprepared m ium insboiling waterbath for 10 minutds.method suffices formedium in tubes up to25X 150 mmt Frequentagitation is neededwith media containing agar.

Altdfnately, orm media can bedirectly heated n a hotplate to the firstbubble oeboiling. Stir the medium .

frequently if direct heat is used, to avoidcharring the medium. -

Do not sterilize in the autoclave.

pla

n an agar medium isdent to make a layeris culture containers

D General Laboratory Procedures withMembrane Filters

'1 Prepare data sheet

Minimum data required are: sampleidentification, test performed includingmedia and methods, sample filtrationvolumes, and the bench numbersassigned to individual membrane filters.

2 Disinfect the laboratory bench surface.

Use a suitable disinfectant solution andallow the surface to dry beforeproceeding.

=

3 Set out. sterile culture containers in anorderly arrangement.

4 Label:the culture containers.'

r

Numbers correspond with the filter,nun ers shown on the data sheet.

5 Place one sterile absorbent Pad* ineach culture container, unless an agarmedium is, being used.

Use sterile forceps for all manipulationsof absorbent pads and membrane filters.Forceps- sterility is maintained bystoring the working tips in about 1 inchof methanol or ethanol.-----Beeause thealcohol deteriorates the filter, dissipateIt by burning before using the forceps.Avoid heating the forceps in the burneras hot metal chars the filter.

used, absorbent pads are not used. The amount of medium should-beapproximately 1/8". deep in the culture container. 1n-the 50 mmthis corresponds to approximately 6-8 ml of culture medium.

,vNOTE: Mention of commercial products and manufacturers does not imply Indorsement by the

Office Of Water Programs, Environmental Protection Agency.,)

W. BA. mem. 86.8.77

114

s.

4.

Detailed Membrane Filter Methods

,

Table 1. EQUIPMENT, SUPPLIES AND MEDIA

Totaj' ,Item

M - EndoBroth

L. E. S. DelayedColifor Coliform1

FecalConform

FecalStreptococeus

VerifiedTests

Funnel unit assembliesRing stand, with about a 3" split ring, tosupport the filtration funnelForceps, smooth tips, type forMF work

Methanol, in small wide-mouthed bottle's,about-20 ml for sterilizing forceps

Suction flasks, glass, 1 liter, mouth tofit No. 4 stopperRubber tubing, 2-3 feet, to connectsuction flask to vacuum services, latexrubber 3/16" I. D. by 3/32" wall ,Pinch clamps strong enough for tight'compression of rubber tubing above

Pipettes, 10 ml, graduated. Mohr type,sterile, diapenae 10 per can per workingspace per day. (Resterilize daily tomeet need).Pipettes, 1 Ill, graduated, Ofohr type,sterile, dispense 24 per can per workingspace per day. (Resterillze daily tomeet need).Pipette boxes, sterile, for 1 ml and10 ml pipittps (sterilize above pipettes .in these boxes).Winders, 100 ml graduated, sterile,(rine ilze daily to meet need).Jars, to receive used pipettesGas burtfer, Bunsen or similarlaboratory typeWax pencils, red, suitable for writingon glass

Sponge in dllute iodine, to disinfect thedesk tops

Membrane filter' (white,Irld marked,sterile,and suitable pore size formicrobiological analysis of water)

Absorbent pads for nutrient, (47 mm indiameter), sterile, in units of 10 padsper package. Not required if medium

Petriagar.

Petri dishee,' disposable, plastic,50 X12 mm, sterile

Waterbath 'incubator 44.5 + 0.2°C

Vegetable crispers, or cake boxes,)1a3nic, wittxtight,fitting covers, formembrane filter incubation,Fluorescent lamp, with extension cord.

...

..

ling stand, with Clamps, utility type

X0

X

X

X

X

X

X

.X

. IX

. X

.

X

X

*

X

X

.

X

X

X ,,,

X

X_

X

X

X

X

X

X

X

X

X

-X

X

.

X. .

X

X

X%

X

X

X-

X

X

1

X

X

4

X

X

X

X.

X

X1,

X

X

X

X

X

X

X

X

XX

.

/

.

.

it

X

X

X

X,

X

X

x,

X

'X

X

X

X

X

X

X

X

X

X,

X

X

X

*

.

,

.

,

.

/

.

.

.

X

.

X

,

.

----.

X.

X

X

X

X

X.

X

X.

X- ,..

X

.X

X

-

X

.

X

X

. X

X

X-

X

X

X

X

X

X

X

X

)

ti

f

Detailed Membrane Filter Methds

Table 1. EQUIPM SUPPLIES AND MEDIA (Cont'd)

t otal Coliforms

Item

Half-round glass paper weights for X X X Xcolony counting,with lower half of a2-ot metal ointment box.

Hand tally, single nit acceptable, X X X X Xhand or desk type

Stereoscopic (dissection) microscope. X X Xmagnification of 10X or 15X, prefer-able binocular wide field type

Bacteriological inoculating needle

Wire racks for culture tubes,10 openings by five openings pgre-ferred, dimensions overall approxi.mately 6" X t2"

Phenol Red Lactose Broth in 16 X150 mm feimentation tubes withmetal caps, 10..ml per tube

Eosin Methylene Blue Agar(Levine) in petri plates, preparedready for use

Nutrient agar slants, in screwcapped tubes, 16 X126 mm

Gram stitin solutions, 4 solutionsper complete set

Microscope, compound, binocular,with oil immersion lens, micro-scope lamp and immersion oil

Microscope slides, new, clean,1" X3" size

*lair proof. plastic bags I X .forfecal cOliform culturedish incubation

4. M-Endo Medium, MF dehydrated X Xmedium in 25 X95 mm fiat bottomedscrew-capped glass vials, 1.44 gper tube, sufficient for 30 ml ofmedium

scre-capped tubes, aboutEthanol, 95% in small bottle X. Xscrew- cappedper tube -

Sodium benzoate solution, 12%aqueous, 'in 25 X150 mm screw-capped tbes, about 10 ml per tubeL.E. S. Endo Apr MF, dehydratedM-Endo medium, 0.38 g pair 25 X95 nun flat bottomed screw-cappedglass vial, plus 0.45 g agar, for 30 mlLactose Lauryl Sulfate Trjittose Broth I Xin 25 X 150.mm test tube withoutincluded gas tubs, about 25 ml, forenrichment in L. E. S. method

M -Endo L. E. S.Broth ColUorm

DelayetrColiforni,

_FecalColiarm

FecalStreptococcus

VerifiedTesta

-r*

, ..116,

X

X

X

1X

X

X

O Detailed Membrane Filter Methods

Table 1: EQUIPMENT, SUPPLIES AND MEDIA (Cony)

Total Conforms

Item

M-FC Broth for fecal conform,dehydrated medium in 25 X95 mmflat bottomed screw-capped glassvials, 'I. 11 g per tube, sufficientfor 30 ml of culture medium

Roso lic acid, 1% solution, in11110.211Na0H, in 25 X 150 mm flat

bottOmed 'screw- capped tubes,about 5 ml per tube, freshlyprepared

RI, Agar. dehydrated medium in 25 X 150 mmscrew-capped tubes, sufficient for 10 nil. 2. isper tube

-.Dilution bottles,. 6-oz, preferableboro-silicate glass, with screw-cap (or rubber stopper protected ..

by paper) , each containing 99 m1,of 'sterilp phosphati buffereddistilled waterElectric hot plate surfaceBeakers, 900 - 600 ml (for water-bath in preparation of membrane .,filter culture media)Crucible tongs, to be used at ,electric hot plates. for `removal

'of hot tubes of culture media forboiling waterbath

11 -4

a

M-EndoBroth

L. E. S.Collform

DelayedColiform

FecalColiform

FeizalStriptococcus

VerifiedTeat

X

4X

X

Xt.

X

....19 .

"....;

X

X

X

X

X

X.

X

X

.

X

X

X

X

X j

04.

xX

X

,,,(1

.5

I.

s

.

. t

to


,7 1. .,..

6 Deliver enough culture medium tosaturate-each absorbent pad,--usinga sterile pipette.

. -

Exact quantities cannot be statedbecause pads and culture containers vary. ,Sufficient medium should be applied sothat when the culture container is tipped,a good- sizeddrop of culture medium'freely drains ai t of the absorbent pad.

I7 Organize supplies and epipment for

convenient sample filtration. In .

training courses, laboratory instructorswill suggest udeful arrangements;eventually the individual will select asystem of bench-top organization mostsuited to his own needs The importantp'oint in any arrangement is, to have all -needed equipment and supplies con-veniently at hand, in such a pattern asto minimize lost time in useless Motions,

- .

8 Lay a sterile membrane filter on thefilter holder, grid-side up; centeredoverthe porous part of the filter

- . support plate.

Membrane filters are eitretnelydelicate and easily- damaged: Formanipulation, the sterile forcepsshould always graspthe outer partof the filter disk, outside the partof the filter through which the samplepasses.

Attach the funnel element to the baseof the filtration unit.

'9

To avoid dame e ta-the membranefilter, locking farces should 2:11y beapplied at the locking arrangement.The funnel element never should beturned or twisted while being seatedand locked to the lower element of thefilter holding unit. Filter holding unitsfeaturing a bayonet joint and lockin,gring to join the upper' eleinentto thellower element require special care onthe part of the operator. The lockingring should be turned sufficiently to itgive a Emig fit, but should not betightened excessively.

0

Shake the sample thoroughly.:

Measure sample into the funnel withvacuum turned off. .

The priniary objectives here are:1) accurate nyeasuIement of sample;and 2) optirkuM distribution of colonieson the filter after incubation. Tomeet thRss objectives, methods ofmeasurement and dispensation to thefiltration assembly are veiled withdifferent Sample filtration volumes.

a With samples grater than 20 ml,measure the sample with a sterilegraduated cylinder and pour it intothe. ftinnel. It is imp?rtant to rinse

- this graduate with sterile buffered;distilledwater-to preclude the lossof %xcessive sample volume. Thisshould be poured into.the funnel.- ,

1

b With eimplei of 10 m-1 to 20 ml,measure the sample with a sterile C1.04n1,-or 20 ml, pipette, and pipetteon b. dry membrane in the filtration'assembly. -

.

c With samples of -2 to 10 ml, pourabout 20m1 of sterile dilutiq,n waterinto the filtration assembly, thenmeasure the'Sazple into the sterile,buffered dilutioiMater with a.10 mlsterile/pipette.

,c1 With samples of 0.5 to 2 xn1, pourabout ZO ml of sterile dilution Faterinto the funnel assembly, thenmeasure the sample into the steriledilution water in the funnel with a '1 ml or a 2 ml pipette.

e If a sample of lees than-0.-54nr1 is tobe filtered, prepare appropriatedilutions in sterile dilution water,and proceed .s Applicable in item cor d above.

When dilutions of samples are needed;"alwhys make-the Tiltrations aloonas possible after dilution of thsample; this never 'should exceed

NOTE: Mention of commercial products and mantgactigers does not imply. endorsenient, by they Office of Water PrograMs, Environniehtal Protection Agency. -

- qv

tk, 1.1-5 --

't


30 minutes. Always shake sampledilutions thoroughly before deliveringmeasured volumes.

--.:.12 Turn On the vacuum.

_Open the appropriate Spring clamp orvalve, and filter the-eample, .

After sample filtration a fevi drbpletsof sample usually remain adhered tothe funnel walls. Unless these dropletsare removed, the bacteria contained inthem will be a source of contaminationof Inter 'samples. (In laboratorypractice the funnel unit is not routinelysterilized between successive filtretionsof a series). The purpose of the funnelrinse is to flush all droplets of a.samplefrom the funnel WallsIto the membranefilter. Extensive tests have shown thatwith proper rinsing technique, bacterialretention on the funnel walls is negligible.

.4o by'

13 Rinse the sample through the filter.

After ell the sample has passecrthroughthe mernbrane filter, rinse down thesides' of the funnel walls with at least20 ml of sterile dilution water. Repeatthe rinse twice after all the first rinsehas passed through the filter. Cut offsuction on the filtration assembly. '

.

14 Remove the funnel element of the filterholding unitt

lfa ring stand with split ring is used,hang the funnel element on the ring;otherwise, place the inverted funnelelemNni on the inner surface of thewrapping material. This; requirescare lit opening the sterilized package,but it is effective as a protection of thefunnel ring from contamination.

15 Take-the membrane filter from thefilter holder and carefully. place itgrid -side up on the medium. .

Check that no air bubbles, have beentrapp betweewthe membrane filterand-the-underlying absorbent pad oragar. Relay the membrane if necessary.

11-6-,

4

5%*

16 Place in incubator after finishingfiltration series.

Invert the containers, s'kThe immediateatmosphere of the incubating membranefilter must be' at or very near 100%relative humidity.

17 Count colonies which have appearedafter incubating for the prescribedtime.

A stereoscopic microscope magnifying10-15 times and careful illiminationgive best counts.

,For reporting results, the computationis

-

bacteria/ 100 ml =

No. colonies counted X100Sample volume filtered in ml

Example:

A total of 36 colonies grewafterfiltering a mI sample. Thenumber reported is:

36 colonies X 100 = 360 per 100 mlmlyo

Report results to two significant figures.

Example:

A total of 40 colonies grew a fter4 filtering.a 3 ml sample.

This calculation gives:

40 colOnfiis X 100 2 1333.33 per 100ml3 ml.

But the number reported should be.. 1'300 per 100 ml. a

5'

4 4


4 \

*II MF LABORkTeltY TESTS FOR

COLIFORM GROUP

A Standard Coliform'Test (Based on M-EndoBroth MF)

'1 Culture medium11"

? a M-Endo Broth MF Difco 0749-02or the equivalent BBL M- ColiformBroth 01-494

P;eParation of Culture Medium'(M--Endo Broth) for Standard MFColiform Test-

0 Yeast extract 1;5". gCasitOne or equivalent 5.0 gThiopeptone or equivalent 5.0 gTryptose 10.0 g .

Lactose 12.5 gSodium desoxycliolat 0.1 gDipotassium phospbat 4.375 gMonopotassium phosp to 11.375 gSodium chloride 5.0 gSodium lauryl sulfate 0.05 gBasic fuchsin (bacteriological) 1.05 g. -

.0Sodium sulfite - ' 2.1 g. .Distilled water (containing 1000 ml20.0 ml ethanol)

This.mediuin is available indehydrated form and it is xec-Ommended that the commerciallyavailable medium be used inpreference to comPoun 0 themedium of its iridividue constituents.

To prepare the medium for use;suspend the dehydrated medium atthe rate of 48 grams per liter of

water containing ethyl alcohol atthe rate of 20 ml per lite,r.

sil

As a time - saving convenience, it isrecommended that the laboratoryworker pre-weigh the dehydratedmedium in closed tithes' for several

:.daye, or even weeks, at one operation,

r'

With this system, a large numberof increments of dehydrated maim(e.g 1.44 grams), sufficient forsome convenient (e. g. ; 30 ml)volume of finishes' culture mediumare weighed and dispensed intoscrew - tapped culture tubes, andstored until needed. Storage shouldpreferably be in a darkened disiccator.

A supply of distilled water containing28 ml stock ethanol per liter can bemaintained,

When the medium is to be used,' itsis reconstituted by adding 30 ml ofthe distilled water-ethanol mixtureper tube of pre-weighed dehydratedcultured medium.

b Medium is "sterilized" as direbtedin I, C.

c Finished medium'can be-retainedup to 96 hours if kept in a cool,dark place, Many workers preferto reconstitute fresh medium daily.

2 nitration and incubationproceduresare as given in I, D.

Special instructions:

a For counting, use the wide field -

binocular, dissecting microscope, orsimple lens. For illumination, usea light source perpendicular to the

c plane of the membrane filter. Asmall fluorescent lamp inteal forthe purpose.

b Coliform colonies have a "metallic"surface sheen under reflected lightwhich may cover the entire colony, orit may appear only-in the center. Non-colifo-rm colonies range fromcolorless to pink, but do not havethe characteristic sheen.

c Record the colony counts on thedata sheet, and 'compute the conformcount per 100 ml of sample.

12o' 11-7

r

4

a,

6

I.


13 Standard Conform Tests (Based on L..E.S;Endo Agar)

The distinction of the 4. E:S. count is atWo -hour enrichment incubation on LSTbroth. M-Endo L.E. S. medium is usedas agar rather than the broth.

1. Preparation of culture medium! . ( L. E. S. Endo Agar) for L. E. S.

,coliform-test

a. Formula from McCarthy, Delaney,and Grasso (2)

/ItieBacto-YZ,ast Extract 1.; gBacto- Casitone 3.7 gBacto-Thiopeptone 3.7 gBacto-Tryptose 7.5 .gBacto- Lactose 9.4 gDipotassium phosphate 3.3 gMono.potassium phosphate 1.0 .gSodium chlorideSodium desoxycholateSodium lauryl sulfateSodittm sulfiteBacto-Basic luthsinAgar ,

Distilled water (containing20 ml ethyl alcohol)

2

3.7 g0.1 ga. 05 a1.6 g0.8 g

15 g

1000 ml

To rehydraie the medium; suspend51 grams in the Water-ethyl alcoholsolutipn. ,

-,,

c -Medium is usterili;ed" as directedin 1, C.

d Pbur 4-6 ml of freshly prepared Agarinto the smaller half of the container.-Allow the medium to cool and solidify.

.

Procedures for filtration and incubation

a a- i out the culture dishes in a rowor series of rows as usual. Placethese with.the upper (lid) or top

. side down.

b Place one sterile absorbsnt pad inthe larger half of each container(lid). Use sterile forceps for all

11-8

4

c

. manipulations of the pads.(Agar occupies smaller half orbottom).

Using a sterile pipette, deliverenough pingle strength laurylsulfate tryptose,broth to saturatethe pad only. Avoid excess medium.

d Follow general procedures forfiltering in 1, D. Place filters onpad with lauryl sulfate .tryptosebroth.

4e Upon 'completion of the filtrations,'invert ttie culture cmqainers acidincub a at 350C for 1 1/2 to 2hours

3 2 -hour pro dures

'a Transfer the membrane filter fromthe. enrichment pa in the upper halfto the agar medium in the lowerhalf af the container.' Carefullyroll the 'membrane onto the agarsurface to avoid trapping airbubbles beneath the membrane..

b Removal of the aletlibfiorbent padis optional.

c The container is inverted andincubated 22 hours + 2 hours + 0.5oC.

4 Counting procedures are as in 1, D.

5 L. E:S. Endo*Agar may be used as asingle-stage medium (no enrichmentstep) in the same manner as M-EndoBroth, MF.

C.- Delayed Incalbatipri Ciliform Test

This technique-le-applicable in situationswhere there is an excessive delay betweensample collection and plating. The procidure,is unnecessary when the interval be-tvieen sample collection and plating iswithin acceptable limits.

Alts

1 Preparation of culture media for.,,delayed incubation conform test

12:1

a Preservative media M-Endo Brothbase

11*

O

1

1

I.

I

I

I.

To 30 ml of M-Endo Broth MFprepared in accordance withdirectio = in II = 1 of thisoutline, a. . 0Fml o sterile12",in'aquedts solution of sodium

_ benzoate,

L. E.S. MF lioldirig Medium-Conform: Dissolve 1'2.7 grams in1 liter of distilled water. No

heating is necessary. Virial.pH7.1 1-0.1. This medium containssodium benzoate. .

b Growth media.

M -Endp Broth MF is used, preparedas described in II, A, 1 earlier inthis outline. Alternately, L. E. S.Endo Medium may be used.

2 General filtration followed is in I, D.

Special procedures are:

a Transfer the membrane filter fromthe filtration apparatus to a padsaturated with benzoated EncloBroth.- r ,

b Close the culture diflies and hold,in a container at ambient temperature.This may-be mailed or transported Ato a centr 1 laboratory. The mailingitor transporting tube should containaccurate transmittal data sheets whichcorrespond to properly labeled dishes.

Transportation time, in t case ofmailed containers, shoo} not exceedthree days to the time of/receptionby the testing laboratory,

c On receipt in the central laboratory,unpack mailing carton, and lay. outthe culture containers on the Tabora -tory bench. ,

d liemovelhe fops from the culturecontainers, Using sterile forceps,remove each membrane and itsabsorbent pad to the other ofthe,culture container.

.

6

Detailed Membrane "Filter Methods

e With 4 sterile pipette or sterileabsor entpad, remove preservativemediurrri from the culture container.

f

g

Place a sterile absorbent pad ineach culture cOntainer, and deliverenough, freshly prepared M-EndoBroth to saturate each pad.

Using sterile forceps, transfer themembrane to the ne* absorbent padcontaining M-Endo Broth. Placethe membrane car eftilly to avoidentrapment of .air, between themembrane 'and the underlyingabsorbent pad. Discard theabsorbent pad containing pre-servative medium.

h After incubation of 20'+ 2" hoursat 350 C count colonies as in theabove section A, 2.

'`i If L. E. S. Endo Agar is used, the

steps beginning with (4) above areomitted; and the membrane filter isremoved frpm the preservativsmedium and transferred to a freshculture container with L. E. S. EndoAgar, incubated, and coloniescounted in the usual way.

D Verified Membrane Filter Coliform /Test -.19,

I

This procedure applies to identificationof colonies growing on Endo -type mediaused for determination.of total conformcounts. isolates from these coloniti arestudied for gas production from lactose -

and typical conform morphology. Ineffect, the procedure corresponds with 'itthe Completed Test stage 'of -the multiplefermentation tube test for Zoliformsi

Procedure:

1 Select a meinbrane filter bearingseveral well-isolated conform-typecolonies. rc - _ .

2 Using sterile technique,, pickiallcoloniesin,a selected area ,with the .inoculation needle, 'matting transfersinto tubes of phenol red lactase-Froth.(or lauryl sulfate tryptose lactose

10;

11-9

I


brOth). Using an appropriate datasheet record the interpretation ofeach colony,- using, for instance,"C" for colonies having the 'typicalcolor. and sheen of coliforms; "NC"for colonies not oonforming tocoliform colony appearance onEndotype media.

3 Incubate tie broth tubes at 350 C4. 0.5°C

4 At 24 hours;

a Read and record the results fromthe lactose broth fermentation tubes.The following code is suggested:

Code0 No indication of acid or gas

prpduction, either with or I

_without evidence of growth.

A Evidence of acid but not gas(applies only when a pH indicatth-is included in the broth medium)

G Growth with production of gas.If pH indicator is used, usesymbol AG to show evidence ofacid. Gas in any quantity is apositive test.

I

b Tubes not showing as production areretUrned to the 350 C incubator.

c Gas-positive tubes are transferredas follows:

1) Prepares streak inoculation onEMB, agar for colony isolation, andUsing The same culture.

'2) Inoculate a nutrient agar slant.

3) Incubate the EMB agar plates andslants at'350C + 0.5 °C.

5 At 48 hours:

a Read and record results of lactosebroth tubes 'whichNvere negative at24 hours and were returned forfurther incubation.

b Gas-positive cultures are subjectedto further transfers as in 4c.Gas-n tivtr cultures are discardedwithout rther study; they areconform; tive.

,c Examine the cultures transferredto EMB agar plates and to nutrientagar slants, as follows:

1) Examine the EMB agar plate forevidence of purity of culture; ifthe culture 'represents more thanone colony type, disbard thenutrient agar culture and reisolateeach of the representative.colonialtypes on the EMB plate and resumeas with 4c for each isolation.If purity of culture appears evident,continue with c (2) below.

2) Prepare a smear and Grain stainfrom each nutrient agar slantculture. The Gram stain shouldbe madeon a culture not morethan 24 hours old. Examine underoil immersion for typical coliformmorphology, and record results.

6 At 72 hours:

Perform procedures described in 5cabove, and record results.

7 Coliform colonies are considered'verified if the procedures demonstratea pure culture of bacteria which aregram negative nonspore-forming rodsand produce galrfrom lactose at 350'C.within 48 hours.

E Fecal Coliform Count (Based on M-FC401taBroth Base)

The count depends upon growth on aspecial medium at 44.5 + 0.2°C.

1 Preparation of Culture Medium(M-FC Broth Base)°for FecalColiforrh Count

123

m.

1

<.-


a Corriposition

Tryptose 10. 0 gProteose Peptone No. 3 , 5.0 gYeast extract 3.0 gSodium chloride 5.0 gLactose 12.5 gBile salts No. 3 1.5 gRosolic acid* (Allied 10.0 ml

Chemical)Aniline blue (Allied Chemical) 0.1 g

Distilled water 1000 ml

b To prepare the medium dissolve37.1 grams,in dater of distilledwater which contains 10 ml of 1%rosolic acid (prepared in 0.2 NNaOH).

Fresh solutions, of rosolic acid givebest results. Discard solutionswhich have chknged from dark redt6 orange.

o To sterilize, heat to boiling asdirected in I, C.

d Prepared medium may be retainedup to 4 days in the dark at 2-80C.

Z.*2 Special supplies

Small Waterproof plastic sacks capableof being sealedagainst water withcapacity of 3 to 6 culturemOntliiners.

.3 Filtration procedures are as given inI, D.

4 Elevated temperature incubation

a Place fecal coliform count mem-branes at44.5 + 1).2°C as rapidlyas possibk..

Filter membranei for fecal coliformcounts consecutively and immediatelyplace them in their culture containers.Insert as mttny as six culture containersall oriented in the same way (i. e:", allgrid sides facing the same direction)into the sacks and seal. Tes. i off theperforated top, .grasp the side wires,and twirl the sack to roll the open endinside the folds of sack. Then submergethe sacks with culture containers in-verteg beneath the Ekurface of a 44.5+ 0. 2 C waterbath,

b Incubate for 22 + 2 hours.

5 Counting procedures

Examine and count colonies as follows:

a Ilse% wide field binocular dissectingmicroscope with 5 - 10X magnification.

b Low angle lighting from the side isadvantageous.

c Fecal conform colonies are blue,461.erally 1-3 mm in diatheter.

a.-

d Record the colony counts on thedata sheet, and report the fecalconform count per 100 ml of simple.(I, D, 17 illustrates method)

III, TESTS FOR FECAL STREPTOCOCCALGROUP-MEMBRANE FILTER METHOD'

A 48 hour incubation period on a choice.of,two different media, 'giving high selectivityfor fecal streptococci, are the-distinctive .

.features of the tests.

41

*Prepare 1%-solution of rosolic acid in 0.2 N N H. This dye is practically insoluble in water.

124

Mailed Membrane Filter Methods

A Test fOr Members of Fecal StreptococcalGroup based on KF-Agar

1 Preparation of the culture medium

a Formula: (The dehydrated formulaof Bacto 0496 is shown, butequivalent constituents from othersources are acceptable). Formulais in grams per liter of reconstitutedmedium. 's

Bacto proteose. peptone #3 10.0 gBacto yeast extract 10.0 gSodium chloride (reagent grade) 5, 0 ,g

Sodium glycerphosphat$ 10.0 gMaltose (CP) 20.0 gLactose (CP) 1.0 gSodium azide (Eastman) 0.4 gSodium carbonate . 0.636 g

(Na 2.CO

3reagent grade)`

Brom cresol purple(wate soluble)

0.015 g 2

Bacto agar 20.0 g3

b Reagent

'2, 3, 5-Triphenyl tetratoliumchloride reagent (T.PTC)

This reagent is prepared by makinga 1% aqueous solution of the abovechemical passing it through a Seitzfilter or, membrane filter. It canbe kept in the refrigerator in ascrew-capped tube until used.

c The dehydrated medium described'above is prepared for laboratoryuse as follows:

Suspend 7.64 grams of the dehydratedmedium in 100 ml of distilled waterin a flask with an alirminuth foilcover.

Place the flask in a boiling water-bath, melt the dehydrated medium,and leave in the 14oiling`waterbathan athlienal 5 Minutes.

Cool the medium to 500-600C, add1.0 ml,of the TPTC reagent, andmix.

For membrane filter studies, pour5-8 ml in each 50 mm glase orPlastic culture dish or enough tomake a layer approxitnately 1/8"thick. Be sure to pOtir plates befoReagar cools and solidifies.

For plate counts, pour as for standard'agar plate counts.

NOTE: Plastic dishes containingmedia may be stored in a dark, coolplace up to 30 days without changein productivity of the medium, pro-yitled that no dehydration occurs.Plastic dishes may be incubated inan ordinary air incubator. Glassdishes must be incubated in anatmosphere with saturated humidity.

Apparatus, and materials ad given inTable 1,

General procedure is as given in I.

Special instructions

a Incubate 48hours, inverted with100% relative humidity afterfiltration. s

b After incubation',, remove thecultures from the incubator, andcount colonies under'wide fieldbinocular dissecting microscope,with magnification Set at 10X or20X. Fecal streptococcus colOniesare, pale pink to dark wine-color.Insize they range from barelyvisible to approximately 2mm indiameter. Colorless colonies arenot counted.

Report fecal streptococcus count'per 100 ml of sample. This iscomputed as follows:

No.- fecal streptococci per 100 ml

No. kcal streptococcus colonies" 100Sample filtration volume in ml

B Verification of Streptococcus Colonies

1 Verification of colony Identificationmay be required in waters containinglarge numbers of Micrococcus orga-nisms. This has been notedparticularly with bathing waters, butthe problem is by no means limited tosuch waters.


3 Sterilize the funnel unit assembly byexposure to formaldehyde or byimmersion in boiling water. If alaboratory autoclave is available, thisis preferred. o

Formaldehyde is produced by soakingan asbestos ring (in the funnel base)with methanol, igniting, and after afew seconds of burning, closing theunit by placing the stainless steelflask over the funnel and base. Thisresults in incomplete combustion O.the methanol, whereby formaldehydeis produced. Leave the unit closedfor 15 minutes .to allow adequateexposure ,to, formaldehyde.

4 Filtration hnd incubation procedurescorrespond with laboratory methods.

5 The unit is supplied with a bookletcontaining detailed step-by-step

e oyerationai procedures. The worker, using the equipment should become

completely versed in its contents andapplication.

C Other commercially available field kitsshould be used according tb manu-facturer!s instructions. It is emphasizedthat the reqUired'standards of performanceare manditory for field devices as forlaboratory equipment.

2 A verification procedure is describedin "Standar&Methoda for the Examinatiodof Water and Wastewater:' 14th ed.(1975). The Worker Should usethis reference for the step'by-step procesiUre.

IV PROCEDURES FOR USE OF'MEMBRANEFILTER FIELD UNITS

A Culture Media'. d5 a .

fl

1 The standard O'liform media used withlaboratory tests are used.

2 To simplify field operations, it issuggested that the medium be sent tothe *ad, preweighed, in vials orcapped culture tubes. The mediumthen requires only the addition of a/suitable volume ofethanol prior to sterilt*tion.

3 Sterilization procedures the fieldare the same as for labor tory methods.-

:4 Laboratory preparation of the media,

ready for use, would be permissible-provided that the`required limitationsOn time and conditions of storage are

%met.

B Operation of Millipore Water Testing Kit,Bacteriological

1 $uppOrting supplies andsquipmeht arethe stage as for the laboratoryprocedures.

2 Set the incubator voltage selectorswitch to the voltage of the 'availableSupply, turn on the unit and adjust:asnecessary to establish operatingincubator temperature at 35 0,50C.

D Counting of.Colonies on Membrane Filters

'1 Equipment and materials

Membrane filter cultures to beexamined

Illumination source

Simple lens, 2X to 6X magnification

Hand tally (optional)

126

2 Protedure

a ReMove the cultures from theincubator and arrange them innumerical sequence. r


b Set up illumination source is thatlight will originate from an areaperpendicular-to the plane ofmembrane filters being examined.A small fluorescent lamp is idealfor the purpose. It is highlydesirable that a simple lens beattached to the light source.

e Examine results. Count all coliformand noncoliform colonies. Coliformand noncoliform colonies. Coliformcolonies have a "metallic" surfaceshun under reflected light, whichmay cover the. entire colony or mayappear only on the center.Noncoliform colonies range fromcolorless to pink or red, but do nothave the characteristic "metaTi"sheen.

d Enter the colony counts in the datasheets:-

e Enter the coliform count per 100 mlof sample for each membrane havinga countable number of coliformcolonies. Computation is as follows:

No. coliform per 100 ml

No. coliEbrm colonies on MFNo. milliliters sample filtered X 100

REFERENCES

1 Standard Methods for the Examination ofWater and Wastewater. APRA, AWWA.WPCF 14th Edition. 1975.

o

jx

2 McCarthy, J.A., 'Blaney, J.E. andGrasso, R. J. Measuring Coliformsin Water. Water and Sewage Works.1961: R-426-31. 1961.

This outline was prepared by H. L.Jeter, Chief,' Program SupportTraining Branch, USEPA, Cincinnati,Ohio 45268

Descriptors: Biological Membranes,Coliforms, Fecal Coliforms, FecalStreptococci, Filters, Indicator Bacteria, ."Laborato.ry Equipment, Laboratory Tests,Membranes, Microbiology, Water Analysis

1 ,2

.

ti

COLONY COUNTING ON MEMBRANE FILTERS .

I INTRODUCTION

On removal of membrane filter cultures fromthe incubator, the worker has several tasksto perform, leading to the reporting of resultsof the bacteriological examination. Thesesteps, together with the selection and use ofassociated equipment, are considered in thisdiscussion. The following topics are included:

A Precautions on removal of membranefilter cultures 'from the incubator.

B Selection of the best membrane filter forcolony counting (when more than-onemembrane filter per sample was prepared,representing-a graded series of sampleincrenients.)

C Use of grid systems on filter surfaces ascounting aids.

D Recognition and counting of desiredcolonies, including selection and use ofoptical equipment.

E Calculations for reporting number of testorganisms per 100 nil of sample.

II REMOVAL OF CULTURE FROM:INCUBATOR

A Incubation time and temperature recom-mendations should-be closely adhered to.This applies particularly to total conform IIIcounts. Some of our earbier trainingmanuals havesuggested counting of coloniesafter as few as 16, hours of incithdtion at A35°C. Currently, 22 + 2 hour's is preferred.

moisture filids its way into the culturecontainer during, incubation. In suchcases, wh'en the culture is removedfrom the incubator, it should be turned"right side up" in such a way as to avoidflooding-the filter with excess liquid. Ifexcessive liquid is present, open theculture container cautiously, and pouroff the excess.

C Drying Filters Before Colony Counts

B All membrane filter cultures should beincubated in the inverted position, withpleasures to avoid loss of culture mediumthrough leakage or evaporation. Some-times. an excessive amount of culture.medium -is applied initially, or additional

1 Some workers advise opening allcultures (especially total conform testswhen Endo-type media are used) fora short time (15 minutes to 1 hour)for partial drying of coliform coloniesbefore counting. Advocates of thisstep report that the typical surfacesheen characteristic of coliformcolonies is improved by this step.

2 Useof preliminary drying'proceduresis a matter of personal preference.In the opinion of the writer, the behefitof preliminary drying is at best debat-able, and at worst, may interfere withsubsequent study of the bacterialcolonies. Correct use of acceptablelighting and optical equipment is afar more important factor in ease andaccuracy of recognition of differentiatedcolonies.

SELECTION OF ACCEPTABLE MEMBRANEFILTER CULTURE FOR EXAMINATION

NNon-Quantitative Tests

In bacteriologic examination of treated'wars, where waters meeting require-ments result in development of very fewor no conform colonies, the typical filtra-tion volume is 100 ml, and but one filtra-tion is made per sample. In this case,there is no problem: the one membrane

NOTE: Mention of, commercial products and manufacturers does not imply endorsement by thealine of Water Programs, Environmental Protection Agency". .

W. BA, ment 85a. 8..,71, '12:4

Colony Counting on Membrane Filters.

filter preparation is the basis of bacteri-ologic evaluation of the sample.

B Quantitative Tests

1 When the bacteriological water qualitystandard is for 'some fixed limitingvalue, such as 70 per 100 ml for shell-fish waters, again only a single samplefiltration.volume may be used. In sucha case, the filtration of a single portionof,50 ml will show directly whether thewater meets bacteriologic standards,or if the limiting standard is being

. .exceeded, . ,

On the other hand, if the objective of thetest was to show how many opliformswere pre.,ent per 100 ml of sample,then' it is necessary to filter a series ofsample indrements from each sample,each increment bein; placed on a .separatemenrane filter. At the end of theineu::ation period, the series of mem-brane filters repregenting each samplemucit- be inspected, vtith selection of themembrane filtei' bearing the number ofcolonies most suitai,le fo'r reportingquantitative results. This is summarizedin Table 1, below:

12 -2 .

The lower limit of 20 is set arbitrarily,as a number, below 'which statisticallyvalid results become increasinglyquestionable with smaller numbers ofcolonies. The upper limits representnumbers above which interference-fromcolony crowding, deposition of extrane:ous material, and other factOrs'appearto result in increasingly questiombleresults. It is emphasized .that theselimiting values are empirical, basedon laboratory. observations alone, Moddo not represent results of theoreticalcalculations. It follows that it is 'quitepossible, with some, sample sources,to obtain acceptable quantitative resultswith colony counts higher than the re-,commendations, but t minimum limitof 20 colonies appe s to apply to the,majority of sample ources. "

3 If no membrane filter bears 'a numberof colonies within the recommendedlimits for the test, the worker has achoice between - a) collecting a newsample and repeating the test; andb) using whatever results actually wereobtained, reporting an "educated guess"as to the number of organisms per100 ml. In the latter case, it is most

1

Table 1. NUMBERS OF COLONIES ACCEPTABLE-) FOR QUANTITATIVE DETERMINATIONS

Test Colony Counting RangeMinimum Maximum

Total coliform 20'

' coliform - 20

Fecal streptococcus 20

-Remarks

80 .200 limit overall

100

12D

11

. .,strongly irged tt t each result of thistype.be specifically identified with aqualifying statement, such as "Estimatedcount, based on non-ideal colony densityon filter. "

4 Sometimes two,or more filters, of aseries Of filtration volumes-from asample, produce colony counts withinthe recommended counting range.'Colony counts should be made on allsuch filters. See Section VI of thisoutline for calculations based on suchresults. These problems may arisefroin the selection of a top-close rangeof sample filtration volumes, fromcolony differentiation failuies relatedto overcrowding on the filters, or fromphysico-chemical interference withcolony deNelopment related to materialin the sample deposited in or on thefilters.'

IV USE OF GRID SYSTEMS IN COLONYCOUNTS

A Most manufacturers provide grid-imprintedmembrane filters for bacteriologic use.The ink used in such filters must be bio-chemi,callyiiiert to the test organisms,and, of course, must be applied in such amanner as not to degrade the quality of thefilter. Examples of suet gridding haveappeared from various manufacturers asfollows:

1 effective filtering area subdividedinto squares equal to 1/100 the effectivefiltering area (when a filtering unitwith funnel-dianieter of 35 mm is used).

2 ... grid markings which subdividethe effective fiAering area intoquaresequal to 1/100 the effective filteringarea (9.6 cm2for 47 mm diameterfilters).

3 ... filters subdivided so that eachsquare orthe grid represents 1/6Q ofthe effective filtering area.

4 - -

. Colony touiltiri4 Qn,Mernbrane Filters

B Some special studies may require use ofmembrane 'filters without grid markings.For example, the ink in some filters pre-.vents`growth of Brucella melitensis. Insuclvcases it maybe necessitry to impro- .vise a viewing grid which can beslacedover the culture after incubation and

Colonydsvelopment.

C Applications of Grids

44-

4

1 The grid dimensions are of no particularsignificance in colony counting, providedthat their size.permits easy and con-tinuous orientation in counting of colonie's...To'besure, a rough estimate of the totalinumber,of differentiated colonies on afilter is possible by counting a repre-sentative number of squares and multi-plying _colony count by the arfpropriatefactor.' For example, with man!filters, colonies in ten squares can becounted, multiplied by 10, and the pro-duct is a rough estimate of the totalnumber, on the entire filter.. It is em-phasized that such procedure is forrough estimates only, and should not, becondoned in quantitative work withmembrane filters.

2 The primary usefulne s of the gridsystem is for orients kin during thecounting procedure. Some colonieswill touch linestimta grid system, anda uniform practice must be establishedto avoid missing some colonies orcounting others twice. The procedureused by the writer is as follows:

a Counts are made in an back-and-forth sweep, front top bottomof the filter. See Figure 1.

b Inevitably, somf colonies viill be incontact with grid lines. A suggsstedroutine procedure for couttingooloniesin contact with lines is 'indicated inFigure 2... Colonies are counted in.the squares indicated by'the arrows,and no effort is made td decidewhether "most of the colony" is inone or the other sqUare.°

t, 13012-3

It

rx

O ;

, f"- COIony Counting or Membrane Filters.

-

lifillE112121511

..., ....AdEZZL1.4.

AMIN MEM,

MICIIIIIIIIIEl 'a EMI11M-2211111IWITMOIFir

Figure 1. The dashed circle indicated thee ective filtering area" The dashed back-and-forth line indicates the colony connting ,

pathway. 6 v o

V COUNTING OF COLONIES

A Equipment

1 A hand tally is a useful cleric% whilecounts are being made.

4

2 Optical assistance in colony counts isstrongly recommended. Dependence

. on naked-eye counts often results intoo-low results. a

. a Preferably, use a wide-field binocu-.lar dissecting microlcope withmagnification of 10)1r 15X.

b Optionally, but.less desirably, .asimple lens with magnification atleast 5X can be ased,- provided thatacceptable illumination also is °

present.

3 Lighting equipment.a For cOliform counting, a large light -*

source is mandator' Fluorescentlamps in housingpermitting 'place-ment* close to and as directly aspossible over the membrane filter isthe best lighting arrangement knownM. the writer. Incandescent lamps,.whether simple light bulbs in a table

t

O

. 4c

grid.

Figure 2. Enlarged portion-of -Inarkedsquare of filter, with various, ways colonie'scan be in contact with grid-lin s. ''Coloniesare counted in squares indicated y the arrows.

lamp or in elaborate microscopelamp housings, are not satisfactoryfo-r coliform colony counting.on

I

membrane filters with Endo-typemedia:

b For fecal coliforms or fecal ttrep-tococci, the lighting requirementsare not so severe; in this case almost ,-- any sufficiently bright light source,

3 which can be placed above the Mier(either at' a high r at a low angle)will suffice,

Lighting arrangement and counting

ft,

a

4%

As above, for conform dounfirig, iothe fluorescent lamp should be at a .high angle (as nearly as possibledirectly over the membrane filter)so 'placed that an linage of the lightsource is reflected off the colony'surfaces into the 'microscope lenssystem. Properly placed, the light -will demonstrate the "golden'metallic: surface luster of coliforxncolonies., which may cover the entirecolony, or may Appcar only in an

. area in the center of the colony.The worker must learn to recognize,the ilifferenceletween the typicalgolden sfieen'Of colifdrm. colonies

- and the.merely-shiny surface of nnn -conform. colonies.

A

cp

0

0Colony Counting or Membrane Filters

11- Other types of colonies (fecal coli-forms, fecal streptococci, etc.) do,not require such rigid control of thelight source. Low-angle lighting- -

can be helpftil, to give a relief ofthe colony profile frci'm the colonysurface. his is valuable with smallcolonies, uch as frequently en-countered in streptococcal studies.IA such cases, almogt any lightsource is acceptable,. provided thatit is bright enough and that it is'applied from some.where above themembrane filter. -. ---''

c The typical appearance of varioustypes of colonies.is related to theculture media applied; therefore,this is not discussed in detail at-this

,point.' See the outlines on culturemedia and on laboratory piaceduresfor specified indicator organismsfor,such information.

. . _

d In colony counting, count all coloniesindividuglly, even if they are incontact with each other (this .is con-trary to usual practice in colony.counting in agar cultures in Petri

o ithes). Such oolonies are recog-ized quite easily when a 4.iicroscope

is Used for colony counting as re-commended. Colonies which havegrown into contact almost in3arfablyshow a very fine lineof contact. Theworker /mist learn.to recognize thedifference between two or morecolonies which have grown into con--tact with each other,, and single,irregularly shaped, colonies which

s sometimes develop on.membranefilters. ' Such colonies almost in-.variably are,associated with g,fiberor particulate material deposited onthe filter, and tend to develop alonga path conforming to the shape andsize of the fiber or particulates.

.CALCULATIONS-

A Counting Units

1 In reporting densities of indicatororganisms ( coliformS, fecal coliforms,

. ,

fecal streptodocci), bacterial countsalways are reported in-numbers per 100ml. In standard pmeigtice, results areexpressed to two Significant figures. Forexample, if the calculation indicates75, 400,or even 75,444 organisms per 100nil, the results would be reported as15, 000 per 10IS ml in each case. (Thedigits 7 and 5 are the-significant figures;the three zeros only locate the decimalpoint.)

2 _When "total': bacterial counts are reported,commonpractice is to report in number

- per ml, not ,thOumbdr per 100 ml.3 'QuantitatiVe work on- enteric pathogen's is,

at thistime, limited to reporting ofoccurrence of designated enteric pathogens,oorrefated with measured density of pollu-tion indicating bacterial groups. At -suchTime as the numerical determination. ofenteric pathogens. becomes feasible, it isanticipated that reports will be in termsof count per 100 ml, or even larger volumeunits.

B Typical Calculations.4.

1 Select the membrane filter bearing-the-----acceptable number of colonies for re-porting, and calculate indicators per100 ml according to the general formula:

No.7 indicator`organisms per 100 ml

N9. colonies of indicator organisinNo. ml of sample filtered

2 Example:

X 100

a Assume that for a total coliformcount, volumes of 50, 15, 5, 1. 5;and 0.5 ml produced coliform colonycounts of -200, 110, 40,-10, and 5,

- respectNely.ti

6 First, the worker. actually would not; have, counted coliform Colonies on

all theskfilters., He would haveselected, by inspection, thee mem-

o, filter(s) most likely to have20-80 coliform cblonieactual counting to such colonies(this does take some practice andskill in making quick estithates, butcomes withexperience).

12-5

132

Colon); Counting or Membrane-Filters

c Having selected the membrane filterprobably" most useful for reportingpurposes, coliform colonies arecounted according to accepted 'pro-cedures, and the general formula.is applied:

Coliforms per 100 ml = 4 5° X 100

Coliforms per 100 ml = 800

C Special Situations in. Calculating Densitiesof Indicator Organisms

1 Assume a conform count in which the' volumes 6(1; "O. 3, 0.1; 0.03, and

0.01 ml, respecti:.;ely, produced coli-form coh?ny eounts of TNTC,'TNTC,75, 30, and 8, respectively:

Aa Hetr. e, two sample vollimgs resulted.

in production of coliform coloiniesin the acceptable counfing range.

b Suggestion: Compile the filtratiolvolumes and colonies from bpthacceptable filters, as follows

Volume, nlil ebtirit

0.1 / 75 '

0:03.00.13 105

i.`

,'

Calculate coliforms per 100 nil fromthe composite result:

Conforms pet..0

'100 ml = 100.

.101

53

X

Coliforms per 100, ml = 81,000

2 ,Assume a coliform count in whichsample volumes of 1, 0.3, and ,01 mlproduced colony .counts of 14;'13',. and0, respectively.

I

a Here, -no colony count falls withinrecommended

b Suggestion: Calculate othe basisof the nost nearly acceptable value,-0

$

, .

s

and report with qualifying remark;thus:

9-.Use 14colonies from 1 ml of sample:

1

14 X 100 = 14000

Report: "Estimated Count, 1400per 100 ml, based on non-idealcolony count"... '

. '3 Assume a coliform 'point in which the

volumes 1, 0. 3, and O. 01 ml- producedcoliform colony counts of 0, 0, and 0,respectively.

0

a Here, no actual calculation is possible;even for "estimate" reports.

b Suggestion: Calculate the-numberof estimated coliforms per 100 mlthat would have been reported ifthere had been 1 coliform colonyon the ilter,repres4iiting the largest'filtration volume: thus:

Use 1 colony, and 1 ml: X 100 = .100

Report: "Less than 100 coliformsper 100 thl".

4 Assume a coliform count in which thevolumes of 1, 0.3, and 0.01 ml pro-duced coliform colony counts of TNC,

,150, and 110 colonies.

a Here, alLoolony counts are abovethe, recommended limits.

b ,Suggestion: Use Example 2, above,and report an estimated count basedon non-ideal colony counts:

0 01 X 100 = 1,100,000.

Report: "Coliform count estimatedat 1,100,000 per 100 ml, based onnon-ideal colony count".

' o

a

5 Assume that, in Example 4, the volurdsof 1. 0, 0. 3, and O. 01 ml, all producedtoo many cOliform colonies to show.separated colonies, and that the labora-tory bench record showed TNTC (TooNumerous to Count).'

Suggestion: Use 80 colonies as the6asis of calculation with the smallest

filtration volume, thus:

0801 X 100 = 800,000

Report: ">)300,000 coliforms per 100ml sample. Filters too crowded."

tr

kr,

. ,Colony Counting or Membrane Filters

VII CONCLUSION

The fore ng discussion has presented anumber oYY factors which determine the quan-titative reliability of membrane filter results.It cannot be too strongly emphasized that thecorrect use of acceptable colony countingequipment is one of the most important singlefactors in successful application of membranefilter methods. Here, there is perhaps agreater exercise of personal skill and judgmentthan in any other aspect of membrane filtermethodology. There is no substitute fortice and experience, supported by liberal use .

of supporting colony verification studies, toprode a skilled wodc.er in colony countson membrane filters.

This outline was prepared by, H. L. Jeter,Chief, Program Support Training Branch,USEPA, Cincinnati, Ohio 45268

Descriptors: Filters, Meinbrane, Bacteria,Microorganisms, Measurement TestingProcedures

14

1 4

ttn

)

0 ?.G=AKC

..

12.47

.

v

I' INTRODUCTION

VERIFIED MEMBRANE FILTER TESTS

k

A .The'pu'rpose of a verified, membrane filtertest procedure is to establish the validityof colony differentiation and interpretationin the applied. Specificall y, averified membrane-filter test may puseful 1) as a self-trainingdevice for newworkers, 2) as a research toel in evalu-ation of new membrane filter tmedia and

-procedures, or 3) to provide supportingevidence of,colony interpretation in cases-where the analytic results may be subjectto_professiona)..orTfficial challenge.

B Reduced to essentials, a verified.mem-hrane filter test consists of 1) interpre-tation of the colorises appearing on aselective, differential medium, 2) re-covery of purified bacterial cultures fromdifferentiated colonies, and 3) application

of supplemental test procedures todetermine the validity of the originalinterpretation of the membrane filter

-colonies.,

C

"

In this discussion, pr imary attention is givento averified membrane filter coliform test.In addition, verificatiorkprocedures arepresented for members btathe fecal coli-form group and for fecal streptococci.

, II. VERIFIED TEST FOR MEMBERS OF THE.COLIFORM'GROUP

,

Reincubate124 hours at i5 °C.

No gas

pi, An abbreviated procedu re 'corresponds to

Confirmed Test of Standard Methods thiouuse of lactbse broth (or-lactose lauryl

A tryptose broth) followed by confirmationin brilliant green lactose bile loioth. Theprocedure is shown diagrammatically asfollows:

..A.

10 - 20 sheen Colonies 'Nom m embran e'filter beach testectVeparately) .

Lactose ior lauryl tryptose )brOthit

No gas

Incubate 24 hours at',350C1,

Gas

Negative colifOrni testColony not coliform

Diagram 1.

1

t

Brilliant Green lactOse bile brOlh..4.Incubate up.to 48 hourS at:35°C

:No gasNegative coliform testColohy not coliform

GasROsitive conform testColony _was coliform

ABBREVIATED COLIFORM VERIFICATION PROCEDURE

W. BA. mem. 83a..8.7'71%3:5

:4

Verified Membran_Siltir Testst

-4B A more elaborate verification of membrane

filter test for coliforms-' resembles clae4;Completed Test of Standard Methods. Thetest is started in exactly the same way_ asthe abbreviated test, and may be repre-sented diagrammatically as a continuationfrom the lactose broth stage of Diagram 1.See Diagram 2. ,

C While 'the diagrams (1 and 2) are pre-sented in terms of sheen colonies (inter-.preted as conforms), the careful workeralso should subject a similarly represen-tative number of non-sheen colonies(judged to be' rioncoliforms) to the' sametest procedure. This will reveal whetherthe medium being studied fails to differ-entiate appreciable numbers of colonieswhich in reality are coliforms, even

Nd gas

.

;though they did not demonstrate thedesired differential characteristic.

III VERIPICATI011 OF FECAL COLIFORM_TESTS ON MEMBRANE FILTERS

A 'Tffe procedure desCribed here is based5 .on the principle that, with use of in:FC

Broth and incubation in a water bath at44. 5°C for' 24 hour, fecal conformcolonies on membrane filters develop ablue color, (sometimes a greenish-blue).Extraneous bacteria are believed to failto develop colonies, or else consist- ofsuch colonies develop some colorotherthan the blue color of fecal conforms(colcirless, buff- or brownish-color, oreven red colonies may develop on themedium). :

Gas-positive lactose broth tubes fromDiagram-1, abov et_

Streak on eosin methylene blue agarplates for colony isolation

Incubate 24 hours at 35°C

Transfer an isolated nucleated colony,(or at least two well-,isolated representativebolonieS in the absence of nucleated colonies)

to

lactose broth (or laCtoselauryl tryptost broth)

Incubate up to 48 hoursat 35°C

NegatiLlre test(Colony ,wad notrtbliform) .

Diagram 2:

.

Gas and

and to nutrient agar slant

1Incubate not more than 24 hoursat 35 °C

Prepare Gram:stained smear andexamine under oil immersion

vsGram- negative, non-sporeforming rods, pure culturebased on morphology

st (Colonyoriginally selected Wks conform).Positive colifOrm

Do"

Lack of asx morpho-;logical feature

. described on left

Negativeboliform teat-(Colony originally chosen .

was not coliform)

EXTENDED COLIFORK-VERIFICATION PROCEDURE'

136

r.

B The verified test for fecal coliformiis indicated in Diagram 3. below:.

No gas

Reincubate 24 hoursat 35°C

Verified Membrane-Filter Tests'

10 - 20 blue colonies from membranefilter (each d separately)

Phenol red lactose broth (or lactosebroth or lauryl tryptose broth

Incubate 24 hours at 35°C4,

No gas)Negative test .forfecal coliforms.Colony was not afecal coliform' colony

Gas

No'

,(1.

EC Broth

Incubate -24 hours at 4.5 °C ± 0.50C in awater bath,

40-

Nogas Gas A

s

I

Negative test for fecal Positive test for'coliforms. Colony was fecal coliforms.not a !ecal coliform Colony was fecal

coliforin

Diagram 3; A VERIFICATION PROCEDURE FOR FECALCOLIFORMS ON MEMBRANE FILTERS

a

IV VERIFICATION OF FECAL: STREPTO-COCCUS'COLOKES ON MEMBRANEFILTERS

'A The procedure is used in the evaluation ofresults from medium similar to them-Enterococcus Agar (Slanetz) de§cribedin the current edition of Standard Methods.

- The membrane filter procedure utilizes48 hour incubation at 35°C, and colonieswhich are pink to red, either in'theirentiretyar only in their, centers, areregarded as. fecal Streptococci. Mostsuchcolonies are 1-2 mm in diameter,an some Jnag be larger.. Occasionally,some samples may be encountered in

aivs

137.

which numerous extremely small colonies,approxithately 0.1 rpm in diameter, arepresent in great numbers. ~Almost in-variably, these are not fecal streptococci.See diagram 4 for a representation ofa verification test.

V 'CALCULATIONS BASEDON VERIFICATIONSTUDIES

A A percent verification can be determinedfor anycolony-validation test:

Percent verifkation =No. -=of colonies meeting verifIcation testNo, of colonies subjected to verification oo

( 13-3 ,

Verified Membrane Filter Tests

.10 - 20 Pink to red colonies from AP

membrane filter (each tested separately)

NI AGA*

(10 20 /I112 10 11111CCOLONIIS 1110MMINSRANE 11111111 IlAC11 0 51 AAAAA EMI

PINK 111D COLONY

'CROWNS IN SIAININAII 1N1USION 11110tH WITHIN DAYS Al 45 C ANDs DAYS Al 10 C WIM C0111121111ItION Al C AAAAAAA .1410MNI

0110WtH Al 45 C AND 10 C

1CONFIRM WITH OlOWTH IN 6 514 NoCI AND pH., 5 IN

SIAIN MALI INFUSION 111101H AND RIDUCTION'OF 01 %.2411H111111 IIUI

/INDUCOON OP 2,103.11C AND

INTIROCOCCUS OROUP

111111INTATION OF D. 501110101 AND OLYCIPOI511206 HYD1101.1515

NIOATIVIPOSPIN( NIOAIIVI POSITIVI

S PAICIUM ANDS DURANS P(PIONIZATION OFPAICALIS ANO VAIN1115 ATYPICAL FAICALIS111111r5 MDR1VIOITATION WWI/ 1.

POS111VI ..10AtIVI

HYDROLYSIS OF MANN .

POS111y1 1110AtIVI11

S PAICALIS S PAICALIS ANDVAR LIOUNACNNS S PAICALIS VAR 2.(11100tNIS

.NIMOLYSIS

POSItNt NIOAtIVI

PAICALIS VAR /Y111001105 S PAtCALIS

1(011111tAtION OF LItAIIIN051\POSItIVI NIOAIIVI

.15 PAICIUM 5 DURAN!

.

S PAICALIS VAR INIUOCOCCILIOUtFACI(145 IWAR11111100011)

NNW, SOUSCII

7ANIMAL SOURC15/-1

1310111tH At 43 C ONLY

S SWISS AND S IOUINUS

5,11 CH HY DROLYSIS

POSIIIVI

L ACTOSI FIRMINTATION

ACID

11

pray NO

S VISIt/ IOUINUS111%1510CP AND POULt2Y

SOUP MI

r.

Diagram 4. FLOW SHEET AND SEQUV10E OF TESTS TO PERFORM VERIFICATIONSTUDIES ON COLONIES BELIEVED TO BE-FECAL STREPTOCOCCI

L

Example: Twenty-five sheen coloniesone Endo-type membrane filter mediumwere subjected to verification studies.wasshown inkDiagram 1." Twenty-two oftheee colonies proved to be conformsaccording to provisions of the test:,Percent verification = 22 X 100

25

= 88

11, A percent verification; figure can beapplied to a direct membrane filtercount per 100 ml to determine the veri-fied membrane-finer count per 100 mlof the test organism.

w

Verified count per 100 mlof the test. organism

Percent verification count per 100 ml100 of test organism''

Example: For-a given.sample,' by adirect membrane filter test, the fecalcoliform count was found to be 42, 000',per 100 ml. Supplemental studies onselected colonies showed 92% verification

t.}

Vef4fied fecal 70793 2T'X 42,000conforni count

= 0 :92 X 42, 000

38, 646

Rounding off: = 39;000 per 100 ml

1 33

Verified Membrane Filter Tests

C A percentage of false-negative test: arai5(V,.1.;can be depermined (See II, C)

Percent false negative, 2- .

No. "negative" colonies found positiveTotal No. "negative" colonies tested

Exaniple: On a total Conform test, -25,nonsheen,(c&iform negative) colony,types were subjected to the conformverification procedure shOwn in Diagram1. Two or these colonies proved'to becoliform Colonies.

X_100

,

Percent false e negatives25

2 X 100

= 8

e

VI SOME APPLICATIONS OF PERCENTVERIFICATION CALCULATIONS

A. In comparisons between two Or more differ-ent membrane filter media, the 'mediumwhich has the highest Percentage of veri-fication, and the lowest percentage offalse negatives (based on a broad rangeof sample types and sourees) is the bettermedium.

B In pr6ductivity comparisons between twoor more different membrane filter-media,the medium which produces the highestverified-membrane filter counts per 100ml (based on a broad range of sampletypes and sources) is' the better medium.

C The worker° is cautioned NOT to applepercentage of Verification determined.from one. sample, to other samples. For'example, do not determine a per bentage4verification on m-Endo broth for a samisfe-taken from,the'Ohio.Riyer on September 6,and then seek to apply that perocntage -

verification to anOher coliform determina-tion from the Little Miami River, on the ,

same date. Even the application of.the.verification percentage to another OhioRiver sample, either, on the' same datefrom a different Station, or on another .<

date from the same station, should beundertaken, with great caution. Such

.. ,

application of verification percentagesi'from'one sample to another should bet en o fter sufficient studies have,been de 'dernonstrate the suitability,

such a procedure.

. ,

VII USE OF VERIFICATION STUDIES,IN`MF-MPtT COMPARISONS

A CompariSons of data obtairiethfrom MF,versus MPN methods have been the sourceof great concern to microbiologithts. Forthe current basis of comparisons, seeStandard Methods (either 11th or 12thedition) with- a proviso-fhat'it shouldbe used for determining the potabilityof drink, water only after paralleltesting had shown that it afforded infor-mation equivalent to that given by thestandard multiple-tube test. "

B Some workers have Ought to apply thisreert1Thlment on the basis of statisticalcalculations, based on comparisons ofnumerical values from membrane filtertests with numerical values obtained fr6mmultiple-tube tests, Further study of thisproblem, and methods different. workershave applied tb the problem, cadbe made,r1 the basis of the appended reference '1st

C Numerical. comparisons between'raw oriferified membrane filter results on splitsamples, compared with multiple-tuberesults, also should take into account thequestion of the reliability of the multiple-4ube test. The numerical results of theCompleted Test for conforms, for example,

,can be compared with the results of theConfirmed Test, to determine a percentageof verification for the multiple-tubetept:

Percent verification

'Completed Test ColifOr'ins per 100 ml X 100Confirmed Test Conforms per 100 ml

Example :: °On a given sample, the testwas carried to the Completed Teststage. Afterward,, both a ConfirmedTest and a Completed Test coliformresult were obtained, consisting of

13913-5

Verified Membrane Filter Test

-Table 1. VALIDITY OF MF ektID MPN "CONFIRMED TEST"*

.

SourceNumber

ofsupplies

MF Coliform Test MPN Confirmed Test°

Minimum MaximumPercentverified

PercentMinimum Maximum verified

Wells - Springs 16' 1.0 7; 600 96. 6 7.0 11,000 64.6

Lakes - Lagoons 23 . 1.6 420,000 79.6 79 490,000 70.9.

Creeks 19 32 260, 000 75, 8 120 460,000 66.4

Rivers 22' 320 890;000 69. 7 7Q0 350,600 75. 7

Sewage 117 1, 400, 000 28,600°,000 68. 6 460,600 49,000,0(4 73. 8

Totals 91 78. 11 70.3

All conform values are per 100 ml of sample

49,060 per 100 ml for the ConfirinedTest and 33,000 per 100 ml for theCompleted Test.

Percent verification', 33, 000 X 10049,000

= 67

See Table 1 for some studies of MFverification studies, and parallelmultiple-tube'werification studies(Confirmed Test carried to CompletedTest). Thee studies have been con-ducted in research laboratories of thisCenter, and demonstrate the difficultyand problems associated comparativeevaluation of membrane filter v*rsusmultiple-tube methods. 'The student is

to, study this table at leisure.

itEFERENCES

Delaney, J. E. , McCarthy, J.A. andGrasso, R.J. Measurement of E. coliType I by the Membrane Filter. Waterand Sewage Works, 109, 289-294. 1962.

2 Geldreich, E. E. , Clark, H. F. , Huff,C. B. and Bost;, b. C. Feder-Conform-Organism Meditim for the MembraneFilter Technique. JAWWA 57,0268-214. 1965. , °

13-6

3 "Geldreich, E. E. ; Jeter, IL L. and Winter ,

pplying the Membrane Filter ro,-cepures. In Press. 1966.

J.A. Technical Consideration in'A

4 Hoffman, D. A. , Kuhns, J, H. , Stewart,R. C. and Crossley, E. I. A .Co rison .of Membrane Filter Counts and t

Probable Numbers of Coliforni\in San' Diego's Sewage and Receiving Waters.

JWPCF 36; 109-117. 1964.

ti McCarthy, , Delaney, J. E. andGrasso, RJ. Measuring Conformsin Watpr. Water and Sewage Works,108, 28-243. 1961.

6 Thomas, H.A. , Jr. and Woodward; R.L.Estimation Of Coliform Density-by theMembrane Filter and the FermentatTube' Methods. American JournalPublic, Health 45, 1431-1437. 1955.

This outline was prepared by H: L. Jeter,Chief, Program Support Training'Branch,USEPA, Cincinnati, -Ohio 45268

. Descriptors: Quality Control, Filters,Membrane, Enteric Bacteria,Microorganisms, Laboratory Tests

A

COLLECTION AND HANDLING OF SA1VTLES.FORBACTERIOLOGICAL EXAMINATION'

INTRODUCTION

The first step in the examination of a watersupply for bacteriological examination isdareful,collection and handling of samples.Information from bacteriological tests isuseful in evaluating water purification,bacteriologic41 potability,. waste disposal,and industrial supply. Topics coveredinclude: representative site selection,frequency, number, size of samples,satisfactory sample bottles, techniques ofsampling, labeling, and transport.

II SELECTION OF SAMPLING LOCATIONS

The basis for locating sampling points iscollection of representative samples.

A Take samples for potability testing fromthe distribution system through taps.Choose representative points covering-the entire system. The tap `itself shouldbe clean and connected direcfly into thesystem. Avoid leaky faucets because ofthe-danger of washing in extraneousbacteria. Wells with pumps may beconsidered similar to distribution systems.

B Grab samples from streams are frequentlycollected for control data or application ofregulatory requirements. A grab samplecan be taken. in the stream near the surface.

C For intensive stream studies on source.'and extent of pollutiOn, representative

,,samples are taken by considering site;method and time of sampling. Thesampling sites may be a compromisebetween physical limitations of thelaboratory, detection of pollution peaks,and frequency of sample collection incertain types of surveys. First, decidehow many sainples.ane needed to beprocessed in a day. Second, decidewhether to measure cycles of immediatepollfftion or more average pollution.Sites for measuring cyclic p,011ution areimmediately,b'elow.the pollution source.Sampling is frequent, for example., everythree hours.

'A site designed to measure more averageconthtj.ons is far enough downstream fora complete mixing of pollution and water.-

W. BA. Stc.1d. 8.78

60

Keep in mind that averaging does notremove el yariatiof but only minimizessharp fluctuations. Downstream sitessampling may not need to be so frequent.

Ssrnpies may be collected' 1/ 4, 1/2 and., 3/4 of the stream width at each site or

othersdistances, depending on surveyobjectives. Often only one sample in thechannel of the stream is collected.

;role.

s are nsually takenenear the surface.

b.,,,Samples from lakes or reservoirs 'arefrequently collected at the drawoff and

..usually about the same depth and may becollected, over this entire surface.

E Collect samples of bathing beach waterat locations and times where the most'bathers swim.

141

III NUMBER, FREQUENCY AN)) SIZEOF SAMPLES

A For determining sampling frequ' enc,y fordrinkingwater, consult the USEPAStandards.

1 The total number, frequency, and siteare established by agreement Witheither state of USEPA authOrities.

2 The minimum number depends upon thenumber of users. Figure 1 indicatesthat the smaller populations tall forrelatively more samples than largerones. The numbers on the left of thegraph refer to actual users and not thepopulation shown by census.

3 'In the event that coliform limits of thestandard are exceeded, daily sample'smust be taken at the same site.'Examinations should continue 'until twoconsecutive samples show coliformlevel is satisfp.ctory. Spcb samplesare to be considered as special samplesand shall not be included in the totalnumber of samples examined.

4 Sampling programs described aboverepresent a minimum number which,may be increased by reviewingauthority.

... ,...$

6 . . ,

14-1

Collections and Handling of Samples for Bacteriological Examination

B For stream investigations the type ofstudy governs frequency of sampling.

C Collect swimming pool samples when useis heavy. .The high chlorine level rapidlyreduces the count when the pool is not

Populationserved:

25 to 1, 0001, 001 to 2, 5002, 501 to 3, 3003, 301 to 4, 1004, 101 to 4, 9004, 901 to 5, 8005, 801 to 6, 7006, 701 to 7, 6007, 601 to 8, 5008, 501 to 9, 4009; 401 to 10, 30010, 301 to 11, 100n, 101 to 12, 00012, 001 to 12, 90012, 901 to 13, 70013, 701 to 14, 60014,.601 to 15, 50015, 501 to 16, 30016, 301 to 17, 20017, 20118, 10118, 90119,80120,_70121;56122, 30123,,20124, 00124, 90125,00128, 00133, 00137, 001

Minimum pumber ofsamples per month

O

to 18, 100to 18, 900to 19, 800to 20, 700to 21, 500to 22, 300to 23, 200to 24, 000to 24, 90Qto 2k 000 .to 28; 000to 33, 000to 37, ocirto 41, 000

41,001. to 46,000 .4

46; 001-o 50, 00050, 061 to 54, 00054, 001 to 59, 00059, 001 to 64, 00064, 001 to 70, 00070, 001 to 76, Q0076, 001 to 83;00085, 001 to 90, 000

-r

2

3

456

78

.91011

1213.1415161718192021

*. 222324252.627282930

.354045505560.657Q758085

' 90

use. Residual chlorine tests arenecessary to check neutraliation ofchlgrine in the sample.

D Lake beaches may be sampled as requireddepending on the water uses.

Populationserved:

,44$),Minimum number dsamples per month

90, 001 to 9 6, 00096, 001 to 111, 000111,001 to 130,000130;001 to 160, 000160, 001 to '190,000190, 001 to 220, 000220, 001 to 250, 000250, 001 to 290, 000

7

290, 001 to 320, Ooo320, 001 to 360,000360; 001 to 4110, 000410, 001 to 450, 000

,/ 450, 001 'to 506; 000

95.10011 0120130140150160170180190200210

520,001 td 550,000 .2205.T0, 001 to 600, 000 230606, 001 to 720, 000 240720, 001 to 780, 000 250786, 001 tio 840, 000 260840, 001 to 910, 000 270910, 001 to 970, 000 280970, 001 to 1, 050,000 2901, 050, 001 to 1, 140, 000 3001, 140, 001 to 1, 230, 000 3101, 230, 001 to 1,.320, 00$ 32111, 320, 001 to 1, 4201 00,E 3301, 420, 001 to 1,520,010 3401, 520, 001 to 1, 630 00 3501, 630, 001 to , 000 3601, 730, 001 1, 850, 000 .3701, 850, 001 to 1, 970, 000 380

' 1, 970, 001 to 2, 060, 000 3902, 060, 001 to 2, 270, 000 4002, 270, 001 to 2, 510, 000 4102, 510, 001 to 2, 750, 000 4202, 750, 001 to 3, 020, 000 4303, 020, 001 to 3, 320, 000 4403, 320, 001 to 3, 620, 000 4503, 620, 001 to 3, 960, 000 4603, 960, 001 to 4, 310, 000 470

. elt 310, 001 to 4, 690, 000 4800690, 001 or more 500

1

1

E

F

A

is)

Collection and Handling of Samples for bacteriological ExaminatiOn

Salt water or estuarine teaches aresampled as needed -with frequencydepending on use.

Size of samples depends upon examinationanticipated..,, Genera 1137,100 ml. is theminimum size.

BOTTLES FOR WATER SAMPLES

The s ple bottles should have capacityfor a least 100 ml of sample, plus anair s ace. The bottle and cap must be ofbacte 'ological inert materials. Resistantglass or heat resistant plastic areacceptable. At the National TrainingCenter, wide mouth ground-glassstoppered bottles (Figure 2) are used.All bottles musebe properly washed andsterilized. Protect the top' of the bottlesand cap from contamination by paper ormetal foil hoods. Both glass and heat

M=IMMAM.ILWIN1

FIGURE 2 z.

resistant plastic bottles may besterilized in an autoclave. Hold plastippoo°at 121°C for at least 10 minutes. Hota:irsterilization. 2 hours at 170°C, maybe ithed,for dry glass bottles.

B Add sodium thiosulfate to bottles intended1/4 for halogenated water samples. A quantity

of 0.1 ml of a.10% soluticili provides 100mg per liter concentration in a 100 mlsample. This level shows no effect uponviability or growth.

143

C Supply catalogs list wide mouth groundglass stoppered bottles of borosilicate-resistance glass, 'specially for watersamples.

TECHNIQUE OF SAMPLE COLLECTION

Follow aseptic technique as early as possible.Nothing but sample,water must touch the insideof the bottle or cap. To avoid loss of sodiumthioslfate, fill the bottle directly and do notrinse. Always remember to leave ap air space.

In sampling from a. distribution system,first run the fauce\t wide open until theservice line is. cleared. A time of 2-3minutes generally is sufficient Reduce ,.

,.the flow and fill the sample bottle without"a-Plashing. Some authorities stress flamingthe tap before collection, but the use of thistechnique is now generally consider4d as value-less. A chlorine determination is often madeon the site

B The bottle may be dipped into some,waters by hand. Avoidjintroductionof bacteria from the hAman hand andfrom surface debris. Some suggestionsfollow. Hold the bottle near the basewith one hand and with the other removethe hood and cap. Push the bottlerapidly into the water mouth down and tgt upup towards the current to fill. A depth/of about4 inchep is satisfaetory.. Whenthere isIno current move the bottlethrough the water horizontally and,awayfrom the hand. Lift the bottle from thewater,' spill a small amount of sampleto provide an air space, and return the

\uncontaminated cap.

14-3

K .

Collection and Handling of Samples for Bacteriological Examination

C Samples may be dipped from swimmingpoolst Determine residual chlorine onthe p water at the site. Test thes 'le at The laboratory to check chlorineeutralization by the thiosulfate.

( D Sample bathing beach water by wading outto the two fobt depth and dipping thesample up from about 6 inches below thesurface. Use the procedure described inV. B.

Wells with pumps ,are similar to

diAtribution systama. With a hand pumpedwell; waste water for about five minutesbefore taking the sample. SaMple a wellwithout a pump by lowering, a sterilebottle attached to a weight. A. device whichopens the bottle underneath the waterwill avoid contamination by surface debris.

F Various types of sampling devices areavailable where the sample point isinaccessible or depth samples are desired.The general problem is to put a samplehottle'in place, open it, close it, andreturn ft to the surface. No bacteria butthose in the sample must enter the bottle.

E

1 The J - Z. sampler described by Zobellin 1941, was designed for deep seasampling but is useful elsevthere (Figure)3). It has a metal frame, breaking ,

device fori&glass tube, and samplebottle. The,teavy metal messenger°strikes the lever arm which breaksthe glass tubing at a file mark. A °bent.rubber- -tube straightens &nd thewater its drawn in several inches from.

-the apparatus. Either glass or collapsiblerubber bottled are sample containers.

Commercial adaptations are available.

2 Note the vane and lever echanism onthe New York State Co servationDepartment's same r in Figure 4.When the apparatus is at proper depththe suspending line is given a sharppull. Water inertia against the vaneraises the stopper, and water; poursinto the bOttle. (Safficient sample iscollected prior to the detachment ofthe 'stopper fronfthe vane arm allowinga closure of the sample bottle.

The New York State ConservationDepartment's sampler is useful forshallow. depths and requties nothingbesides glass stoppered sample bottles.

.14-4 ,

ti

4

FIGURE 3

Reproduced with permission of the Journalof Marine Research 4:3, 173-188 (1941) bythe Department of Health, Education andWtlfare.

for

Collection and Handling of Samples for BaCteriologicalExamination

FIGURE 4

3 A commercial sampler 4s availablewhich is an evacuated sealed tube witha capillary tip. When a lever on thesupport rack breaks the tip, the tube'fills. Other 'samplers exist with alever for pulling the stopper, whileanother uses an electromagnet.

VI DATA RECORDING

A Information 'generally includes: _dale, timeof collection, temperature of water, locaticof sampling' point, and name of the samplecolleCtor. Godes are often used. Thelocation description must be exact enoughto guide another person to the site.

- Reference to J:Iridges, roads, distance tothe nearest town may help. Use of thesurveyors' description and maps arerecommended. Mark identification tql thebottles or on securely fastened tags.Gummed tags may soak off and areinadvisable.

("4'

#a

B While a sanitary survey is an indispensablepart of the evaluation of a water supply, itsdiscussion is not within the scope of thislecture. The sample collector could supplymuch information if desired.

VI SHIPPING 'CONDITIONS

The examination should commence as soonas possible, preferably within one hour. Amaximum elapsed time between collection andexamination is 30 hours for potable watersamples and 6 hours for other water samples(time from collection tollaboratory delivery).An additional 2 houis is allowed from deliveryto laboratory, to the completion of first-daylaboratory procedures. Standard Methods(14th Edition) recommends icing of samplesbetween collection and testing.

VII PHOTOGRAPHS.

A photograph is a sample in that it ,is evidencerepresenting water quality. Sample collectorsand field engineers may carry cameras torecord what-they see. Pictures help the generalpublic and legal courts to better understandlaboratory data. -

REFERENCES

1 APHA, AWWA, WPCF, Standard MethOd;for the Examination of Water and Wastewater.-(12 Ed. ) 1965.

2 Prescott, Sre....WintloW, C. E. A. , andMgCradY, .M. H. Water Bacteriology. 6th Ed.,368 pp. John Wiley and Sons, Inc. , New York.1946.

3 Haney, P. D; , and Schmidt, J. RepresentativeSampling and Analytical Methods in StreamStudies. Oxygen Relationships in Streams, .

Technical Report W58-2 pp. 133-42. U. S.Department of Health, Education and Welfare,Public Health Servije, Robert A. TaftSanitaryEngineering Center, Cincinnati, Ohio. 1958.

4 Velz, C. J. Sampling for Effective Evaluationof Stream Pollution. Sewage apd IndustrialWastes, 22;666-84: 1950.

. ,

145 14-5

ti

Collerction and Handling of Samples for Bacteriological Examin

5 Bathing Water Quality and Health III. Coastal Waters- .134 pp. U. S

11116partment of Health, Education, and leWelfare, Public -Health Service, RobertA. Taft Sanitary EngineeriCincinnati, Ohio. 1961.

6 Zobell, C. E. Apparatus for CollectingWater Samples from DifferenVepths

r

14-6

5

-

o e

O

. g

for Bacteriblogical Analysis. Journal, of Marine Research,,4:3:173-88. 1941

This outline was Originally pre-pared byA. G. Jose, former Microbiologist FVFPCATraining Activities, SEC and vpsjEcted bythe Training. Staff, Nationar-Training Center,DTTB;\MDS, .WPO, EPA, Cincinnati, , OH45268:

Descriptors: Equipment Miciobiology,iamplirieVater Sampling

V4

V

o

-c

146

t

1

I INTROE/UCTION

TESTING THE SUITABILITY OF DISTIJJLED WATERFOR THE BACTERIOLOGY LABORATORY",

tA Standard Methods for the Examination of

Water and Wastewater (12th Edition) states;

'Only distilled water or-deMineralizedwater which has been testediTh:n1---faund freefrom traces of dissolved metals and bac-tericidal and inhibitory compounds maybe used for the preparation of cultureand reagents. B c 'tidal co ds maybe measured b a bioib 'c.test procedure... outline deSo ibes a suitableprocedure.

II A need for such a test has been Shown in'the lack of reproducibility of plate countsand'a possible,cause of inconsistent re-sults in split sample examinations.

II THEORY OF THE TEST PROCEDURE

A Growth of Aerobacter aerogenes in a.chemically defined minimal growth medium.The addition of a toxic agent-br a growth

. promoting substance will alter the 24 hrpopul4tion by an increase or decreage of20% or more, when compared to a control,.

AGENTS

A Use, reagents of the highest purity. Somebrands of potassium dihydrogen phosphate(KH2PO4) haire large amounts of impurities.-The sensitivity,ofthe test is controlled inpart by.the purity of the, reagents eniployed.

1 .Carbon source - Sodium citrate, reagent,,crystals (Na3C6H5017 2H20) 0.29 gdisSolved in 5Q0 ml of redistilled water.

III APPARATUS AND MATERIALS

A Glassware - rinse all glassware in freshly' redistilled water from a glass still. Thesensitivity of the 'test depends upon thecleanliness of the sample containers,flasks, tubes, and pipettes: - Use onlybordsiliscate glassware.

B Culture - any strain of coliform IMViCtype --++ (A. aerogenes). This.can beeasily obtained from any polluted river orsewage sample.

e *r

2 Nitrogen source - issolve 0.60 g ofammoniuM.sulfate, reagent, crystals,,(NH4) 2504) in 500 1 of redistilledwater.

3 Salt mixture solution - Dissolve thefollowing compounds in 500 ml of re-distilled water.

Magnesium sulfate, reagent, crystals(MgSO4 7H20) 0.26 g.

,

Calcium chloride, reagent, crystals(CaC1

22H 0)-0:17 g.

rrous sulfate, reagent, -crystals .

(FeS04 H2O) 0.23 g.

Sodium chloride, reagent, crystals(NaC1) 2.50 g.

4 Phosphate buffer 'solution - Use p. 1 to25 dilution of a stock phosphate solutionprepare&by dissolving 34.0 gm ofpotassium dihydrogen phosphate(ICH2PO4) inc.500 ml--OT distilled water,adjusting to pH 7.2 with 1 N NaOH anddiluting to 1 liter with distilled water.

5 Toxic control - dissolVe 0.40 gramsCuSO4 5H20 in 100 ml of redistilledwater. Dilute 1:1000 for 1 mg per literCu before use.

ri'estilig the Suitability of Distilled Water

Aoseo-

:B Sterilization Of Reagents

4

Unknown distilled water sample - eitherboilfor oblninute or sterilize by meth-bran filtration.

Prepare reagents with rediptilled waterheated to boiling for 1 to.2 minutes.Phosphate buffer solution may be sterililed,by MF filtration or.botling.

Sollttionsare useful up to two weeks when-stored at 5°C in sterilized) glass stopperedbottles. salts solution must be storedin the ,dark because sunlight results incopious ferric ion precipitation. A slightturbidity arising in the first 3 - daysdoes not detract from the u ness ofthe reagents.

y PROCEDURE4

A , C011ect 150 - 200 ml of water sample in 'asterile bOrosilic ate glass flask and sterilize.Label 3 flasks or tubes: A, B, and F. .

Add water Samples and redistilledwater to each flask as indicated at thebottom of the 'page.

N

MediaReagenti

STANDARD TEST

°Int rol UnknownDist. Water

'A

B Add a susperision ofAerobacter aerogenes4IMV,iC type ---14) of such density, that eachflask will contain 25 4 75 cells per ml.Makle an bacterial.count by'platinga 1 ml sample"Th,plate count agar. Incu-bate tests A-F at 32° pr 35 °C for 20 - 2(hr. Make plate counts using dilutions of1, 0. 1, 0.01, 0.001 and 0.0001 ml.

VI PREPARATION OF A BACTERIALSUSPENSION

A Bacterial'Growth-

On the day prior to perning the distilled"water suitability test, 05culate a strain of \Aerobacter aerogenes onto a nutrient agar,slant with a slope of approximately 2 - 1/2inches j length contained in a 125 mm X16 min screw cap tube. Streak the entireagar surface to develop a continuousgrowth film and incubate 18 - 24 hrs at35 °C:

°B Harvesting Viable Cells

Pipette 1 - 2ml of sterile dilution;waterfrom -a 99 ml water blank onto the 18 - 24 'hr culture., Emulsifrthe grow i op the

ToxicControl

^

Citrate 2.5 2. 5 2.5Ammonium sulfate . 2:5 2.5 2.5Salt mixture 2; 5 2: 5 2.5

hosphate buffer (TA + . 1):.---.....1. 5 1.5 11 5

Water, I mg per liter Cu X X ,' `2.1.0 d. __ ----.Unknown water. ..

X ...."....- ,, 21: 0 ......--

Itedistilled water .41.--...mr.. 21.4...--.

co

TOTAL VOLUME. 30.0 304

148`

4

OPTIONAL TEST

Food Nitrogen' CarbonAvailable Source ' -Source

C

X 2.5 X

2.5 .2. 5 2. 51. 5 1.5. 1.5

i% X X

21.0 21.0 2 1, 05.0 2,5 2.5

30.6 30.0 qo. o

OPTIONAL TEST

1

A

If

I.

I.

N

testing the Suitability of Dis tilled ater

D TESTE

slant by gently rubbing the bacterial fainwith the pipette, being careful not to tearthe agar, and pour the contents back intothe original 99 ml water blank. .

C Dilution of Bacterial Suspension

Make a 1 - 100 dilution of the originalbottle into a second water blank; and afurther 1 - 1.00 'dilution of the secondbottle into a third water blank, shakingNigorpusly after each transfer. Thenpipette 0.1 ml of the third dilution

000, 000) into each of the flasks A.B, and F (see Standard Methods forExamination of Dairy. PrOlucts, 12th ed.).This procedbre slmuld result in a finaldilution of the organisms to a range of25.-75 viable cells, for each iil of testsolution.

0

D Verification of Bacterial Depsitys ,

Variations_ among strains of the sameorganism, different organisms, media,and surface area of agar slopes will._possibly necessitate adjustment of thedilution procedure to a.rrive,at a specificdensity range between 25 - 75 viable cells..To estahlist tbegrowth range' numerically.far a specific organism and'Mediuin, makeasserias of plate otints from the,thirddilution to determine ths,bacteriat density.Then4hoose the proper volume from thisthird 'dilkdon'Which when diluted by the 30144 in the flasks. A, B, and F will

:'fc

,-

contain 25 - 75 viable cells per ml. Ifthe procedures are standardized as tosurface area of the

chsl n1 and laboratory.

etechnique, it is possi le to reproduce rsults on repeated with "the

-

same strain of microorganisxns.:

°E Procedural Difficulties:I

ChlOrine or chloramine distilling overinto receiver. Distilled water should bechecked by a suitable quantitative pro-ee c.4 re like the starch-iodide titration.If chlorine is found, sufficient sodium thio:.sulfate or sodium sulfite must be adlied.

2 -Unknown water sample stored irrsoft.'glass containers or glass containers

. without liners for metal taps.

3 Contamination of reagents of distilledwater with a bacterial background.

4 Incorrect dilution of A. aerogenes toget 25 - 75 cells pe'r ml.

- 5 Gross contamination of the sample de-termine,d by the initial colony count be-fore incubation.

F Calculation:

1 Fofigt.owth inhibiting substances:

colony couritper ml Flask B. '

colony count per ml Flask A

a Ratio 0.8 to 1.2 (inclusive) showsno toxic substances.

15-3

-1

4

1

-

iTesting. or Suitability of Distilled

C

e

alter

b 'Ratio less than 0. a'shows g thinhibiting substances in watt sample.

.

2 For tox4 control

colony count per ml Flask F.,' Ratiocolony count per ml Flask A .

OPTIONAL TEST

31*For nitrogen and carbon sources thatpromote growth** .

colony count per ml Flask. C ;Ratiocolony count per ml Flask A -

4 For nitrogen sources that promotegrowth**

5

colony count per ml Flask Dcolony count per ml Flask A Ratio

*For carbon sources that promotebacterial growth**

colon'', count per ml Flask Ecolony count per ml Flask A Ratio

.v.G Interpretation of Results:

1 The colony count frofn Flask A after20 - -24 hours, at 356C will depend on

-_the_maqb&r of organisms initiallyplanted in Flask A and on the strain of A.aerogenes used in the test procedures.

--This isihe reason the control Flask Amust be run for each individual seriesof tests. However, for a given strainoft. aerogenes under identical,environmental conditions, theterminalcount should be 'reasonably constantwhen,the.iiiitial plant is the same.

.Thus, it is essential that the initialcolony count on Flask A and Flask 13should'be aperoximately equal to secureaccurate data.

I c.

2 When the ratio exceeds 1.2, it maassumed that growth stimulating sustances tare present. However,Tthisproce(re is an extremely sensitivetest and-ratios up to 2.6 would havelittle significance in actual practice.Therefore) Test C, D, and E-do notappear necessary except in specialcircumstances, when the ratio, itsbetween 1.2 and 3.0.

3 Usually Flask C will be very low andflasks D and E will haire a ratio of lessthan 1.2 when the ratio of Flask B/Flask A is between 0.8 and LI. Thelimiting factors of growth in -Flak A_are the nitrogen and organic carbolf-:present. An extremely_large, amountof ammonia nitrogen with no organiccarbon could increase the redo 'inFlask D above 1.2 or the +absence ofnitrogen with high cater on concentrationcould give_ ratios above 1.2 in FlaskE with an IB ratio between 0.8 and1. 2.

4 A ratio below 0.8 indicates' the water 'contains toxic subst4nces and this ratioincludes all? allowable tolerances-. Asindicated in item 2 (above), the 1.2ratio could go as high as, 3.0 withoutany undesirable results.

5 We are unable to recommend coiAectivmeasures in specific cases of defectivedistillation apparatus. However, ac

n of t a nequipment an a review of productand handling of the distilled watershould enable the-17566.1 laboratorypersonnelo correct the cause of thedifficulty.

*to not attempt tn.e Iculat.e 7ratios, 3, 4, or- 5 when ratio 1-irklicates a toxic reaction...**Ratib in excess of 1.2 indicates availablesource for bicterial growth;

115

O

Testing fbr Suitability of'Distilled Water

CASE EXAMPLES

Test results for various diStilled'water samples

SOURCETEST CONTROL

`COUNT COUNT1

0001

2

3

4

5 310, 000

< ' 100120,

74, 000

18, 000

21, 000

850, 000 37,

170,

14,

14,

60,

000

000

000

000

RATIO

0.4

3

1.

5. 2

000 22. 9

INTERPRETATION

Toxic Substance

Toxic Substance

Excellent water

E>icellent water

Growth Substance

Growth Substance

REFEnNCES `°

1 Standard MethoCiNfor Examination of Water. and Wastewater. 12th .Edition. 1965".p 578.

LI.

2 Geldreich, E. E. and H. F. 'Clark. Dis-tilled Water Suitability for Microbiologi-cal Applications. Journal Milk and FoodTechnical. In Press. 1965. te

This outline was brennrad by E. E GeldrPieh.Chief Bacteriologist, Water bupply ProgramsDivision, WPO, EPA, Cincinnati, OH 45268.

Descriptors: Bacfe Microbiology,Laboratory, Water Supply, Distillation, ,Water Quality Control

I. INTRODUCTION

.

-

RESIDUAL CHLORINE AND TURBIDITY

0 '. )

-0.,

The Interim Primary Drinking Water Regulations (Federal,Register, December 247N1975) permi s, the options of substitution ofllp to 75 percent:of the bac-

teriolqgtca samples with residual chlorine determinations. Any'community

or non-c pity water system,may avail themselves df this option with

approval f m the State based upon resul of sanitary surveys. Residual

tS'\

chlorine d erminations must be carried o C t at the frequency of at least

fourior each substituted microbiological mule.

Simce many potable' lintssarry out their own microbiolOgical deter:minations, it will be neceSsa-ry that these laboratories be certified. for

the bacteriological parameters. Residual chlorine determinations may be .

carried out by any person accept#ble to the Stkte and the'analytical

method and techniqueqused must be evaluated'in some manner to assure.that

reliable fdlorpticin'is obtained.

Since the presence 6f high turbidity can interfere with the-disinfectioncapability of chlorine,.a maximum all wable limit has been set for turbidity 4

as follows:

A. One turbi sty unit (TU) as determined by a monthly average exceptthat five or.fewer turbidity units may be allowed if the supplier

of waterOan demonstrate to the State that thehigher turbidity

does not

. 1. Interfere with disinfection,

2. Prevent maintenance of residual of disinfectant throughout.

distribution system, or,

3. Interfere with microbiological determinations.

B. Five turbidity units based on an average of two consecutive days.

The 'Criteria and Procedures Document for Water Supply Laboratory Certifi-.

cation suggests thatsome quality controlguidelines,W instituted for

.' the residual chlorine and turbidity measurements at 89/State level for

the purpose-of:ensuring data validity for these critical measurements.

In respons- o public comments regariifng the proposed Primary Regulations

(Federal Register,' December 24, 1975) it, is stated, that Operators per-

forming residual'chlorinOnd turbidity analyses "....be certified,approved, or at least minimally trained to perform the analyeitartasks

before a State could accept their analytical determinations:..."

.'TI.TRi3. 3.9:77

152

II. RESIDUAL CHLORINE

Since_residual chlorine anajysis 'would be carried out in "field'? conditions

or in the small laboratories of treatment plants, perhaps by unskilledoper'ators,cit:is necessary to keep the analytical method as simple as

For,a number of years, operators had utilized the orthotolidine-technique in'a kit form to determine the-chlorine residual. Recentstudies and regulatory guidelines have dictated against this test procedure.The acceptable test procedure is now the DPD Test (13th Ed,, Standard Methodsfor the Examination of Water and Wastewater, pgs. 129-132), for which kitsare available from at least two companies and which meet requirements for '

accuracy and reliability. These (Pits are capable of measuring, both free

...and-combined chlorine of which only the free chlorine is measured to meet

codOliance requirements. Kit procedures call for a premeasured single

powder or tablet reagent added'to theitest cell with the sample and aresultant color development measures by comparison the standardized colors

within one minute. Standard. Methods includes cautions regarding temperatureand pH,control regarding this test parameter and.this test procedure, theDPD Test, is least effected by temperature and the pH is adjusted by the

added reagents. The only interfering substance,eoxidized manganese, canbe determined in a preliminary step and compensaIedsor in the final.test

value.

III. TURBIDITY

- Turbidity as long been used in the water supply industry for indicating proper

operational techniques. Turbidity shoOld be clearly understood to be an ex-

pression of the optical property of a sample which cau's'es light to be Scattered

and absorbed rather than transmitted in straight lines through the,sample.4,

The standard method for the determination of turbidity has been based on theJackson candle turbidimeter.% However, the lowest turbidity value which canbe measured directly on .the Jackson turbidimeter is 25 units which is well

above the monitoring level. Because of these low level requirements, thenephelometric method was chosen and procedures are given'in Standard Methods

.-- (13th Ed., 1971).

IV. NEPHELOMETkIC MEASUREMENTS FOR COMPLIANCE MONITORING

The subjectivity and apparatus deficiencies involved'in visual methods of

measuring turbidity make each unsuitable as a standard method.

Since turbidity is arAxpression of the optical property of scattering orabsorbing :Light, it was natural that optical instruments with photometers

would be dei'Eloped for this measurement:

The type of equipment specified for compliance monitoring(3

'

6)utilizes

nephe'!ometry.

Basic Principle( 7), /

The intensity of light scattered the sample is compared (under defined

conditions) with the intensity of light scattered by 'a standard reference

solution (formazin). The greater, the intensity of scattered light, the

greater the turbidity. Readings are made and reported in NTUS (Nephelometric

Turbidity Units).

153.

B. Schematic

Meter

Photocells)

Lamp Lens

)c

44 4

Turbidity ParticlesParticlesScatter Light

Sample tell(Top View)

Figure 2 'NEPHELOMETER

(90° Scatter)

Light passes through a Wan ing lens and n to the sample 9.,e cell.Suspended particles (turbidity in the sample tter the light.

.Photocell(s) detect light scattered by the, particles at a 90° angle to the

'path of the incident light. This light energy is'converted-to an electric -

signal for the meter to measure.

1. Direction of Entryof Incident Light to Cell-

a. The lamp might be positioned as shown in the schematic so thebeam enters a sample horizontally.

b. Another. instrument design has the light beam entering thesample(in a flat-bottom cell) in a vertical direction, with the photocellpositioned accordingly at a 90° angle to the paih,of incident light.

2. Number of Photocells

The schematic shows the photocell(s) at one 90° angle to the path of

the incident light. An instrument might utilize more than one photo-cell position, with each final position being at a 90° angle to the

sample liquid.

3. Meter Systems

a. The meter might measure the signal from the scattered light in-

tensity only.

b. Themeter might measure the signal from a ratio of the' ..scattered

light versus light, transmitted directly through,the sample to a'

photocell.

S r, ,

154

,

41 Meter Scales and Calibration

," a. The meter may already be calibrated in NTUs. In this case;at least one standard is run in each instrument range to beused in order to check the accuracy of the calibration scales.

b. If a.pre-calibrated scale is not'supOied, a'caltbration curveis prepared for each range of the instrument by using appropriate

dilutions of the standard turbiditvs0Cension.

C. EPA Specifications for Instrument Design-7)

Even when the same suspension is usedfor calibration of differentnephelompters, differences'in physical design of the turbidimeters willcause differences-in measured values for the turbidity of the same sample.To minimize such differences, the following design variables have beenspecified by the U. S. Environmental Protection Agency.

1. Defined Specifications

a. Light Source

Tungsten lamp operated at not less than 85% of rated voltageand at not more than ratedcoltage.

b. DistanCe Traveled by Light

The total of the distance traversed by the incident light -plus.scattered light within the sample tube hould not exceed 10 cm.

. Angle of Light Acceptance of the DetectorIc.

' Detector centered at 90° to the incident light path and not toexceed-4:10 ° from 90°.

,,

(Ninety degree scatter is specified because the amount of scattervaries with size of particles at different scatter angles).

d. Applicable 'Range

. The maximum-turbidity-to be measured-is 40 units,. Several rangeswill be necessary to obtain adequate coverage. Use dilution'forsamples if their turbidityexceeds 40 units.

2. Other EPA Design Specifications

a. Stray Light416

Minimal stray 1. ht thould reach the photocell(s) in the absenceof turbtdity.'

4

Some causes of stray light reaching the photocell(0- are:

1) Scratches or, imperfections in glass cell windows.

2) Dirt, film or condensation on the glass.

3),-Light leakages in the'instrument system.

A schematic of these causes is shown in Figure 3.

Meter

Light LebkagefroM Lens System

Lamp - Lens

N

11

UKIIMMIPAMAMMEIMUMMEMMV\V/

LightScatterty glass tube(Top View)

.Figure 3 t*EPKI.O METER.

.. 'SOURCES OF STRAY LIGHT. ,

Stray light error can be as much as 0.5 NTU., Remedies are ..-close inspection of sample cells for imperfections and dirt,and good design°which can minimize the effect of stray lightby controlling the angle at which it reaches the sampq.

0

. 1

Photocell(s)

Light Leakage fromTransmitted Light

R as

bt. Drift

The turbidimeter should be free from significant drift after a

short warmzup period. This is imperative if the inafirst.is

- relying -on a manufacturer's solid scattering standard for settingoverall instrument sensitivity for all ranges.

.

c. Sensitivity

In waters having turbidities less than 'one unit, the instrumentshoulddetect turbidity differences of 0.02 unit dr less.Several ranges will be necessary to obtain sufficient,sensitivity

. for low turbidities......--ph

3. ;141es of instruments meeting the speCifications lied in 1 and 2

nor include:

,a.. Hach" 2100 and 2100A.

b. Hydroflow Instruments DRT 100, 200, and 1000%

J.

156 16-5 , .

0

16.6

4. Other turbidimeters meeting the listed specifications are also

acceptable. A

D. Sources of Error

CElls

a. Discard Scratched or etched cells.

b. Do not touch cells where light strikes them in instrument.

c. Keep cells scrupulously clean, inside and out.(a)

ly Use detergent solution.

2) Organic solvents may also be used,

`3) Use deionized water rinses.

4). Rinse and dry with alcohol or acetone.

2. Standdrdizing.SuspensionsP)

a. Use turbidity - free water for pre arations. Filter distilled

water through a O.45um pore size me rane filter if such filtered

water shows a lower .turbidity than t distilled water..

b. Prepare a new stock suspension of Formazin each month.

. c. Prepare a new standard suspension and dilutions of Formazin

each week.

3. Sample Interferences

a. Positive

1) Finely divided air bubbles

( b. Negative

- 1) Floating debris

4 2) Coarse sediments (settle)

3) Colored dissolved substances(absorb light)

,

5.

E. Reporting-Results (7)

NTU RECORD TO NEAREST

0.0-1.0

1-10

10 -40'

40-100

100-400

- 400-1000 '

0.05

0.1

1

5

10

50

>1000

F. Precision and Accuracy(7)

1. In a single laboratory-(EMS6, using surface water samples atlevels of 26, 41', 75 and180 NTW, the standard diAations were+O. ; +0:94, +1.2 and +4.7 units, respectively.

Accuracy data is not available at this time.

V. STANDARD SUSPENSIONS AND RELATEDUNITS(9').

One of the critical problems in measuring turbidity has been to find amaterial which can.be made into a reproducible suspension with uniform sized

particles. Various materials have been used.

A. Natural Materials

1. Diatomaceous earth

2. Fuller's earth,'

3. Kaolin

4. Naturally turbid waters.

Such suspensions are not suitable as reproducible standards becausethere is no way to control the size of the suspended particles.

B. Other materials

'1. Ground glass

2. MicroorgahisMs

3. Barium Sulfate 15.84. Lates spheres

SusVensions of these also provedinadequaid.

Formazin

1. A polymer formed by reacting hydrazine sulfate and hexamethylenete-

tramine sulfate.

. It is more reproducible than previoUsly,used standards. Accuracy

of 1. one percent for replicate solutions has been reported.

3. In 1958:0 the Association of Analyti cal Chemists initiated a standard-

ized system of turbidity measurements for the brewing industry by:

; a. Defining a standard formula for making stock Formazin solutions

and

b, Designating a unit of measurement based on Formazin, i.e., the

Formazin Turbidity Unit (FTU).

4. During the 1960's Formazin was increasingly used for water quality_

turbidity testing. It is the currently recognized standard for

compliance turbidity measurements.

D. Units

1. At first results were translated into Jackson Turbidity Units (JTU).However, the JTU was derived from a visual measurement using con,centrationi-(Mg/liter) of silica suspensions prepared by Jackson.They have no direct relationship to the intensity of light scattered

at 90,degrees in a nephelometer.

2. For a few years, res ults of nephelometric measurements using' specifiedForMazin standards were reported directly as Turbidity Units (TUs).

3. Currently, the unit used is named according to the 'used for -

measuring turbidity. SpeWied Formazin standards-are used to calibrate

the instrument and results are reported as Nephelometric Turbidity

Units,(NTUs).

VI. SUMMARY1

i4 iThe importance of residual,chlorine determination' can be seen n ts,poSsible

effect on .the health of the consumers. The Criteria and Procedures for '

Laboratory Certification suggests that some form of quality 'assurance should .

be instituted on a state level to assure valid data foi= both the chlorine and

turbidity. measurements. The comments on the pu&lic respows to the proposedInterim Primary Regulations also suggests some form of quality assurance on

the state level lo-be instituted. Consequently, the Regional Certification team

should point out to the principal laboratories the importance of some kind of

effort being instituted. States might wish to offer some kind of formal

training effort as' part of the approval mechanism for the operators doing

the chlorine and/or turbidity measurements. .

6'8

1,0

159 .

.1

314****************AO3k - ERIC

Documents

Transcript of 314****************AO3k - ERIC

314**********************AO******3k - ERIC

Documents

Transcript of 314**********************AO******3k - ERIC

314****************AO3k - ERIC

Transcript of 314****************AO3k - ERIC