Analisis Air ( teknik lingkungan) ist akprind yogyakarta
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Transcript of Analisis Air ( teknik lingkungan) ist akprind yogyakarta
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ANALISIS AIR DANAUOleh
Sri SunarsihJurusan Teknik Lingkungan, Fak Sains Terapan,
IST AKPRIND Yogyakarta
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Alkalinitas dan kesadahan Alkalinitas: ukuran kemampuan air untuk menetralkan
asam kuat (menerima dan dinetralkan oleh proton) Dinyatakan dalam mg CaCO3 per liter mikroekivalen Alkalinitas air alamiah berkisar 20 - 200 mg/L
Dalam air alamiah yang dominan bikarbonat dan karbonat Kesadahan : Ukuran total konsentrasi ion Ca dan Mg
Dinyatakan dalam mg CaCO3 per liter Sebenarnya ion Ca dan Mg diperlukan untuk pertumbuhan
normal dan daya tahan tumbuhan dan binatang. Kesadahan dapat mempengaruhi toleransi ikan terhadap
logam toksik
Alkalinitas: ukuran kemampuan air untuk menetralkanasam kuat (menerima dan dinetralkan oleh proton) Dinyatakan dalam mg CaCO3 per liter mikroekivalen Alkalinitas air alamiah berkisar 20 - 200 mg/L
Dalam air alamiah yang dominan bikarbonat dan karbonat Kesadahan : Ukuran total konsentrasi ion Ca dan Mg
Dinyatakan dalam mg CaCO3 per liter Sebenarnya ion Ca dan Mg diperlukan untuk pertumbuhan
normal dan daya tahan tumbuhan dan binatang. Kesadahan dapat mempengaruhi toleransi ikan terhadap
logam toksik
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Analisis Alkalinitas pH meter Buret* Termometer Pengaduk Magnetik
dan stirer Top loading balance
pH meter Buret* Termometer Pengaduk Magnetik
dan stirer Top loading balance
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Analisis Alkalinitas Reagen
0.04 N H2SO4 Analisis total alkalinitas dengan titrasi sampai sampel
mencapai pH tertentu (disebut titik akhir titrasi) Pada pH tersebut, semua senyawa basa dalam sampel
sudah habis Jumlah asam yang digunakan sesuai dengan
alkalinitas total sampel Hasilnya dinyatakan dalam milligram per liter kalsium
karbonat (mg/L CaCO3) Juga dapat dinyatakan dalam milliekuivalen dengan
dibagi 50
Reagen 0.04 N H2SO4
Analisis total alkalinitas dengan titrasi sampai sampelmencapai pH tertentu (disebut titik akhir titrasi)
Pada pH tersebut, semua senyawa basa dalam sampelsudah habis
Jumlah asam yang digunakan sesuai denganalkalinitas total sampel
Hasilnya dinyatakan dalam milligram per liter kalsiumkarbonat (mg/L CaCO3)
Juga dapat dinyatakan dalam milliekuivalen dengandibagi 50
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Analisis Alkalinitas
samplemLNCBLCaCOmgalkalinitytotal 50000)2(/, 3
samplemLNCBLeqalkalinitytotal 999100)2(/,
or
samplemLNCBLeqalkalinitytotal 999100)2(/,
Where:B = mL titrant first recorded pH (i.e., to pH = 4.5)C = total mL titrant to reach pH 0.3 unit lower, andN = normality of acid (titrant)
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Analisis kesadahan Idealnya kesadahan ditentukan dengan perhitungan secara
terpisah antara kalsium dan magnesium.
Satuan kesadahan adalah mg CaCO3/L][118.4][497.2 MgCa
Dengan Ca dan Mg dalam mg/L
][118.4][497.2 MgCa
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Analisis alkalinitas dan kesadahanAda kit tes titrasi yangtersedia untukalkalinitas dan kesadahan
Ada kit tes titrasi yangtersedia untukalkalinitas dan kesadahan
www.hach.com
www.lamotte.com
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ANALISIS NUTRIEN Kolorimetri & spektrofotometri nitrogen and phosphorus using
spectrophotometry Specific techniques for students to review
in or out of class included: developing calibration curves QA/QC : standards, spikes, etc
Kolorimetri & spektrofotometri nitrogen and phosphorus using
spectrophotometry Specific techniques for students to review
in or out of class included: developing calibration curves QA/QC : standards, spikes, etc
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Kolorimetri & spektrofotometri
Prinsip:1.Makin tinggi kosentrasi
warna = absorbansimakin tinggi, add a dye that binds
specifically to nutrientof interest
measure the increasein color as anestimate of analyteconcentration
2. Menyiapkan larutankalibrasi standar(konsentrasi sampelharus berada dlmkisaran kalibrasi
3. Membandingknabsorbansi sampeldengan absoebansistandard danmengestimasikonsentrasi sampel
Prinsip:1.Makin tinggi kosentrasi
warna = absorbansimakin tinggi, add a dye that binds
specifically to nutrientof interest
measure the increasein color as anestimate of analyteconcentration
2. Menyiapkan larutankalibrasi standar(konsentrasi sampelharus berada dlmkisaran kalibrasi
3. Membandingknabsorbansi sampeldengan absoebansistandard danmengestimasikonsentrasi sampel
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Kolorimetri & spektrofotometri4. Menambahkan
reagen untukmembentuk warna
5. Membandingkan Menggunakan peta
warna Mengguakan
kolorimeter Menentuan
absorbansinya denganspektrofotometer
4. Menambahkanreagen untukmembentuk warna
5. Membandingkan Menggunakan peta
warna Mengguakan
kolorimeter Menentuan
absorbansinya denganspektrofotometer
rendah.. ke . tinggiKonsentrasi fosfat
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Komparator warna dankolorimetri
Test Kits Ada banyak merek
Images from www.hach.com
Tabung warna Cakram warna Kolorimeter saku
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Instrumen pengukur warna
Hach DR2400 portablespectrophotometer
Bausch & Lombspectrophotometer 20
Hach DR2400 portablespectrophotometer
Bausch & Lombspectrophotometer 20
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Standard Kalibrasi
Standard dibuat dari lrutn stndr yang lebih pekat, diencerkandengan presisi tinggi
Ortho-P:Use dried KH2PO4, K2HPO4,NaH2PO4 or Na2HPO4
NH4-N dan NO3-N:Menggunakan NH4NO3 keringsebagai standar (masing-masing50%)
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Water chemistry 101Procedure: See specific analyses
Reagents are added to eachsample and standardidentically
Mix after each step
Incubate at room temp or inwater bath for 20 min to ~ 2hrs, depending on the analyte
Procedure: See specific analyses
Reagents are added to eachsample and standardidentically
Mix after each step
Incubate at room temp or inwater bath for 20 min to ~ 2hrs, depending on the analyte
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Kurva kalibrasi Standar
NH4-N standards
Garis lurus:A = a + b*c
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Estimasi konsentrasi
maka, jika sampel memilikiabsorbansi 0.290
N
Konsentrasinya kira-kira~ 0.33 ppm N
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#2
Standard curves troubleshooting
Errors in preparing the 0.25 and 0.50ppm standards perhaps ?
Example #1 Live with it or re-run the batch#1
The line becomes non-linear after ABS ~ 1.0 (~
1000 ugN/L)
Example #2 Fit a straight line from 0-1000 and a 2nd line from 1200-2000ugN/L
Use non-linear quadratic instead ofa line for 0-2000 ugN/L
Re-read in smaller cuvette or diluteand re-run
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Some data from northern Minnesotalakes
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0 50 100 150 200 250
ortho-P (ug/L)
Abs
orba
nce
@ 8
80 n
m Calibration curve
= std
ABS = (-0.0010) + (0.00254)* PR2 = 0.9997 n=12
Sample #1 = 11.2 ugP/LSample #1 - Replicate = 12.6 ugP/LSample #1 + 50 Spike = 59.4 ugP/L
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0 50 100 150 200 250
ortho-P (ug/L)
Abs
orba
nce
@ 8
80 n
m
Sample #1 = 11.2 ugP/LSample #1 - Replicate = 12.6 ugP/LSample #1 + 50 Spike = 59.4 ugP/L
% RPD = 100* (1.4)/ 11.9 = 12%% R = 100* (59.4-11.9)/50 = 95%
Conclusion:The data are valid
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ANALISIS TSS DAN KEKERUHAN
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Analisis total padatan tersuspensi1. Air yang volumenya tertentu
disaring dengan kertas saringyang sudah dicuci, dikeringkan(pada 103-105oC), dan ditimbang(~ + 0.5 mg)
2. dibilas, dikeringkan dalam oven,ditimbang dengan neraca analitisuntuk mengukur berat TSS mg/L(ppm)
3. Kertas saring disimpan untukanalisis lain misal padatantersuspensi volatil (VSS) yangmemprediksi senyawa organik
1. Air yang volumenya tertentudisaring dengan kertas saringyang sudah dicuci, dikeringkan(pada 103-105oC), dan ditimbang(~ + 0.5 mg)
2. dibilas, dikeringkan dalam oven,ditimbang dengan neraca analitisuntuk mengukur berat TSS mg/L(ppm)
3. Kertas saring disimpan untukanalisis lain misal padatantersuspensi volatil (VSS) yangmemprediksi senyawa organik
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Analisis total padatan tersuspensiPenyaring jenis apa yang
digunakan?
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Analisis total padatan tersuspensi Jenis penyaring:
Filter membranmenahan partikulat danorganisme sub-mikron
Filter gelas mikrofiber100% terbuat dari gelasborosilikat.
Polikarbonat memilikiukuran pori yang tepattetapi alirannya lambat
Jenis penyaring: Filter membran
menahan partikulat danorganisme sub-mikron
Filter gelas mikrofiber100% terbuat dari gelasborosilikat.
Polikarbonat memilikiukuran pori yang tepattetapi alirannya lambat
www.whatman.com
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Total padatan tersuspensi Ada beberapa set peralatan
Corong yang terikat klem, disekrup, atau dgn magnetic Peralatan plstik yg bermanfaat unt di lapangan
multiple towers
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Peralatan yg diperlukan
oven pengering
Neraca Analitik
Total padtn tersuspensi
oven pengering
Penyaring dan petri dish
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Menghitung TSS:
Total padatan tersuspensi
TSS (mg/L) = ([A-B]*1000)/Cdengan
A = berat akhir kertas saring (mg)B = berat awal kertas saring (mg)C = Volume air yang disaring (Liter)
TSS (mg/L) = ([A-B]*1000)/Cdengan
A = berat akhir kertas saring (mg)B = berat awal kertas saring (mg)C = Volume air yang disaring (Liter)
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Hubungan kekeruhan denganTSS
Aturan umum :1 mg TSS/L ~ 1.0 - 1.5 NTU kekeruhan
BUT hamburan oleh kekeruhan bergantung pada ukuranpartikel sehingga perkiraan tersebut hanya merupakanpendekatan kasar
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Turbidity - meter Kebanyakan menggunakan nefelometrik
optik dan daibaca dlm satuan NTU(nephelometric turbidity units)
Pengukuran kekeruhan dilakukan dengan: Turbidimeter (untuk sampel diskrit) Sensor kekeruhan yg dapat dibenamkan
(USGS menganggap sbg metoda qualitative)
Instrumen Laboratorium : Turbidimeter
Kebanyakan menggunakan nefelometrikoptik dan daibaca dlm satuan NTU(nephelometric turbidity units)
Pengukuran kekeruhan dilakukan dengan: Turbidimeter (untuk sampel diskrit) Sensor kekeruhan yg dapat dibenamkan
(USGS menganggap sbg metoda qualitative)
Instrumen Laboratorium : Turbidimeter
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KekeruhanTurbidimeter
Nefelometrik optik Kekeruhan nefelometrik
ditaksir menggunakan efekhamburan sinar oleh partikeltersuspensi
detektor terletak pada sudut 90oterhadap sumber sinar
http://www.bradwoods.org/eagles/turbidity.htm
TurbidimeterNefelometrik optik
Kekeruhan nefelometrikditaksir menggunakan efekhamburan sinar oleh partikeltersuspensi
detektor terletak pada sudut 90oterhadap sumber sinar
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Satuan Kekeruhan Nephelometric Turbidity
Units (NTU) Standardnya adalah
formazin atau material lainyg tersertifikasi
Satuan JTU berasal dariteknologi yang lebih awalmenggunakan nyala lilin ygdilihat melalui tabung air
1 NTU = 1 JTU (JacksonTurbidity Unit)
Nephelometric TurbidityUnits (NTU)
Standardnya adalahformazin atau material lainyg tersertifikasi
Satuan JTU berasal dariteknologi yang lebih awalmenggunakan nyala lilin ygdilihat melalui tabung air
1 NTU = 1 JTU (JacksonTurbidity Unit)
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Kekeruhan standard formazin
Contoh 1 set standard formazin
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Kekeruhan
Here is a range of NTUs using clay
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Bench and portable instruments and kits vs.Turbidi meters dan probes
Submersible Turbidimeters
YSI wiping turbidityYSI 6820 withunwiped turbidity
Hydrolab
Submersible Turbidimeters
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Turbidity - methods
Comparability of different methods: With the proliferation of automated in situ turbidity
sensors there is concern about the comparabilityof measurements taken using very differentoptical geometries, light sources and lightsensors.
The US Geological Survey and US EnvironmentalProtection Agency are currently (August 2002)developing testing procedures for a fieldcomparison of a number of instruments producedby different manufacturers.
Comparability of different methods: With the proliferation of automated in situ turbidity
sensors there is concern about the comparabilityof measurements taken using very differentoptical geometries, light sources and lightsensors.
The US Geological Survey and US EnvironmentalProtection Agency are currently (August 2002)developing testing procedures for a fieldcomparison of a number of instruments producedby different manufacturers.
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Turbidity - calibration Turbidity free water = zero (0
NTU) standard USGS recommends filtering
either sample water ordeionized water through a 0.2um or smaller filter to removeparticles
WOW uses deionized waterthat is degassed by sparging(bubbling) with helium, tominimize air bubbles that maygive false turbidity readings
Turbidity free water = zero (0NTU) standard USGS recommends filtering
either sample water ordeionized water through a 0.2um or smaller filter to removeparticles
WOW uses deionized waterthat is degassed by sparging(bubbling) with helium, tominimize air bubbles that maygive false turbidity readings
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Turbidity - standards Standards range depends on anticipated sample
values Lakes - typically 0-20 NTU Streams and wetlands - 0-20, 0-50 or 0-100 NTU 2 non-zero standards typically adequate
(response is linear) Types of standards
Formazin particles (either from a recipe orpurchase a certified, concentrated stock solution -usually 4000 NTU)
Other commercially available materials, e.g.,polystyrene
Standards range depends on anticipated samplevalues Lakes - typically 0-20 NTU Streams and wetlands - 0-20, 0-50 or 0-100 NTU 2 non-zero standards typically adequate
(response is linear) Types of standards
Formazin particles (either from a recipe orpurchase a certified, concentrated stock solution -usually 4000 NTU)
Other commercially available materials, e.g.,polystyrene
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Table of standards Prepare daily2 to 20 NTUHach Company
Prepare dailyAll dilutionsStandard Methods(APHA 1995)
Prepare monthly20 to 40 NTU
Suggested holding timesConcentrationsSourceTurbidity standards
Prepare weeklyAll dilutionsEPA Region 5
Prepare dailyAll dilutionsStandard Methods(APHA 1995)
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ANALISIS BOD
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BOD
BOD mengukur jumlah oksigen yangdiperlukn oleh mikroorganisme untukmenguraikan senyawa organik, termasukuntuk mengoksidasi senyawa anorganik
Uji BOD mengukur jumlah oksigen ygdiperlukan selama waktu tertentu(biasanya 5 days at 20o C)
BOD mengukur jumlah oksigen yangdiperlukn oleh mikroorganisme untukmenguraikan senyawa organik, termasukuntuk mengoksidasi senyawa anorganik
Uji BOD mengukur jumlah oksigen ygdiperlukan selama waktu tertentu(biasanya 5 days at 20o C)
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BOD 5 DO is measured initially and again after a
5-day incubation at 20o C BOD is computed from the difference
between initial and final DO The rate of oxygen consumption is
affected by a number of variables: temperature pH the presence of certain kinds of
microorganisms the type of organic and inorganic material in
the water
DO is measured initially and again after a5-day incubation at 20o C BOD is computed from the difference
between initial and final DO The rate of oxygen consumption is
affected by a number of variables: temperature pH the presence of certain kinds of
microorganisms the type of organic and inorganic material in
the water
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BOD - analysis Equipment needed:
Incubation bottles Air incubator or water bath
thermostatically controlled at20 +/- 1o C
DO meter and probe
Equipment needed: Incubation bottles Air incubator or water bath
thermostatically controlled at20 +/- 1o C
DO meter and probe
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BOD Reagents:
Dilution water provides nutrients necessaryfor microorganism growth
Seed a population of microorganismscapable of oxidizing the organic matter in thesample
Commercially available or freeze-driedculture
A conditioned bacteria source (effluentfrom a biological treatment source such asa wastewater treatment plant).
Glucose-glutamic acid standard
Reagents: Dilution water provides nutrients necessary
for microorganism growth Seed a population of microorganisms
capable of oxidizing the organic matter in thesample
Commercially available or freeze-driedculture
A conditioned bacteria source (effluentfrom a biological treatment source such asa wastewater treatment plant).
Glucose-glutamic acid standard
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BOD QA/QC Assure quality with:
Seed control determine the BOD of the seedingsource
Dilution water blank used to check for quality ofunseeded dilution water and incubation bottlecleanliness
Steps to Include: Read and record temperature of incubator Prepare replicate bottles for dilution water blanks and
seed controls Include at least one set of replicate samples per
analysis
Assure quality with: Seed control determine the BOD of the seeding
source Dilution water blank used to check for quality of
unseeded dilution water and incubation bottlecleanliness
Steps to Include: Read and record temperature of incubator Prepare replicate bottles for dilution water blanks and
seed controls Include at least one set of replicate samples per
analysis
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BOD - procedure
Blanks Prepare dilution water, bring to 20o C and
aerate Add sufficient seeding material to produce a
DO uptake of 0.05 to 0.1 mg/L in 5 d (dilutionwater)
Samples Add sample to bottle and dilute. Dilutions should result in a residual DO of at
least 1 mg/L and DO uptake of at least 2 mg/Lafter 5 day incubation
Blanks Prepare dilution water, bring to 20o C and
aerate Add sufficient seeding material to produce a
DO uptake of 0.05 to 0.1 mg/L in 5 d (dilutionwater)
Samples Add sample to bottle and dilute. Dilutions should result in a residual DO of at
least 1 mg/L and DO uptake of at least 2 mg/Lafter 5 day incubation
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BOD procedure Steps in procedure:
Fill bottles with enough dilution water so thestopper displaces all of the air, leaving NO airbubbles
Read initial DO Incubate for 5 days at 20o C Read final DO Calculate BOD5 correcting for the exact duration
Steps in procedure: Fill bottles with enough dilution water so the
stopper displaces all of the air, leaving NO airbubbles
Read initial DO Incubate for 5 days at 20o C Read final DO Calculate BOD5 correcting for the exact duration
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Phytoplankton/Algae countingmethods
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BOD
Calculations When dilution water is not seeded:
When dilution water is seeded:
PDDLmgBOD day 215 )/(
Calculations When dilution water is not seeded:
When dilution water is seeded:
PDDLmgBOD day 215 )/(
PfBBDDLmgBOD day )()()/( 21215
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Algae chlorophyllinstrumentation
Spectrophotometer: Visible with 1-2 nm
bandwidth Matched cuvettes, 1-5
cm
Fluorometer: Requires excitation and
emission filtersspecifically forchlorophyll measurement
Spectrophotometer: Visible with 1-2 nm
bandwidth Matched cuvettes, 1-5
cm
Fluorometer: Requires excitation and
emission filtersspecifically forchlorophyll measurement
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Algae chlorophyll filtration
Apparatus - extraction Prewashed 47 mm glass fiber filters (GF/C,
GF/F, AE, or equivalent) Gelman polycarbonate filtration tower or
equivalent Vacuum pump (5 to 7.5 psi) Centrifuge (clinical) DIW/acetone (90%) washed 15 mL Corex
centrifuge tubes with caps
Apparatus - extraction Prewashed 47 mm glass fiber filters (GF/C,
GF/F, AE, or equivalent) Gelman polycarbonate filtration tower or
equivalent Vacuum pump (5 to 7.5 psi) Centrifuge (clinical) DIW/acetone (90%) washed 15 mL Corex
centrifuge tubes with caps
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Algae chlorophyll filtration(cont.)
Filter a known volume ofwater through a GF/C filter
Volume filtered dependsupon algal density
Add a few drops of saturatedMgCO3 solution near the end
When all the water has beenpulled through, fold the filterinto quarters and wrap in foil
Filter a known volume ofwater through a GF/C filter
Volume filtered dependsupon algal density
Add a few drops of saturatedMgCO3 solution near the end
When all the water has beenpulled through, fold the filterinto quarters and wrap in foil
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Algae chlorophyll storage Wrap the folded filter in a
square of foil, label, thenfreeze
Record the volume filtered,date, site, depth, replicate# all with permanentmarker
Store the filter in thefreezer at < 20o C
EPA holding time for afrozen chlorophyll filter is 2weeks
Wrap the folded filter in asquare of foil, label, thenfreeze
Record the volume filtered,date, site, depth, replicate# all with permanentmarker
Store the filter in thefreezer at < 20o C
EPA holding time for afrozen chlorophyll filter is 2weeks
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Algae chlorophyll extraction &analysis
Chlorophyll extraction: Tear filter into several pieces Place in a test tube Add 10 mLs of 90% acetone Extract overnight at 4oC
Chlorophyll analysis: After 18-24 hr extraction,
centrifuge to settle filter debris Read absorbance or
fluorescence of the supernatant
Chlorophyll extraction: Tear filter into several pieces Place in a test tube Add 10 mLs of 90% acetone Extract overnight at 4oC
Chlorophyll analysis: After 18-24 hr extraction,
centrifuge to settle filter debris Read absorbance or
fluorescence of the supernatant
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Algae chlorophyllmeasurement
Measure absorbance of a 90% acetone solutionblank at 750 nm and at 664 nm to correct forprimary pigment absorbance
Record sample absorbance at 750 nm and 664nm
Estimate phaeophytin by acidifying the sample.Record the absorbance at 665 nm and again at750 nm
Run working standard solutions of purifiedchlorophyll-a (Sigma Chemical Co. Anacystisnidulans by the procedure used for the blank)
Measure absorbance of a 90% acetone solutionblank at 750 nm and at 664 nm to correct forprimary pigment absorbance
Record sample absorbance at 750 nm and 664nm
Estimate phaeophytin by acidifying the sample.Record the absorbance at 665 nm and again at750 nm
Run working standard solutions of purifiedchlorophyll-a (Sigma Chemical Co. Anacystisnidulans by the procedure used for the blank)
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Algae chlorophyll andphaeophytin
What is phaeophytin? Degradation product of
chlorophyll Absorbance wavelength
(665 nm) is very close tothat of chlorophyll (664nm)
acid
What is phaeophytin? Degradation product of
chlorophyll Absorbance wavelength
(665 nm) is very close tothat of chlorophyll (664nm)
H
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Algae spectrophotometrycalculations
LVVEELgalchlorophyl
sample
extab
][7.26)/( 665664
LVVEELgnphaeophyti
sample
extab
]7.1[7.26)/( 664665
Where:b = before acidificationa = after acidificationE664b - [{Abs664b(sample)Abs664b(blank)}-{Abs750b(sample)Abs750b(blank)}]E665a - [{A665a(sample)-Abs665a(blank)}-{Abs750a(sample)-Abs750a(blank)}]Vext = Volume of 90% Acetone used in the extraction (mL)Vsample = Volume of water filtered (L)L = Cuvette path length (cm)
LVVEELgnphaeophyti
sample
extab
]7.1[7.26)/( 664665
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Algae chlorophyll QA
Quality assurance There are no commercial QA check standards Lab replicates are usually not done Essentially, the analysis is a one-shot deal,
you dont get a second chance, so be careful Field replicates should be done every 10
samples Cut filters in half and save one half if nervous
Quality assurance There are no commercial QA check standards Lab replicates are usually not done Essentially, the analysis is a one-shot deal,
you dont get a second chance, so be careful Field replicates should be done every 10
samples Cut filters in half and save one half if nervous
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Algae- counting methods Wet mounts Filter Counting chambers Utermohl
requires an invertedmicroscope (light fromabove)
Sedgewick rafterchamber
Hemocytometer
Wet mounts Filter Counting chambers Utermohl
requires an invertedmicroscope (light fromabove)
Sedgewick rafterchamber
Hemocytometer
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Microscopes capable of magnifications of 100X to 1000X
Algae counting methods
Inverted microscopeCompound microscope Less expensiveinverted microscope
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Algae- taxonomy Use an algal taxonomic key that shows
species from your geographical area
Phytoplankton are continually beingdescribed and re-classified so its essentialfor a good taxonomist to keep current (noteasy by any means)
Its a good idea to take photographs ofslides for cataloging
Use an algal taxonomic key that showsspecies from your geographical area
Phytoplankton are continually beingdescribed and re-classified so its essentialfor a good taxonomist to keep current (noteasy by any means)
Its a good idea to take photographs ofslides for cataloging
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Algae determining biomass Algal biomass (standing crop):
A quantitative estimate of the total mass of livingorganisms within a given area or volume
Biovolume estimates: Identification to genus and species level Calculate cell volume by approximation to
nearest geometrical shape Count cells over a known area of the slide so
cells per unit volume can be determined Chlorophyll
Algal biomass (standing crop): A quantitative estimate of the total mass of living
organisms within a given area or volume Biovolume estimates:
Identification to genus and species level Calculate cell volume by approximation to
nearest geometrical shape Count cells over a known area of the slide so
cells per unit volume can be determined Chlorophyll
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Algae determining biovolume
Taxonomic keys often include questionsabout size
Determining size is basically like using aruler. The standard ruler for a microscope is called
an "ocular micrometer," which is fitted into theeyepiece of your microscope
Taxonomic keys often include questionsabout size
Determining size is basically like using aruler. The standard ruler for a microscope is called
an "ocular micrometer," which is fitted into theeyepiece of your microscope
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Algae determining biovolume
Some formulas to estimate biovolume fromcell dimensions (Wetzel & Likens 2000)
B
A
Rod4/2AB
BA
6/3ASphere
A
Ellipsoid6/2AB
A
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Algae chlorophylldetermination
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Algae chlorophylldetermination
Measuring chlorophyll-a concentrationremains the most common method forestimating algal biomass
Chlorophyll-a concentration has also beenshown generally, when comparing lakes,to relate to primary productivity (Wetzel1983)
Can be used to assess the physiologicalhealth of algae by examining itsdegradation product, phaeophytin
Measuring chlorophyll-a concentrationremains the most common method forestimating algal biomass
Chlorophyll-a concentration has also beenshown generally, when comparing lakes,to relate to primary productivity (Wetzel1983)
Can be used to assess the physiologicalhealth of algae by examining itsdegradation product, phaeophytin
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Algae chlorophyll basics Algal biomass is most commonly
estimated by chlorophyll-a. Units are ug/L or mg/L (ppb and ppm) Detection limit depends upon method used
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Algae chlorophyllmethodology
Spectrophotometry and fluorometry,utilizing 90% acetone extraction, remainthe most commonly used methods
Spectrophotometry is most widely usedbut fluorometry is more sensitive and maybe used when low levels of chlorophyll areanticipated or when handling largevolumes of water is logistically difficult
Spectrophotometry and fluorometry,utilizing 90% acetone extraction, remainthe most commonly used methods
Spectrophotometry is most widely usedbut fluorometry is more sensitive and maybe used when low levels of chlorophyll areanticipated or when handling largevolumes of water is logistically difficult
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Microscopes capable of magnifications of 100X to 1000X
Algae counting methods
Inverted microscopeCompound microscope Less expensiveinverted microscope
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Bacteria 2 indicator methodsTwo basic methods:1. membrane filtration 2. multiple-tube
fermentation
http://www.intelligence.gov/2-community_examples.shtml
http://picturethis.pnl.gov/picturet.nsf/f/uv?open&SMAA-3V9T37
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Bacteria membrane filtertechnique
The fecal coliform MF procedure uses anenriched lactose medium and incubationtemperature of 44.5 0.2o C for selectivity.
Results in 93% accuracy (APHA 1995) indifferentiating between coliforms found in thefeces of warm-blooded animals and thosefrom other environmental sources.
Fecal Coliform is reported as colony formingunits per 100 mL (CFU/100 mL).
The fecal coliform MF procedure uses anenriched lactose medium and incubationtemperature of 44.5 0.2o C for selectivity.
Results in 93% accuracy (APHA 1995) indifferentiating between coliforms found in thefeces of warm-blooded animals and thosefrom other environmental sources.
Fecal Coliform is reported as colony formingunits per 100 mL (CFU/100 mL).
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Bacteria membrane filter equipment Materials needed for MF
method: Air incubator or water
bath Non-corrugated forceps Heat sterilizer (Bacti-
Cinerator) Filter flask and tower
(Autoclavable) Vacuum pump or water
aspirator
Materials needed for MFmethod: Air incubator or water
bath Non-corrugated forceps Heat sterilizer (Bacti-
Cinerator) Filter flask and tower
(Autoclavable) Vacuum pump or water
aspirator
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Bacteria membrane filter equipment MF materials
(continued): Sterile 50 mm petri
plates (with tight-fittinglids)
Sterile 0.45 um griddedmembrane filters
Sterile absorbent pads Autoclave (121o C at
15-17 psi)
MF materials(continued):
Sterile 50 mm petriplates (with tight-fittinglids)
Sterile 0.45 um griddedmembrane filters
Sterile absorbent pads Autoclave (121o C at
15-17 psi)
http://www.nbtc.cornell.edu/biofacility/autoclave.html
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Bacteria membrane filterprocedureProcedure:
Saturate the absorbent pad with M-FC brothSelect a sample volume that will yield 20-60
colonies/filterFilter sample and dilution water through padPlace pad into petri dishInvert plates and place in incubator for 24 hrs
Procedure:Saturate the absorbent pad with M-FC brothSelect a sample volume that will yield 20-60
colonies/filterFilter sample and dilution water through padPlace pad into petri dishInvert plates and place in incubator for 24 hrs
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Bacteria membrane filtercounting
Fecal coliformcolonies bacteria arevarious shades ofblue.
Non-fecal coloniesare gray to creamcolored. normally, few of these
are present.
Fecal coliformcolonies bacteria arevarious shades ofblue.
Non-fecal coloniesare gray to creamcolored. normally, few of these
are present.
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image showing method of countinghttp://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
Bacteria MF counting (cont.)
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MTF image process
Bacteria multiple tubefermentation
http://water.usgs.gov/owq/FieldManual/Chapter7.1/images/Fig7.1-3.gif
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Bacteria cleaning and sterilizingAll equipment Wash equipment thoroughly with dilute nonphosphate, laboratory-grade detergent.
Rinse 3 X with hot tap waterRinse again 3-5 X with deionized or glass-distilled water.
Glass,polypropylene,or Teflonbottles
If sample will contain residual chlorine or other halogens, add Na2S2O3.If sample will contain > 10 ug/L trace elements, add EDTA.Autoclave at 121 C for 15 min or bake glass jars at 170 C for 2 hrs.
Stainless-steelfield units
Flame sterilize with methanol (Millipore Hydrosol units only), or autoclave, orbake at 170 C for 2 hrs
Portablesubmersiblepumps andpump tubing
Autoclavable equipment (preferred): autoclave at 121 C for 15 min.Non-autoclavable equipment:Submerge sampling system in a 200 mg/L laundry bleah solution and circulatesolution through pump and tubing for 30 min; follow with thorough rinsing, insideand out, with sample water pumped from the well. **SEE NOTES
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Bacteria USGS summaryTest (media type) Ideal count range
(colonies per filter)Typical colony color, size, and morphology
Total coliform bacteria(m-Endo)
20-80 Colonies are round, raised and smooth; 1 to 4 mm di; andred with golden-green metallic sheen.
Escherichia coliAfter primary culture astotal coliform colonieson m-Endo (NA-MUG)
None given but muchfewer in number thantotal coliformson the same filter
Colonies are cultured on m-Endo media as total coliformcolonies. After incubation on NA-MUG, colonies have blueflorescent margins with a dark center. Count under a longwave ultra violet lamp in a completely dark room.
Fecal coliform bacteria(m-TEC)
20-60 Colonies are round, raised and smooth with even to lobatemargins; 1 to 6 mm di; light to dark blue in whole or part.Some may have brown or cream colored centers.
Fecal coliform bacteria(m-TEC)
20-60 Colonies are round, raised and smooth with even to lobatemargins; 1 to 6 mm di; light to dark blue in whole or part.Some may have brown or cream colored centers.
Escherichia coli(m-TEC)
20-80 Colonies are round, raised and smooth; 1 to 4 mm di;yellow to yellow brown; many have darker raised centers.
Fecal streptococci(KF media)
20-100 Colonies are small, raised, and spherical; about 0.5 to 3mm di; glossy pink or red in color.
Enterococci(m-E and EIA)
20-60 Colonies are round, smooth and raised; 1 to 6 mm di; pinkto red with a black or red dish brown precipitate onunderside.
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Uneven; not mixedwell; low volume
Fecal coliforms troubleshooting
No matter which assay is used, after incubation there should be ~20-60 colonies evenlydistributed across the Petri dish
poor seal around theedges; poorly seated
with air bubble Dry spot frompoor seating
Uneven; not mixedwell; low volume
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Fecal coliforms troubleshooting(cont.)
Too many useless sample Too few use more sample
Looks good