1948, 56(2):157. J. Bacteriol. 

Albert Kelner Antibiotic ActivityMicrobial Populations for A Method for Investigating Large

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A METHOD FOR INVESTIGATING LARGE MICROBIALPOPULATIONS FOR ANTIBIOTIC ACTIVITY'

ALBERT KELNER2.8Laboratories of the Medical School, University of Pennsylvania, Philadelphia, Pennsylvania

Received for publication March 31, 1948

In their search for new antibiotics, investigators throughout the world haveisolated from nature and tested thousands of microbial strains. Neverthelessonly a few useful antibiotics have been found and, from this, it is obvious thatonly a minute fraction of the microflora of nature produce desirable antibiotics.A method that would drastically increase the number of strains screened withoutproportionate increase in labor would improve the chances of finding new anti-biotics. It would also be useful for the selection of high-yielding variants in apure culture of an antibiotic producer.The most serious drawback of earlier methods for testing large populations

was that they detected only certain definite types of antagonists. Thus the"crowded plate" method (Waksman, 1945) detected only soil organisms inhibit-ing other soil organisms and producing an antibiotic after not more than one ortwo days of incubation. Foster and Woodruff's method (1946) detected onlyantagonists that produced antibiotics on media supporting the growth of sapro-phytic mycobacteria and that inhibited such bacteria. The development(Williston et al., 1947) of various media on which both pathogenic tubercle bacilliand antagonists will grow further extends the usefulness of Foster and Woodruff'smethod (1946) but does not remove its fundamental limitations.

Stansly (1947) and Wilska (1947) described methods for spraying sensitivebacteria onto plates on which colonies of soil organisms had grown for severaldays. It was thus possible to test antibiotic action against a greater variety ofbacteria than was possible before. Spray methods are still, however, restrictedto media on which both antagonistic colonies and sensitive bacteria can grow.In addition, there is the ever-present possibility that inhibition zones would becaused by the exhaustion of nutrients around a colony, or by an inhibitory pH,rather than by true antibiotic production. The latter objection applies also tomethods that add test bacteria by flooding the surface of the plate with a liquidsuspension of sensitive organisms.Fleming (1942) suggested coating a plate on which an antagonist has grown

with a layer of sterile agar, on which a sensitive bacterium could be streaked.He sought to test individual isolates, and did not describe methods for testinglarge populations. With Fleming's agar layer suggestion as a basis, a method hasbeen developed whereby large populations can be tested for many types of

1 This work was aided by a grant from Smith, Kline, and French Laboratories.2 Present address: The Biological Laboratory, Cold Spring Harbor, Long Island, New

York.8 With the technical assistance of Betty Morgan.

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antagonists capable of inhibiting various microorganisms, or even antagonistsinhibiting nonmicrobial organisms.

EXPERIMENTAL PROCEDURES

The material serving as the source of antagonists is plated on agar so as to have30 to 50 evenly distributed isolated colonies. After suitable incubation thecolonies are covered with a layer of sterile agar, followed by a layer of agar con-taining the organism whose inhibition is to be tested. After further incubationclear zones over certain colonies disclose the antagonists. The addition of agarlayers is a tecbnique remarkably well adapted to replication, and if each step inthe procedure is made simple enough, 200 to 1,000 plates can be prepared in afew hours by two workers. From 6,000 to 50,000 colonies are screened in anafternoon.The agar layers, in the order to which they are added to the plate, are: (1)

Foundation layer: a layer of about 15 ml sterile agar medium, of a compositionsuitable for the growth of the antagonist; e.g., peptone beef extract, corn meal,Czapek, wort agar, etc. (2) Seeding layer: a layer of 0.5 ml soft agar medium(0.25 per cent agar), in which is diluted the microflora to be tested. The dilutionis calculated to give 30 to 50 colonies per plate.. The layer of soft agar soon driesdown to such a thinness that all colonies are surface colonies. If it is not neces-sary to have all the colonies absolutely on the surface, 5 ml of a nutrient agarcontaining 1.5 per cent agar can be substituted for the softer medium; this is evenbetter adapted for mass replication. (3) Diffusion, or barrier, layer: 5 to 10 mlsterile agar of special composition, whose use is illustrated in problem 3. (4)Test layer: 3 or more ml of agar medium (0.5 to 1.5 per cent agar) containing asuspension of bacteria whose inhibition is to be tested.The actual working out of the method and its flexibility will be illustrated by

describing its use in four diverse problems in antibiotic screening.Problem 1. To test soil for antagonists against Escherichia coli-the antago-

nists in the soil to grow on nutrient agar. A suspension of soil in distilled waterwas centrifuged lightly to remove coarse particles. The supernatant was asayedon nutrient agar for viable cells, then stored at 5 C. The colony count wasdetermined after incubation for 2 to 4 days at 28 C. Foundation layers of 15 mlnutrient agar were added to 200 plates. Since it was not imperative to have thesoil colonies strictly surface colonies, a seed layer of 5 ml nutrient agar (1.5 percent agar) was used. The soil suspension was diluted so that 5 ml contained 50viable cells. The last step in the dilution was made into 500-ml portions ofmelted nutrient agar, kept in a 45 C water bath. Five-ml seed layers werepipetted onto the foundation layer, and the plates were then incubated at roomtemperature for 1 week. There were now 200 plates containing about 50 colonieseach, or a total of 10,000 colonies.The diffusion layer was omitted, and a test layer was added directly over the

soil colonies. To prepare the test layer, 20 ml of a 24-hour broth culture ofEscherichia coli were added to 1,000 ml nutrient crystal violet agar kept in a 45 0water bath. Five ml of this material were pipetted to each of 200 plates to form

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ANTIBIOTIC ACTIVITY IN MICROBIAL POPULATIONS

the test layer. The small concentration of crystal violet, 1:750,000, preventedsoil fungi, actinomycetes, and gram-positive bacteria from growing up into thetest layer, but it did not appreciably inhibit the growth of E. coli. After incu-bation at 37 C overnight numerous clear zones were found (figure 1).

Problem 2. To test soil for antagonists against Staphylococcus aureus theantagonists in the soil to grow on corn meal agar. A suspension of soil wvas pre-pared as described for problem 1 and assayed on corn meal agar. Use of thismedium would promote the growth of colonies that may have been suppressedor overgrown on nutrient agar, and, in addition, a different nutritive medium forantibiotic production would probably reveal antagonists missed on nutrient agar.The foundation and seed-layer medium was corn meal agar. Twenty ml of a24-hour broth culture of S. aureus w-ere added to 1,000 ml melted nutrient agar at

Figure 1 Typical plates of soil organisms growin1on nutrient agar and tested by theaddition of a test layer of crystal violet nutrient agar containing Escherichia coti. Left:Plate showing large inhibitioni zone. In most cases the active colony is the, one at themathematical center of the zone. Right: Typical plate with no active colonies.

45 C, and test layers of 5 ml each were added to the plates. Since crystal violetinhibits S. aureus it could not be uised; howiever, by incuibating the test layers for6 hours instead of over-night, the soil colonies did not grow up into the test layerto any appreciable extent, whereas the S. aureits colonies in this period grewsufficiently to make inhibition zones clearly visible. It was convenient to addtest layers in the afternoon, store the plates overnight at 5 C, then incubate at37 C for 6 to 8 hours. This experiment illustrates the use of different media inthe foundation and test layers.

Problem 3. To study the variation in antibiotic activity of strains of a puire cultutreof an actinomycete (A-13) which was a good antibiotic producer. If the spores ofactinomycete A-13 were plated on nutrient agar and tested against E. coli, thesize of the zones of inhibition around individual colonies could be taken as a meas-ure of the antibiotic activity of the organisms in each colony. Those colonieswith exceptionally large zones of inhibition could be isolated for further study.

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A preliminary experiment showed (1) that all colonies had to be on the surface,for even partly buried colonies had distinctly smaller zones than the surface colo-nies, and (2) when a plate contained more than 2 or 3 colonies the large (60-mmradius) inhibition zones overlapped, making impossible the comparison of zonesize.The use of a 0.5-ml seed layer of soft (0.25 per cent agar) nutrient agar readily

ensured surface colonies. The spores were diluted in the melted agar kept at45 C in a water bath. Aided by tilting and shaking of the plates, the 0.5-mlinoculum spread easily, especially over freshly poured, solidified but still warmfoundation layers. Two people working as a team could inoculate 200 to 300plates in an hour.The large zones of inhibition caused a more serious difficulty, for unless plates

containing 30 to 50 colonies could be tested, the aim of testing large populationswas defeated.

Smaller average zone size might be obtained by (1) using a medium less favor-able for antibiotic production than nutrient agar, (2) growing all of the coloniessubsurface, or (3) testing the colonies after only 2 or 3 days of incubation, beforethe peak of activity was reached. Such measures were discarded because it wasfeared that suboptimum conditions for antibiotic production would result in anonsignificant increase in variability of zone sizes.The use of organisms more resistant to the antibiotic than E. coli might solve

the problem. Serratia marcescens, Eberthella typhosa, Alcaligenes faecalis, Esch-erichia communior, and Bacillus mycoides were tried, but none gave suitable smallzones. Continued research with other bacterial species might have disclosedstrains more suitable than those tried. A simple physical method was dis-covered, however, by which one could at will decrease the zone size to any desireddegree. A 10-ml sterile diffusion layer was interposed between the A-13 coloniesand the test layer. If the diffusion layer was simple nutrient agar, the additionaldistance the antibiotic had to diffuse resulted in a decrease in zone size of a fewmillimeters at most. But incorporation into the diffusion layer of substances thatwould partially adsorb the antibiotic would result in a sharp decrease in zonesize. Several adsorbents were tried, and decolorizing carbon (norit A) was foundsatisfactory. The zone size varied inversely with the concentration of norit Ain the diffusion layer (table 1).

The decrease in zone size varied from experiment to experiment, and in actualpractice 0.75 per cent norit A in a 10-ml diffusion layer of nutrient agar gavesatisfactorily small zones. Norit A was added to the nutrient agar before auto-claving. Since the heavy powder settled rapidly in melted agar, it was necessaryto shake the suspension just before use. The use of a diffusion layer as outlinedis capable of widespread application, since most antibiotics can be adsorbed withone substance or another.The procedure finally adopted for study of this active actinomycete A-13, was

as follows: A suspension of conidia of A-13 was filtered six times through absorb-ent cotton, assayed on nutrient agar, and then stored in the refrigerator until the

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assay colonies had grown. To petri dishes were then added foundation layersof nutrient agar, and 0.5 ml of soft (0.25 per cent agar) nutrient agar suspensionof conidia calculated to contain 40 viable spores per 0.5 ml. The plates wereincubated at 28 C for 5 to 7 days. A diffusion layer of 10 ml nutrient agar con-taining 0.75 per cent norit A was added, followed by a test layer of 3 ml nutrientagar (0.5 per cent agar) containing 1 to 2 ml of 24-hour broth culture of E. coliper 100 ml agar. After incubation overnight at 37 C, zone sizes of the actino-mycete colonies were measured.Problem 4. To test soil for antagonists against a green plant. By substituting

small plant seeds for the bacteria in the test layer, the agar overlay methodmight be used for detecting microorganisms inhibiting (or stimulating, or affect-ing in another specific way) germinating seeds. The inhibition of germinatingseeds by javanicin and oxyjavanicin (Anstein et al., 1946), polyporin (Bose et at.,1948), and penicillin (Ribeiro, 1946; Smith, 1946) has been reported.

TABLE 1Effect of norit A on zone size

CONCENRAION OF NORIT A IN DIPMSION LAYZR AVERAGE ZONE SIZE (RADIUS)

% N

0 540.3 250.5 180.7 160.8 131.0 8

The several days necessary for seed germination (as compared to the 6- to18-hour incubation period for bacteria) complicated the method by allowing timefor soil organisms to grow up into the test layer; and no simple means for testinglarge populations was found. Nevertheless, the success obtained by the follow-ing procedures illustrates the adaptability of the method.

Sterile filter paper was placed in the bottom of petri dishes, followed by a 20-mlfoundation layer of corn meal agar, and a 3-ml seed layer of a soil suspensiondiluted in corn meal agar to give 40 colonies per plate. The plates were incu-bated at room temperature for 1 week. Before adding the seeds, the entire agarlayer was carefully overturned into a sterile petri dish, so that the underside ofthe foundation layer was now on top and the soil colonies on the bottom. Ithelped to use a spatula inserted between the petri dish rim and the agar layer,and under the filter paper. By peeling off the filter paper a clean sterile surfacewas uncovered on which to add the test layer, with lessened danger of contamina-tion by soil organisms.Redtop grass seeds (Agrostis alba) were surface-sterilized by being soaked in a

solution of sodium hypochlorite for 10 minutes, then washed in sterile distilledwater. The test layer medium consisted of 7 ml of half-strength Shive solution(Miller, 1931) in melted agar at 45 C, to which had been added about 1 part in 5

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of the seeds. The test layer was pipetted onto the foundation layer, and theseeds were spread uniformly over the surface by gentle tilting of the plate. Theplates were incubated in diffuse light at room temperature for 3 days. Despitesome contamination around the edge of the plates, most of the seeds germinatedcleanly. Clear-cut circular zones of inhibition were seen around some soilcolonies.

SUMMARY

A general method is described whereby large numbers of microbial coloniesfrom diverse sources can be tested for antibiotic activity. Use of the method isillustrated by descriptions of experiments in which (1) a soil flora is tested forstrains producing antibiotics against Escherichia coli, or Staphylococcus aureus;(2) a pure culture of a highly active actinomycete is tested for variants withincreased activity; and (3) a soil flora is tested for strains inhibiting the germina-tion of redtop seeds.

REFERENCESARNSTEIN, H. R. V., COOK, A. H., AND LACEY, M. S. 1946 Production of antibiotics by

fungi. II. Production by Fusarium javanicum and other Fusaria. Brit. J. Exptl.Path., 27, 349-355.

BOSE, S. R., BOSE, A. B., AND DEY, K. L. 1948 Effect of crude polyporin on seed germi-nation and root growth: a preliminary study. Science, 107, 63.

FLEMING, A. 1942 In vitro tests of penicillin potency. Lancet, I, 732-733.FOSTER, J. W., AND WOODRUFF, H. B. 1946 Bacillin, a new antibiotic substance from a

soil isolate of Bacillus subtilis. J. Bact., 51, 363-369.MILLER, E. C. 1931 Plant physiology. McGraw-Hill Book Co., New York.RIBEIRO, D. F. 1946 Penicillin action on germination of seeds. Science, 104, 18.SMITH, W. J. 1946 Effect of penicillin on seed germination. Science, 104, 411-412.STANSLY, P. G. 1947 A bacterial spray apparatus useful in searching for antibiotic-pro-

ducing microorganisms. J. Bact., 54, 443-446.WAKSMAN, S. A. 1945 Microbial antagonisms and antibiotic substances. The Common-

wealth Fund, New York.WILLISTON, E. H., ZIA-WALRATH, P., AND YoumANs, G. P. 1947 Plate methods for

testing antibiotic activity of actinomycetes against virulent human type tuberclebacilli. J. Bact., 54, 563-568.

WILSKA, A. 1947 Spray inoculation of plates in the detection of antagonistic microor-ganisms. J. Gen. Microbiol., 1, 368.

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