ecological relationships selecting · geographical distribution, and their ecological relationships...

PROBLEMS IN THE SEARCH FOR MICROORGANISMSPRODUCING ANTIBIOTICS'

J. B. ROUTIEN AND A. C. FINLAY

Biochemical Research Laboratories, Chas. Pfizer and Co., Inc., 11 Bartlett Street, Brooklyn 6,New York

An entirely new field of microbiological research was opened to investigationwhen, approximately ten years ago, the concept of antibiosis found clinicalapplication in the use of penicillin against microbial agents of disease. Thepossibility of discovering new antibiotics varying in their antimicrobial powers,their chemical properties, and their toxicities attracted numerous investigators.Their numbers rapidly increased when several industrial firms with facilities forconducting microbial fermentations organized research groups specifically toseek and characterize new and potentially useful antibiotics.As a result of an increased interest in antibiotics, a rapidly expanding band

of researchers has reported on the discovery of many new antibiotics. In 1951Rouatt et al. (42), for example, stated that since 1940 there have been reportedover 50 antibiotics produced by actinomycetes alone. Baron (3) in 1950 enu-merated 141 antibiotics reported to be produced by various groups of micro-organisms and by higher plants. Florey et al. (16) have compiled long lists ofthe results of tests reported in the literature, and Brian (5) has collected theavailable information on the nearly 100 antibiotics ascribed to fungi. Kareland Roach (23) have recently compiled A Dictionary of Antibiosis consisting ofshort descriptions of antibiotics, organisms producing antibiotics, and technicalterms used in the study of antibiosis. It is interesting to note that they haveextended the definition of "antibiotics" to include products of green plants andanimals. Exhaustive references cover the literature through 1950. Most of thesereports have originated from nonindustrial sources. The industrial laboratories,generally more specifically organized for mass screening, have discarded manymore antibiotics that are weak or that are too toxic for human use.Much valuable information concerning the several aspects of locating, pro-

ducing, and using antibiotics has accumulated. Included are the types of organ-isms capable in either natural or artificial milieux of producing antibiotics, theirgeographical distribution, and their ecological relationships with other micro-organisms; the criteria for selecting the organisms to be tested and the pro-

' At the 51st general meeting of the Society of American Bacteriologists held in Chicago,May 27-31, 1951, the first Commercial Solvents Award in Antibiotics was made. The awardwas for the research by a team of eleven scientists at Chas. Pfizer and Co., Inc. that ledto the discovery of terramycin. The members of this team were the following: A. C. Finlay,G. L. Hobby, S. Y. P'an, P. P. Regna, J. B. Routien, D. B.. Seeley, G. M. Shull, B. A.Sobin, I. A. Solomons, J. W. Vinson, and J. H. Kane. On behalf of this team of researchworkers the authors have written this review to commemorate the award. We gratefullyacknowledge the assistance of Mr. J. W. Vinson for helpful criticism and suggestions inpreparation of the manuscript.

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52 J. B. ROUTIEN AND A. C. FINLAY [VOL. 16

cedures employed; the optimal conditions of growth for inducing high potencies;the theory and application of mutation toward augmented antibiotic production;the chemical isolation and definition of the antibiotic; the complexities of invitro and in vivo evaluation of the antibiotic; and, finally, the mode of action ofthe antibiotic and the pattern of resistance to be anticipated on its clinical use.Since the search for new agents effective against bacterial, fungal, and viralcauses of disease in man, animals, and plants continues undiminished, it wouldseem worthwhile to survey research programs and results. The objective of thispaper will be limited to a discussion of those groups of microorganisms which,judged by their previous performance, can be regarded as potential producersof new antibiotics. By reviewing the methods formulated in research programs,where these are available in published form, and by attempting to evaluate theinformation we possess about the organisms so far studied, we may find that thesedata can suggest more rational approaches in our quest for new antibiotics. Thisreview will not furnish references to all papers on the subject; only enough areused to oriefit our thinking about certain topics.

In designing a research program one of the first problems to arise is the sourceof the cultures to be tested. Existing culture collections represent an immediatesolution. The limited number of species and strains in such repositories, however,obviously makes imperative the initiation of a program of isolating desiredcultures from such natural sources as soil, water, muds, composts, and leaf-litter. It is then necessary to decide whether to test all bacteria, fungi, andactinomycetes or whether to concentrate on one group. Since most reportsdealing with antibiotics useful against human pathogens refer to the actino-mycetes, it follows that most investigators have considered this group to be themost promising. Information about the bacteria and fungi has already beenpresented in the excellent work of Florey et al. (16). Accordingly the data andopinions expressed in the present paper will pertain chiefly to the actinomycetes.

Groups of Organisms Tested

Within the Actinomycetales antibiotic powers have been demonstrated formembers of the genera Streptomyces, Nocardia, and Micromonospora (62). Ofthese, Streptomyces has so far been the most rewarding genus investigated.Recently Waksman et al. (64) have stated that the majority of antibiotic-producing cultures so far known can be included in certain groups of actino-mycetes. These are as follows: S. lavendulae group producing streptothricin;S. griseus group producing streptomycin and others; S. fradiae group producingneomycin and fradicin; S. flavus group producing aureomycin and terramycin;S. albus, S. antibioticus and other groups. In this work, because of the un-satisfactory state of the taxonomy of the genus Streptomyces, the descriptionwith regard to species is intended to be only provisional. It is the authors'belief that no natural groups of species have as yet been set up; until that hasbeen accomplished and the natural relationships indicated, we can speak ofgroups of species only provisionally and of the extent of variability within speciesonly with reference to given cultures deposited in culture collections.


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19521 SEARCH FOR MICROORGANISMS PRODUCING ANTIBIOTICS 53

Attempts have been made to enumerate and classify by various means theactinomycetes isolated from obvious sources. In 1942 Waksman et al. (63) setforth a tabular evaluation of a number of species with regard to activity againstBacillus msbtilis. A group of 16 species was highly bacteriostatic, while a secondgroup of 23 showed moderate activity. The authors pointed out that membersof the genera Proactinomyces and Micromonospora also displayed some anti-biotic power. In his recent book, The Actinomycetes, Waksman (58) lists 60species occurring typically in soils and composts. Although the list includes anumber of actinomycetes that produce well-known antibiotics, it was publishedprior to the announcement of several new, useful ones. The fact that these newantibiotics are produced by new species may, of course, reflect taxonomicdifficulties or divergence of opinion on speciation within this group. On theother hand, it may indicate that in the future the more common species shouldbe avoided.

Various biological characteristics have been used in attempts to distinguishorganisms producing antibiotics from those that are inactive forms. In hisintriguing paper von Plotho (37), working with actinomycetes, correlated pig-ment production with antibiotic activity. Two hundred and ninety-one culturesgrown in colorless media could be classified into four groups on the basis ofpigments produced either in the mycelium or in the medium. Activity wasdetermined by testing against Mycobacterium eos. Of the 61 cultures (21 percent) showing activity, 21 were in the colorless group, 20 were in the red-yellow,12 in the red-blue, and 8 in the red-brown group. By using media in whichpigments could be seen readily, investigators might well try such a correlationto learn whether particular groups could be eliminated without further testing.Furthermore, since only forms within the Penicllium chrysogenum-notatum groupare valuable for penicillin production it is interesting to speculate whethercertain species or groups of species of the actinomycetes or cultures with aparticular color or some other unique biological property would be the mostsuitable for testing.To date there has been published no information dealing with the isolation of

actinomycetes on any basis such as colony color. Presumably, workers eitherisolate at random from the soil plates or select particular types. In some fewsamples one can exercise almost no selection because only a very few differenttypes of actinomycetes will appear. For example, one sample from an ant-hillin Africa yielded almost a pure culture of an organism that looked like Strepto-myces californicus. By far the more common thing, however, is to find a numberof different-appearing cultures, and mention in this paper of numbers of actino-mycetes refers just as much to diversity of organisms as to total numbers ofcolonies. It is logical to assume that workers strive to secure not only a largenumber of cultures but also to select all colonies that appear to be different insome manner from other colonies. The differences would obviously be onlythose that could be seen easily in the petri dish, i.e., differences in the rate ofgrowth, contour of surface of colony, edge of colony, color of vegetative myce-lium, color of any soluble pigment, and color of the spores.


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Sources of Organisms in NatureOrganisms have been isolated from composts, decaying vegetable matter, lake

mud, and other miscellaneous materials. The commonest source, however, hasbeen soil. The choice of soils appears to have been random, some investigatorsusing local soils and others preferring world-wide collections. Waksman et al.(59, 64), for example, cited Texas soils as the source of certain cultures. Duggar(12) stated that the organism producing aureomycin was obtained from atimothy field in Missouri. Ehrlich et al. (14) reported that chloromycetin waselaborated by two isolates: one from near Caracas, Venezuela, and the otherfrom the vicinity of Urbana, Illinois. Chichester (7) reported that E. R. Squibband Sons, because of the difficulty of determining which soils are preferable,isolated organisms from miscellaneous soils. In the process of screening thousandsof soil samples from widely scattered geographical areas we have found organ-isms producing certain antibiotics to be extremely common. Actinomyceteselaborating streptomycin, streptothricin, chloromycetin, actinomycin and xan-thomycin-like antibiotics apparently have a world-wide distribution. Terra-mycin- and aureomycin-producing cultures have been isolated only a few times,and one interesting antibiotic has been observed from only one culture from oneparticular soil. We have found that certain antibiotics are produced by strainsof actinomycetes quite common in soils in certain somewhat localized areas.On the other hand, from some soils collected within a restricted area we havefound a number of different antibiotics. In this connection a paper by Umezawaet al. (52) is of interest. By using the technique reported by Waksman et al. (56)of adding a particular antibiotic (in this case chloromycetin) to the mediumbefore plating out soil samples they were able to demonstrate the presence of arather large number of chloromycetin-producing strains. They found suchstrains to be present in four of the five soils tested. This would seem to indicatea wide distribution of the chloromycetin-producing organism. It should bepointed out, however, that the soil samples were

".... gathered from Yono in Saitama prefecture . ..

and on the basis of our experience we would guess that the sites of collection ofthe samples were not far apart.The presence and function of antibiotics in the soil have provoked study and

speculation. Waksman and Woodruff (62) demonstrated that a substance re-sembling actinomycin, which inhibited the growth of certain bacteria, could beextracted from the soil. Actinomycin added to the soil in quantities muchgreater than needed in artificial medium, however, was ineffective. They pointedout that the a-humus in soils, peats, and composts reduced the activity ofactinomycin even in artificial culture media. A few years later Brian et al. (6)reviewed their study of the fungous flora of the Wareham Heath, Dorset, whereno conifers would grow. Some toxic substance, presumably of biological origin,prevented the growth of mycorrhizal fungi and thereby, apparently, the growthof conifers. They found that the mold flora consisted almost entirely of a num-ber of strains of three species of Penicillium. All isolates of the penicillia when


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1952] SEARCH FOR MICROORGANISMS PRODUCING ANTIBIOTICS 55

grown in liquid media produced gliotoxin which was subsequently found toinhibit mycorrhizal fungi.

In another example, one organism produced in the soil a substance inhibitinganother soil form. Hessayon (20) allowed soil seeded with Trichothecium roseumto incubate for 10 days at 25 C. Quantities of this soil were placed in petridishes and flooded with two layers of agar; the agar was then planted withFusarium oxysporum var. cubense. A decrease of nearly 10 per cent occurred inthe growth of the test organism. With streptomycin, however, Siminoff andGottlieb (46) reported different results. A portion of a certain soil was seededwith a streptomycin-producing strain of S. griseus and another portion with anonstreptomycin-producer, and two portions of the same soil to which tryptonehad been added were likewise seeded. After 17 days' growth B. subtilis wasadded to all soils. Both strains equally depressed the growth of this organism,and the authors concluded that the inhibition obviously was not due to strepto-mycin elaborated in the soil. Studies have shown that streptomycin itself addedto soil was adsorbed onto clays and organic matter and thus rendered inactive.It seemed probable that basic antibiotics like streptomycin, if produced in thesoil, would be inactivated and consequently would have no biological effect. Theprevalence of streptomycin- and streptothricin-producing cultures makes onewonder, however, whether there may not be some survival value in the abilityof cultures to produce them. Or perhaps these antibiotics represent merely thefinal (?) products of some metabolic pathway common in actinomycetes, thesteps of which are, of course, subject to mutation.One may also wonder whether those bacterial cultures in the soil that are

resistant to the antagonistic properties of their neighbors may not represent thedescendants of resistant mutants that developed long ago. Perhaps differentpatterns of resistance, like the penicillin type and streptomycin type of re-sistance, represent "lines" of mutation that occurred in the past. If sensitivestrains of these resistant forms were available, it would be interesting to seewhether resistance would develop to a particular antibiotic in a manner likeresistance to streptomycin or to penicillin and whether the physiologicalproperties causing resistance are the same as in the normal resistant form. Onewonders, further, what factors of the microbial balance in the soil allow thepersistence of those forms that are demonstrably sensitive to antibiotics producedby other soil forms.That cuJtures producing various antibiotics have in some cases widely scattered

geographical distributions and that morphologically distinct species can syn-thesize the same antibiotic lead one to believe that mutation to production ofthese substances must have occurred many times in many places. On the otherhand, mutation to production of other, less common antibiotics must havehappened rarely or recently or, if frequently, in organisms less successful inpropagating themselves. The possibility that new antibiotic-producing strainsmay exist in well-isolated areas will lead to further scouring of the earth'ssurface in exotic places.The types of soils from which cultures may be isolated have been investigated


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by several workers. If the assumption can be made that diversity of antibioticsis directly correlated with numbers of antibiotic-producing cultures, then theinvestigator can ignore soils that are poor in antibiotic producers even thoughthey may be rich in numbers of cultures and thus increase the chances of de-tecting a new antibiotic by a study of soils harboring antibiotic producers.The magnitude of the task of adequately examining soils for desirable micro-

organisms can be envisioned by considering an area such as the continentalUnited States. Any one of the 1,903,000,000 acres in the United States (44)might, theoretically, contain a desirable culture that has arisen by mutation. Inaddition to this, one can point out that certain data from our laboratory showthat the distribution of antibiotic-producing cultures may Gary greatly in areasfar less than an acre in size. Information which would guide an investigator inchoosing one kind of soil in preference to another is scanty, but a few papers areworthy of mention.As early as 1937, Nakhimovskaia (34) compared the antibiotic capacities of

actinomycetes from different soil types in Russia. The validity of her conclu-sions, however, may be limited by the small number of her samples. Of the 80isolates secured, all four strains from a solonetz chernozem produced a solubleantibiotic. Calculations from her data of the other soils yielding organismsproducing diffusible antibiotics give the following percentages: first products ofrock weathering of aPairsoil at an altitude of 3,808 m above sea level, 70;chernozem of the Ukraine, 36; cultivated Pamir soils, 30; podzol near Moscow,27; dry desert Pamir soil 4 m above sea level, 20; sand, 16; river bank, meadow14; products of more advanced weathering of the Pamir soil, 0.Waksman et at. (63) described an experiment involving 244 actinomycetes

isolated at random from certain soils. The cultures were classified as "inactive","moderately active", and "highly active", and the soils were graded accordingto the numbers of cultures obtained from them which showed some degree ofactivity. The most "active" of these was potted soil which had been enrichedwith mixtures of bacteria. The remaining soils were rated as follows in de-creasing order of activity: stable-manure compost; lake mud; potted soil en-riched with Escherichia coli; fertile manured limed soil; infertile manured limedsoil; potted soil.

Landerkin et al. (28) demonstrated that 61.2 per cent of 660 cultures ofactinomycetes isolated from soils from five localities in northern Canada showedfrom slight to strong activity against the eight test organisms used. Devising an"antibiotic index" to give a numerical evaluation to the various soils, theyshowed that the soils from the Yukon River and Dawson sites gave higherantibiotic indices than did the others and that samples obtained six inches belowthe surface had a greater index than the surface inch or 11-inch-deep samples.They presented evidence that the number of antagonistic actinomycetes in therhizosphere of some plants was lower than that in the control soil, and theybelieved that this higher "antibiotic index" of the two soils might have been dueto the sparse vegetation of the collection sites.

Recently Rouatt et al. (42) examined 544 actinomycetes isolated from ten


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different sources. The percentages of cultures showing activity against either thegram-negative or gram-positive bacteria used as test organisms ranged from 17per cent from decayed redwood to 64 per cent from earthworm castings withseveral soils showing 53 to 58 per cent of cultures active. They remarked that agreater percentage of cultures isolated from the rhizosphere displayed activitythan did the cultures isolated from control soil. Only a few of the culturesshowing zones on streak plates displayed activity when grown in liquid media.

In a comparison of a few soils Schatz and Hazen (45) reported that forest soilwas the best source of actinomycetes showing activity against certain humanpathogens used as test organis. A higher percentage of active cultures wassecured by flooding a petri dish containing the growing organism from the soilsample with a fungus-seeded glucose-tryptone agar than by isolating the actino-mycetes at random.Few data are available regarding distribution of antibiotic-producing organ-

isms in bogs or muds from lakes, rivers, or oceans. Waksman (58) found theactinomycetes in water basins to be chiefly of the genus Micromonospora. Hefound a very high percentage of actinomycetes showing antibiotic powers inlake mud and that this group is fairly common in undrained peat bogs in Florida.Perhaps because they are not acidic, sedge and reed bogs commonly containactinomycetes. Commenting on an address by Waksman (57) at the FourthInternational Congress of Microbiology, Ziemi~gcka (57) stated that in Polandmany antagonistic actinomycetes have been found in peaty soils that had beendrained during the war. Of 58 species of marine microorganisms tested againstnonmaine forms by Rosenfeld and ZoBell (41) nine showed antibiotic activity.Many ecological factors must be considered in deciding which soil to choose

for examination. Where in a plot of ground a soil sample should be taken, atwhat depth, at what season, and when in relation to rainfall, temperaturechanges and application of fertilizers may be important. Cover crops may beinfluential, and one may be superior to another. These points are involved intwo general aspects of the distribution of actinomycetes: (a) areas encouragingrapidity of growth of the actinomycetes with concomitant increase in rate ofmutation will favor the formation of new antibiotic-producing cultures; and(b) any peculiarity of metabolism of the actinomycetes may be correlated withsome odd habitat.Many of these factors have been discussed to some extent in The Soil and the

Microbe by Waksman and Starkey (60). A few points might be emphasizedfurther.

1. Actinomycetes develop better in soils of low than in soils of high moisturecontent. The observations of Waksman and Starkey were expanded by Jensen(21) in a series of experiments in which, using the contact-slide technique, heshowed that growth and sporuIation of actinomycetes were greater when thesoil moisture content was 18 per cent of capacity than when it was higher. Hereferred to Cholodny's demonstration of this same fact and von Plotho's findingthat sporulation in this group is favored by a dry atmosphere.

2. Jensen (-1 ) showed that the optimum temperature for vegetative growth


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of the actinomycetes was 28-37 C, with less growth above 37 C, while bacteriaand fungi were more numerous at a temperature range of 5-15 C.

3. Neutral to alkaline media favor the growth of actinomycetes according toWaksman and Starkey (60). In a survey of the alkaline and soils of Bikini andRongelap Islands (pH 8-9.3) Johnstone (22) found the microbial population tobe chiefly actinomycetes; occasionally 95 per cent of the organisms were repre-sentatives of this group. The authors have found tremendous numbers of actino-mycetes in several soil samples obtained from limestone cliffs. Of special interestis the observation of Cooper and Chilton (9), in their studies of actinomycetesin soil active against Pythium arrhenomanes, that the average antagonistic levelof the actinomycete population increased with the pH value of the soil until apH of 7.5 was reached.

4. Food supply clearly influences the number of actinomycetes present in asample. Conn (8) has mentioned, for instance, that a larger number of actino-mycetes was associated with old sods than with new.

5. Rainfall appears to exert an interesting influence. Cooper and Chilton (9)found that while rainfall during a two-week period increased the numbers ofactinomycetes, as detected in plate counts, the isolated cultures showed nogreater antibiotic powers than those previously isolated.

6. It is commonly believed that the numbers of actinomycetes increaserelative to other organisms with increasing depth of soil sampling. Warcup (68),however, has shown recently that in five dry, sandy, grassland soils the actino-mycetes were dominant in the upper levels in three soils but that in the othertwo they were much more numerous in the B horizon, where the soil has a pHof 6-7, a low humus and a low moisture content. This B horizon was 5-11inches deep in one soil and 12-28 inches deep in the other. In a paper mentionedearlier, Landerkin et al. (28) stated that in soils from northern Canada thegeneral level of antibiotic activity of the cultures isolated at the 6 inch depthwas greater than that of cultures obtained at either 1 or 11 inch depths.

7. Chemical treatment of the soil can seriously modify the microbial popula-tion. For example, Warren et al. (69) recently reported that spraying plants inpotted soil with 2,4-D reduced the number of actinomycetes as compared withthe control-pot population. A large number of colonies of an actinomycete notdetected before treatment was isolated from the "treated" soil, and this culturepossessed antifungal properties.

8. Cover crops affect the number of actinomycetes showing antibiotic prop-erties in the soils beneath them. Lochhead and Landerkin (29), for example,have reported that the number of actinomycetes per gram of dry soil was greaterin samples from the potato rhizosphere from untreated soil than from treatedsoil but that a greater percentage of cultures antagonistic to Streptomyce8scabies and to two human pathogens was obtained in samples from the potatorhizosphere of soil having a soybean cover crop.

9. The microbial population of a soil is subject to continuous variation. Thevalidity of the results of the paper just cited (29) depends, consequently, uponthe degree to which the sampling from the two fields revealed a true picture of


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the general actinomycete populations. Pointing out that the microbial popula-tion of a soil could change even in a few days, Waksman and Starkey (60) statedthat distribution of organisms is not uniform and that pronounced differencesmight occur in the numbers of microorganisms in samples taken just a few inchesapart. Although these remarks seem to refer chiefly to bacteria, they raisequestions that should be kept in mind in attempting to choose the superiorsamples of soil for isolating actinomycetes and fungi.The increasing interest in the "rhizosphere effect" of various plants has been

reflected in the reviews by Harley (19) and by Katznelson et al. (24). Althoughthe effect varies with different species of plants and with different soils, the totalnumbers of bacteria are generally much greater in samples in contact with theroots of plants than in the control soil distant from the roots. The bacteria mostaffected appear to be those with special requirements for amino acids and/orvitamins. Harley (19) stated that fungi and actinomycetes were little influencedby the rhizosphere. Katznelson and Richardson (25), however, were able todemonstrate an increased number of actinomycetes, fungi, and bacteria aroundthe roots of tomato plants following sterilization of the soil by certain agents.Formaldehyde treatment, however, reduced the numbers of actinomycetes.Landerkin et al. (28), in interpreting their work referred to earlier, ascribed thehigh "antibiotic index" of Yukon River and Dawson soils to the fact that thesoils were from a region sparsely covered with plants. Some plants, they believed,actually prevent the growth of actinomycetes.Timonin (50) grew plants aseptically so that any root secretions would diffuse

through a collodion membrane into the soil. Microbes near the membrane werestimulated or retarded just as they were in the natural rhizosphere of the plant.Thaysen (49) found the antibiotic-producing microflora of the rhizosphere to bequite active against plant and animal pathogens. He had evidence that some ofthe organisms

". . . are specifically adapted to their habitat and depend for their normal develop-ment on conditions prevailing there, including, perhaps, secretions from the rootsystem itself."

This presumably referred chiefly to bacteria. It should be recalled here that thereare several instances in which the roots of various plants have been shown tosecrete particular chemical substances. For example, West (70) reported thatthiamin and biotin were secreted by roots of flax when they were grown in aliquid solution. McKee (30) mentioned the excretion of ammonia (data ofPrianishnikov) and of sugar, adenylic acid nucleotides, and wheat flavones(data of LundegArdh and Stenlid). Vallance (53), in a study of germination ofseeds of Striga hermonthica, used as the germinating solution a liquid in whichroots of Sorghum vulgare had grown; the stimulative power of such liquid foroptimum germination of seeds of certain angiospermous root parasites has beenknown for some time.Hoping to find a plant the roots of which would secrete some substance en-

hancing the growth of actinomycetes, especially antibiotically active ones, we


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sterilized the surface and germinated on agar the seeds of sweet clover, tomato,eggplant, mangold, cabbage, beet, okra, and radish. After germination theywere placed on the constricted end of a long tube with the roots dangling in theautoclaved nutrient and with only the plant tops exposed to sunlight. Water lostby evaporation was replaced aseptically. After a few weeks of growth the liquidsfrom tubes containing the same species of plant were pooled. Unsterilized soilwas moistened with these pooled liquids, as well as with a control of nutrientliquid, and incubated at 28 C. Sufficient quantities of these liquids were addedwhen necessary to maintain moisture at the desired level. At the end of theincubation period, determinations of the numbers of organisms in each soilper gram of dry weight were made and compared with the determinations madeon each soil before the initiation of treatment. The increase in the numbers ofactinomycetes always was less than the increase in soil containing the controlnutrient solution alone. No stimulation of antibiotically active actinomycetesoccurred, the treated soils had a smaller percentage of active actinomycetesthan did the control soil, and the control soil had a lower percentage of activecultures than did the soil before treatment.The filamentous fungi have been studied so much more than the actinomycetes

that in a recent paper Garrett (17) could discuss where particular groups offungi are most abundant and why they are so common in some places but miss-ing elsewhere. Warcup (67), likewise, has shown that the mycelial zone of certainmushrooms restricts the population of microfungi but that certain genera aremore common there than in the rest of the soil. Further work may enable us tovisualize the distribution of particular groups of actinomycetes as we can nowvisualize to some extent that of the other forms of fungi.

Theoretically it might seem that antagonists against any particular parasiteshould be sought in the habitat of the parasite, and this hypothesis has beensubstantiated by several workers. Nickell and Burkholder (36), for example,isolated from the soil actinomycetes that inhibited Azotobacter; Landerkin andLochhead (27) found actinomycetes in soil active against 12 soil bacteria; andWallhiiusser (65) found that each of 23 bacteria and 16 fungi isolated from thesame soil sample showed antagonism to some extent against many of the others.Cooper and Chilton (9) found that 18.5-31.5 per cent of the 8,302 actinomycetesisolated from 181 sugarcane soil samples showed activity against Pythium ar-rhenomanes, an organism sometimes causing a root-rot of sugar cane. Meredith(32) found actinomycetes with antagonistic properties against Fusarium oxy-sporum var. cubense in soils from banana plots.

Consideration of the concept just discussed but with reference to organismspathogenic to humans might be of importance. Relevant studies might include(a) isolating all orga isms from mixed infections and testing, each for antag-onismn against the primary invader; and (b) isolating organisms from soils orwater frequently contaminated with infectious discharges or bodies of personswho died because of infectious diseases.

Florey et al. (16) quoted from one old case history that suggested possibilitiesof isolating an antibiotic-producing organism from an infection undergoing


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19521 SEARCH FOR MICROORGANISMS PRODUCING ANTIBIOTICS 61

healing. In a recent paper Rountree and Barbour (43) give data that are ofinterest in connection with this problem. They isolated Staphylococcus pyogenes(Micrococcus pyogenes) from the noses of nurses at the time of their first employ-ment at a hospital and at several subsequent times. The strains that were isolatedwere studied in regard to the type of phage capable of lysing them, whetherthey were penicillin-resistant and whether they produced any antibiotic sub-stance. They learned that it was chiefly among those nurses who were notnormally carriers of S. pyogenes that a change in the staphylococcic flora oc-curred. This change was in the direction of a higher rate of penicillin-resistantstrains. They found that once a strain became established it was displaced byanother only rarely. The dominant strains were not dominant, however, byvirtue of producing an antibiotic active against other strains since the produc-tion of antibiotics could be demonstrated in only a small proportion of thecultures that were isolated.The writers on two occasions have attempted to isolate antagonistic or-

ganisms from sources which fit the second category mentioned previously. Soilfrom an orthodox Jewish cemetery, where bodies are interred soon after deathwithout embalming, was examined for antagonists against the pathogens intro-duced into the soil. Soil samples and earthworms from graves being dug ad-jacent to older graves were collected, but the cultures obtained from the soilsand the earthworm guts were disappointingly devoid of interesting antagonists,At another time soil samples were secured along the route of dispersion of thesewage effluent from a sanatorium for tuberculous patients. From the septictank, in operation for many years, fairly clear effluent ran through a systemof branching tile drains downhill to a small stream. Biological activities in thesystem ensured almost complete destruction of the tubercle bacilli. Samples ofthe soil taken along this channel of treatment from the raw sewage to a point50 yards downhill yielded no organism of high antagonistic powers.

Methods of Isolating and Testing CulturesBoth Waksman (55) and Florey et al. (16) have summarized in an excellent

manner the methods available and in common use for the isolation of antibiotic-producing microorganisms from the soil. In addition, Duggar (13) in his reviewemphasized the variable physical and chemical modifications that could beemployed for the isolation of fungi. Methods for isolating bacteria are foundin many texts and papers. Several reports deal with various methods and ad-ditives to the medium used to enhance the isolation of fungi. Warcup (66)claimed excellent results for his method of pouring buffered Czapek-Dox agarof pH 4.0 onto a small quantity of moistened soil and mixing the two; Milleret at. (33) recommended the use of dehydrated bile; Tyner (51) preferred boricacid at the rate of 0.3 g per 100 ml of medium; Smith and Dawson (47) advo-cated the superiority of rose bengal at a concentration of 1 part in 15,000 ofthe medium; while Martin (31) gave a higher rating to rose bengal used at therate of 1 part to 30,000 parts of the medium plus 30 micrograms of streptomycinper ml of medium. Recently Crook et al. (10) reported that sodium propionate


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at 0.4 per cent concentration favored the isolation of actinomycetes by prevent-ing the growth of bacteria and molds.Dubos (11), Woodruff and Foster (71), and others have enriched soil with

bacteria in order to favor the growth of organisms antagonistic to the addedbacteria. Risler (40) removed half the volume from liquid cultures of eitherMycobacterium tuberculosis or E. coli and reconstituted the liquid to the originalvolume with a medium suitable for the growth of aspergilli. Aspergillus Jfavusor A. oryzae was then seeded onto the bacterial culture. Both species producedan antibiotic against the original "host", and the potency of the antibioticcould be increased by serial transfer of the Aspergillus to new cultures of theM. tuberculosis or E. coli.

It would seem impossible to judge which specific method of isolating po-tentially antagonistic cultures is superior to the others. The writers know ofno published comparative data. Expediency or personal bias appears to havedictated the method used by each person or group concerned with the problem.Although Waksman (55) and Florey et al. (16) have discussed conditions or

methods of testing for antibiosis and pitfalls to be avoided, a few remarks mightbe appended.

Failure to use the proper medium, correctly prepared, can lead to failure ofthe organism to produce an antibiotic. The importance of this is brought outin the patents (12, 14, 48) covering several antibiotics in which the formulaeof the media for industrial fermentations are listed. The medium used for crossstreak or other tests for activity is also important. In addition, many culturesproduce antibiotics on solid, but not in liquid, media.Although most papers refer to the use of solid media for testing, Baker (2)

suggested that tubes containing liquid media, planted with cultures, tilted ata slight angle to provide aeration and rotated at 100-200 rpm could be usedfor producing small quantities of fermentation liquor.The temperature and length of incubation are important. In screening actino-

micetes for the production of antibacteriophagic substances Asheshov (1)found that by incubating shake-flask cultures for from two to three weeks hecould detect those antibiotics produced late or slowly in the course of metabolism.The actual testing of cultures for antibiotic activity involves the critical

choice of test-organisms. General activity may be detected by employing asensitive organism like B. subtilis. In seeking an antibiotic specific for a par-ticular parasite, such as M. tuberculosis, that particular organism is logicallythe sole test-organism. When inherent dangers in using such an organism com-pel its substitution by a suitable innocuous organism, the imperfection of anysubstitute should be constantly kept in mind. Most investigators appear toemploy a wide variety of organisms: gram-positive and gram-negative bacteria,fungi pathogenic to man, and fungi saprophytic or parasitic to plants.

Antibiotics displaying interesting properties should be identified. In additionto the methods listed by Florey et al. (16), two further tools have proved useful.Vanderline and Yegian (54) developed the idea of using streptomycin-dependentbacteria in streak-plate tests to identify cultures producing streptomycin. Paper


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strip chromatography has been used in the separation and identification ofproducts obtained during the chemical treatment of polymyxins (39) and inthe separation of polymyxin hydrochlorides (35). In isolating the new hydroxy-streptomycin Benedict et at. (4) at first were able to distinguish it from strep-tomycin only by its different chromatographic pattern. Studying the sameantibiotic, Grundy et al. (18) found that the fermentation liquor from shakeflask cultures showed a spot of inhibition differing from neomycin, strepto-thricin, mannosido-streptomycin, streptomycin, and dihydrostreptomycin. Ac-cording to Chichester (7), workers at E. R. Squibb and Sons use paper chro-matography in their screening program in

". . . concentrating unknown factors with antibiotic powers",

but the use of the chromatopile for such concentrations appears not to havebeen popular. It is evident from information in patents, however, that paperchromatography is extensively used for identification and characterization ofnew antibiotics.

Variations in the degree of activity of different antagonistic cultures arewell known. The potency of cultures may inexplicably decline. Antibiotics maybe labeled weak because the particular strain may produce them in small quanti-ties. If concentrated, however, these antibiotics might possess new and usefulproperties. Deciding into which category a particular antibiotic falls becomesa time-consuming, perplexing, and vexatious problem, and its ultimate solutionmay depend solely upon the tenacity of the investigator.The study of antibiotics produced by mixtures of two or more cultures may

well be worth consideration. Preliminary trials in our laboratory several yearsago showed that interesting cross-antagonisms exist, and recently Redmondand Cutter (38) have demonstrated a synergistic effect of two cultures. Eachof two nonsporulating fungi produced in liquid medium an antibiotic activeagainst Mortierella alpina. Both substances (or one?) were thermolabile. Whenthe two fungi were grown together in liquid medium or when they were grownin separate cellulose sausage casings immersed in a common liquid medium,they produced another, different, thermostable antibiotic having greater ac-tivity against the test organism than either of the original antibiotics. Thesestudies suggest a new approach in the various possible combinations of cultures.

Induction of Antibiotically Active MutantsAll searches for antibiotics are based upon the belief that mutations have

resulted in new strains differing in antibiotic capacities. These presumablyarose either from completely inactive strains or else from strains showing somekind of antagonistic property.

In 1949 Kelner (26) published the interesting results of X-ray and ultravioletirradiation of spores of actinomycetes showing no antibiotic activity. Of theseven species tested five after irradiation gave rise to antibiotically-activestrains. Some species produced active mutants at a rate of only 0.01 per centof cells surviving treatment, but others yielded a mutation rate of 1.9 per cent.


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One of the test organisms was E. coli, an organism generally most resistant toantibiotics. Kelner pointed out, further, that since many of the mutants grewslowly they might not survive in natural habitats. That mutants from anyparticular species frequently differed in their antibacterial spectra suggeststhat cultures producing different antibiotics might be secured by irradiatinga single culture and that further treatment of these cultures might yield a newantibiotic. Considerable information on the origin and relationship of variousantibiotics might be gathered by this technique.

ProspectsIt is unlikely that interest in the field of antibiotics will abate. A satisfactory

agent against the smaller viruses is yet to be discovered, and it is conceivablethat an antibiotic will be useful in the treatment of cancer. Better agents againstprotozoa and mycobacteria would be highly desirable. In the search for chemo-therapeutic agents the rational approach, as exemplified by the theory pro-pounded by Woods (72) and Fildes (15) and expanded and refined by Woolley(73), has an irresistible attraction to the scientific mind. The history of medicalscience, however, teaches us that more often than not rational theory followsempirical discovery. Even at best the rational approach must await the discoveryand characterization of essential metabolites by empirical methods. The ap-plication of the empirical method directly to research in antibiotics has yieldeda series of agents, some useful clinically, which interfere with enzyme systemsin ways still dimly understood. It may be confidently expected that greaternumbers of agents will literally be "unearthed" and that eventually theirmodes of chemical activity will reveal essential metabolic pathways. Until thetime when knowledge of these metabolic pathways permits exact prediction ofthe antibiotic to be synthesized by the organic chemist, the present empiricalmethods must be used. It is already apparent that with each discovery of anew and clinically valuable antibiotic it becomes more difficult to find another.To find a new antibiotic will tax to the limit the resources of the mycologist inisolating microorganisms capable of producing them, the ability of the bac-teriologist in characterizing them, and the skill of the biochemist in securingpure crystalline antibiotics.

REFERENCES1. ASHESHOV, I. N. 1951 Address before the New York Chapter of the Society of Ameri-

can Bacteriologists. October 15, 1951.2. BAKER, P. B. 1949 A rotating test-tube apparatus for experimental fermentation.

Nature, 163, 732.3. BARON, A. L. 1950 Handbook of Antibiotics. Reinhold Publishing Co., New York,

New York. 303 pp.4. BENEDICT, R. G., STODOLA, F. H., SHOTWELL, 0. L., BOWD, A. M., AND LINDENFELSER,

L. A. 1950 A new streptomycin. Science, 112, 77-78.5. BRIAN, P. W. 1951 Antibiotics produced by fungi. Botan. Rev., 17, 357-430.6. BRIAN, P. W., HEMMING, H. G., AND MCGOWAN, J. C. 1945 Origin of a toxicity to

mycorrhiza in Wareham Heath soil. Nature, 155, 637-638.7. CICHESTER, D. F. 1950 Soil samples are screened for antibiotics on mass basis by

Squibb pilot installation. Drug Trade News, 25, 29, 46.


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8. CONN, J. H. 1917 Soil flora studies. V. Actinomycetes in soil. N. Y. Agr. Exptl.Sta. Tech. Bull. No. 60, 25 pp.

9. COOPER, W. E., AND CHILTON, S. J. P. 1950 Studies on antibiotic soil organisms.1. Actinomycetes antibiotic to Pythium arrhenomanees in sugar-cane soils of Lou-isiana. Phytopath., 40, 544-552.

10. CROOK, P., CARPENTER, C. C., AND KLENS, P. F. 1950 The use of sodium propionatein isolating actinomycetes from soils. Science, 112, 656.

11. Dueos, R. J. 1939 Studies on a bactericidal agent extracted from a soil bacillus.J. Exptl. Med., 70, 1-10, 11-17.

12. DUGGAR, B. M. 1949 Aureomycin and preparation of same. U. S. Patent 2,482,055.Assignor to Lederle Laboratories.

13. DUGGAR, B. M. 1950 Isolation of cultufable fungi from diverse habitats. Trans.N. Y. Acad. Sci., Ser. II, 12, 168-171.

14. EHRLICH, J., SmITH, R. M., AND PENNER, M. A. 1949 Process for the manufacture ofchloramphenicol. U. S. Patent 2,483,892. Assignors to Parke, Davis and Co.

15. FILDEs, P. 1940 A rational approach to research in chemotherapy. Lancet, I,955-957.

16. FLOURY, H. W., CHAIN, E., HEATLEY, N. G., JENNINGS, M. A., SANDERS,A. G., ABRAHAM, E. P., AND FLOREY, M. E. 1949 Antibiotics. Vol. 1. OxfordUniversity Press, New York, N. Y. 628 pp.

17. GARRETT, S. D. 1951 Ecological groups of soil fungi: a survey of substrate relation-ships. New Phytol., 50, 149-166.

18. GRUNDY, W. E., WHIMAN, A. L., HANES, M. E., AND SYLVESTER, J. C. 1951 A studyof Streptomycee NA232-M1 and hydroxystreptomycin. Antibiotics and Chemo-therapy, 1, 309-317.

19. HARE Y, J. L. 1948 Mycorrhiza and soil ecology. Biol. Revs. Cambridge Phil. Soc.,23, 127-158.

20. HESSATON, D. G. 1951 "Double-action" of trichothecin and its production in soil.Nature, 168, 998-999.

21. JENSEN, H. L. 1943 Observations on the vegetative growth of Actinomycetes in thesoil. Proc. Linnean Soc. N. S. Wales, 68, 67-71.

22. JOHNsTONE, D. B. 1947 Actinomycetes of Bikini atoll, with special reference to theirantagonistic properties. Soil Sci., 64, 453-458.

23. KAREL, L., AND ROACH, ELIZABETH S. 1951 A Dictionary of Antibiosis. ColumbiaUniversity Press, New York, N. Y. 373 pp.

24. KATzNELSON, H., LocmwAD, A. G., AND TiMoNIN, M. I. 1948 Soil microorganismsand the rhizosphere. Botan. Rev., 14, 543-588.

25. KATZNELSON, H., AND RICHARDSON, L. T. 1943 The microflora of the rhizosphere oftomato roots in relation to soil sterilization. Can. J. Research (C), 21, 249-255.

26. KELNER, A. 1949 Studies on the genetics of antibiotic formation: the induction ofantibiotic-forming mutants in actinomycetes. J. Bact., 57, 73-92.

27. LANDERKIN, G. B., AND LOCHHEAD, A. G. 1948 A comparative study of the activityof fifty antibiotic actinomycetes against a variety of soil bacteria. Can. J. Research(C), 26, 501-506.

28. LANDERKIN, G. B., SMITH, J. R. G., AND LOCHHEAD, A. G. 1950 A study of the anti-biotic activity of actinomycetes from soils of northern Canada. Can. J. Research(C), 28, 690-698.

29. LOCHHEAD, A. G., AND LANDERKIN, G. B. 1949 Aspects of antagonisms betweenmicroorganisms in soil. Plant and Soil, 1, 271-276.

30. MCKEE, H. S. 1949 Review of recent work on nitrogen metabolism. New Phytol.,48, 1-83.

31. MARTIN, J. P. 1950 Use of acid, rose bengal, and streptomycin in the plate methodfor estimating soil fungi. Soil Sci., 69, 215-232.

32. MEREDITH, C. H. 1946 Soil actinomycetes applied to banana plants in the field.Phytopath., 36, 983-987.


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66 J. B. ROUTIEN AD A. C. FINLAY [VOL. 16

33. MILLER, J. J., PEERS, D. J., AND NEAL, R. W. 1951 A comparison of the effects ofseveral concentrations of oxgall in platings of soil fungi. Can. J. Botany, 29, 26-31.

34. NAKHIMOVSKAIA, M. I. 1937 The antagonism between actinomycetes and soil bac-teria. Microbiology (U. S. S. R.), 6, 131-157.

35. NASH, H. A., AND SMASHEY, A. R. 1951 Application of buffered paper strips to thechromatography of polymyxins. Arch. Biochem., 30, 237-240.

36. NICKELL, L. G., AND BURKHOLDER, P. R. 1947 Inhibition of azotobacter by soilactinomycetes. J. Am. Soc. Agron., 39, 771-779.

37. PLOTHO VON, 0. 1947 Farbstoffe und Antibiotica bei Actinomyceten. Arch. Mikro-biol., 14, 142-153.

38. REDMOND, D. R., AND CUTTER, V. M., JR. 1951 An example of synergistic growthinhibition between root-inhabiting fungi. Mycologia, 43, 723-726.

39. REGNA, P. P., SOLOMONS, I. A., FORSCHER, B. K., AND TIMRECK, A. E. 1949 Chemicalstudies on polymyxin B. J. Clin. Investigation, 28, 1022-1027.

40. RISLER, J. 1949 Antibiotics and the production thereof. British Patent Applica-tion 20028.

41. ROSENFELD, W. D., AND ZOBELL, C. E. 1947 Antibiotic production by marine micro-organisms. J. Bact., 54, 393-398.

42. ROuATT, J. W., LECHEVALIER, M., AND WAKSMAN, S. A. 1951 Distribution of an-tagonistic properties among actinomycetes isolated from different soils. Antibioticsand Chemotherapy, 1, 185-192.

43. ROUNTREE, P. M., AND BARBOuR, R. G. H. 1951 Nasal carrier rates of Staphylococcuspyogenes in hospital nurses. J. Path. Bact., 63, 313-324.

44. SCHANTZ, H. L., AND ZON, R. 1924 Natural Vegetation. Atlas of American Agri-culture. U. S. Department of Agriculture. Government Printing Office, Washing-ton, D. C. 26 pp.

45. SCHATZ, A., AND HAZEN, E. L. 1948 The distribution of soil microorganisms an-tagonistic to fungi pathogenic for man. Mycologia, 40, 461-478.

46. SIMINOFF, P., AND GOTTLIEB, D. 1951 The production and role of antibiotics in thesoil. I. The fate of streptomycin. Phytopath., 41, 420-430.

47. SMITH, N. R., AND DAWSON, V. T. 1944 The bacteriostatic action of rose bengal inmedia used for plate counts of soil fungi. Soil Sci., 48, 467-471.

48. SOBIN, B. A., FINLAY, A. C., AND KANE, J. H. 1950 Terramycin and its production.U. S. Patent 2,516,080. Assignors to Chas. Pfizer and Co., Inc.

49. THAYSEN, A. C. 1950 Antibiotics in the soil. Nature, 166, 93.50. TIMONIN, M. I. 1941 The interaction of higher plants and soil microorganisms.

III. The effect of by-products of plant growth on activity of fungi and actinomycetes.Soil Sci., 52, 395-416.

51. TYNER, L. E. 1944 Effect of media composition on the numbers of bacterial andfungal colonies developing in petri plates. Soil Sci., 57, 271-274.

52. UMEZAWA, H., TAZAKI, T., AND FUKUYAMA, S. 1949 Resistances of antibiotic strainsof Streptomyces to chloromycetin and a rapid isolation method of chloromycetin-producing strains. J. Antibiotics, II Supplement B, 91-94.

53. VALLANCE, K. B. 1950 Studies on the germination of the seeds of Striga hernwthica.I. The influence of moisture-treatment, stimulant-dilution, and after-ripening ongermination. Ann. Botany, 14, 347-363.

54. VANDERLINE, R. J., AND YEGIAN, D. 1948 Streptomycin-dependent bacteria in theidentification of streptomycin-producing microorganisms. J. Bact., 56, 357-361.

55. WAKSMAN, S. A. 1945 Microbial Antagonisms and Antibiotic Substances. TheCommonwealth Fund, New York, N. Y. 350 pp.

56. WAKSMAN, S. A., REILLY, H. C., AND JOHNSTONE, D. B. 1946 Isolation of strep-tomycin-producing strains of S. griseus. J. Bact., 52, 363-397.

57. WAKSMAN, S. A. 1949 Distribution of antagonistic microorganisms in the soil andtheir possible significance in soil processes. Fourth International Congress of


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Microbiology, Copenhagen. 1947, 468-471. Rosenkilde and Bagger, Copenhagen,Denmark.

58. WAKSMAN, S. A. 1950 The Actinomycetes. Chronica Botanica Company, Waltham,Massachusetts. 230 pp.

59. WAKSMAN, S. A., AND LECHEVALIER, H. A. 1951 The principle of screening anti-biotic-producing organisms. Antibiotics and Chemotherapy, 1, 125-132.

60. WAK5MAN, S. A., AND STARKEY, R. L. 1931 The Soil and the Microbe. John Wileyand Sons, New York. 260 pp.

61. WAKSMAN, S. A., AND WOODRUFF, H. B. 1940 The soil as a source of microorganismsantagonistic to disease-producing bacteria. J. Bact., 40, 581-600.

62. WAK8MAN, S. A., AND WOODRUFF, H. B. 1942 The occurrence of bacteriostatic andbactericidal substances in the soil. Soil Sci., 53, 233-239.

63. WAKSMAN, S. A., HORNING, E. S., WELSCH, M., AND WOODRUFF, H. B. 1942 Distri-bution of antagonistic actinomycetes in nature. Soil Sci., 54, 281-296.

64. WAKSMAN, S. A., KOCHI, M., AND LECHEVALIER, H. A. 1951 Actinomycetes as pro-ducers of antibiotics, with special reference to the flavus group. Bact. Proc., p. 30.

65. WALLHUXUSSER, K. H. 1951 Die antibiotischen Beziehungen einer natfarlichen Mikro-flora. Arch. Mikrobiol., 16, 201-236.

66. WARCUP, J. H. 1950 The soil-plate method for isolation of fungi from soil. Nature,166, 117.

67. WARCUP, J. H. 1951 Studies on the growth of basidiomycetes in soil. Ann. Botany,15, 305-317.

68. WA~cup, J. H. 1951 The ecology of soil fungi. Brit. Mycol. Soc. Trans., 34, 377-399.69. WARREN, J. R., GRAHA, F., AND GALE, G. 1951 Dominance of an actinomycete in

a soil microflora after 2,4-D treatment of plants. Phytopath., 41, 1037-1039.70. WEST, P. M. 1939 Excretion of thiamin and biotin by the roots of higher plants.

Nature, 144, 1050-1051.71. WOODRUFF, H. F., AND FOSTER, J. W. 1946 Streptin, an antibiotic from a species of

Streptomyces. J. Bact., 52, 502.72. WOODS, D. D. 1940 The relation of p-aminobenzoic acid to the mechanism of the

action of sulphanilamide. Brit. J. Exptl. Path., 21, 74-90.73. WOOLLEY, D. W. 1952 A Study of Antimetabolites. John Wiley and Sons, New

York. 269 pp.


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