H. A. LECHEVALIER AND C. T. CORKE

lose to the hay diet of a sheep increased the rate offermentation of both cellulose and hemicellulose bythe rumen fluid of the sheep. The addition of hemi-cellulose to the diet, however, increased the rate ofhemicellulose fermentation but not that of cellulose.Both increases were lost 48 hours after stopping thediet supplementations.

REFERENCES

HUNGATE, R. E. 1950 The anaerobic mesophilic cellulolyticbacteria. Bact. Rev. 14, 1-49.

MITCHELL, R. L., ROGERS, S. C., AND RITTER, G. J. 1948Hemicelluloses from maple holocellulose. Ind. Eng.Chem. 40, 1528-1529.

QUIN, J. I. 1943 Studies on the alimentary tract of Merinosheep in South Africa. VII. Fermentation in the fore-stomachs of sheep. Onderstepoort J. Vet. Sci. AnimalInd., 18, 91-112.

REID, J. T., AND HUFFMAN, C. F. 1949 Some physical andchemical properties of bovine saliva which may affectrumen digestion and synthesis. J. Dairy Sci., 32, 123-132.

WALTER, W. G. 1952 A study of microbial activities in theovine rumen. Thesis, Michigan State College.

The Replica Plate Method for Screening Antibiotic-ProducingOrganisms'

H. A. LECHEVALIER AND C. T. CORKE

Received for publication December 8, 1952

The various screening methods available for isolationof antibiotic-producing organisms have been reviewedin the past (Waksman 1947). The most reliable of thesemethods has been the laborious isolation of micro-organisms, more or less at random, and the subsequenttesting for antibiotic activity, either on solid or inliquid media. Other methods, such as the bacterial agar

plate, -the crowded plate, and the direct soil inoculationmethods, are of questionable value. Attempts were

made in the past to differentiate at an early stage be-tween antibiotic-producing organisms and nonproducersby inoculating a test organism directly on the plates inwhich the microorganism-rich substrate (such as soil)had been plated out. This inoculation was done eitherby spraying the test organism (Stansly 1947) on thesurface of the plates or by flooding this surface with a

water or agar suspension of the test organism. Thesemethods have the disadvantages of being laborious andof giving only the activity against a single test organismon one medium. Another difficulty encountered in thescreening of actinomycetes is that media selective foractinomycetes are not good for antibiotic production.

Lederberg and Lederberg (1952) have described a

method of replica plating and demonstrated its applica-tion in the selection of bacterial mutants. In the presentnote, the authors present a method of screening forantibiotic-producing organisms which is an adaptationof Lederberg and Lederberg's method.

This method permits selection, in a relatively shorttime, of isolates having the desired antagonistic proper-

ties against various test organisms.Paper of the Journal Series, New Jersey Agricultural

Experiment Stati6n, Rutgers University, The State Universityof New Jersey, Department of Microbiology.

On a medium selective for actinomycetes, such as anegg albumin agar (Waksman 1950) or KenKnight'sagar, (KenKnight 1939) a natural substrate, such assoil, can be plated out at high enough dilutiorns to givewell-isolated colonies. By means of a sterile velveteenstamp, four or more replica plates on yeast extractdextrose agar (containing 20 ml of medium per plate)can be made of every suitable soil plate.

TABLE 1. Comparison between antibiotic spectra determined byreplica plate method and by standard cross-streak method*

REPLICATE. CROSS-STREAK INEIBITION ZONESCULTURE

NO. Candida Bacillus Escker- Caudida Bacillus Escker-albicans ceresS ichia Cali albcaus cereu icsia ccli

mm mm mm

1 ++ + _ 13 6 02 ++ ++ - 13 29 53 ++ ++ ++ 12 27 204 ++ ++ + 15 32 5

* Both tests run on yeast-dextrose agar.++ = strong inhibition.+ = weak inhibition.- = no inhibition.

The velveteen stamps used in our laboratories weremade by covering with a velveteen sheath heavy disksof glass 8.5 cm in diameter and 1.5 cm thick. Woodenhandles were screwed on top of the glass disks to facili-tate handling of the stamps as transfers are made fromone plate to another. The velveteen stamps werewrapped in paper and were sterilizedin the autoclave.

After proper incubation (figure 1), one of these fourreplica plates is kept in the refrigerator for isolationpurposes, and the contents of the other three are un-

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REPLICA PLATE SCREENING METHOD

molded upside down, with a flamed spatula, into large(14 mm) sterile Petri dishes. The top of the unmolded

PLATE 1 PLATE

activity (figure 2) it can be isolated for further studiesfrom the plate kept in the refrigerator.

COLONY 1

S~~~ OLNil ......... OLONY 4PLATE 4 PLATE 6

FIG. 1. Replica plating of soil plate rich in actinomycetes. Six replica plates were made on yeast dextrose agar. This pic-ture shows plates 1, 2, 4, and 6. Colonies 1, 2, 3, and 4 were isolated and their antibiotic spectra were determined (see figure 2and Table 1).

CCOLONY O

,~~~~~~~~~~~~~~~~ _s--.-KY-_L~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ,1l........~........ . .....FIG. 2. Effect of the actinomycetes of a replica plate on Candida albicans. The content of the plate has been unmolded up-

side down. The positions of colonies 1, 2, 3 and 4 (figure 1) are reserved. They all have activity against C. albicans.

agar can be seeded in each dish with a different testorganism and incubated. If any one of the coloniespresent in the soil plates has interesting antimicrobial

Table 1 shows that the "crude" antimicrobial spec-trum obtained by this method corresponds with themore accurate spectrum obtained by cross-streaking.

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FIELD, MORRIS AND POE

Various modifications of this method are obvious.By use of the proper selective media, this method canbe adapted to the screening of bacteria or fungi. Varioussolid media can also be compared for the productionof the antibiotics.

SUMMARYA time-saving method of screening for antibiotic-

producing actinomycetes is described. The method,an adaptation of Lederberg and Lederberg's replicaplate method, permits the rapid recognition of anti-biotic-producers and gives a partial antimicrobialspectrum of a culture before the culture is actuallysolated.

REFERENCES

KENKNIGHT, G., AND MUNCIE, J. H. 1939 Isolation ofphytopathogenic actinomycetes. Phytopath. 29, 1000-1001.

LEDERBERG, J., AND LEDERBERG, E. M. 1952 Replica platingand indirect selection of bacterial mutants. J. Bact.68, 399-406.

STANSLY, P. G. 1947 A bacterial spray apparatus useful insearching for antibiotic producing microorganisms. J.Bact. 54, 443-445.

WAXSMAN, S. A. 1947 Microbial antagonisms and antibioticsubstances. 2nd ed. The Commonwealth Fund. NewYork.

WAKSMAN, S. A. 1950 The Actinomycetes. Chronica Bo-tanica Co., Waltham, Mass.

Fermentation of Polyhydric Alcohols By the GeneraEscherichia and Aerobacter II. i-Inositol

J. THOMAS FIELD, RUSSELL C. MoRas, AND CHARLES F. POE*

College of Pharmacy, University of Colorado, Boulder, Colorado

Received for publication December 15, 1952

INTRODUCTION

The metabolic products (especially their quantita-tive estimation), which are produced by species ofEscherichia and Aerobacter in media containing thehigher polyhydric alcohols, have been the subject oflittle study. Several references may be found relativeto the fermentation of mannitol, inositol, and dulcitol.Most investigators have dealt with the qualitativedetection of the products formed by bacteria.Harden (1901) was one of the pioneers in the study

of the fermentation of alcohols and sugars by genusEscherichia. He made a quantitative study of theproducts formed from several common sugars. Hardenand Walpole (1906) reported a similar study withAerobacter aerogenes by using media containing glucoseand mannitol. Fred et al. (1919), Peterson, et al. (1924),and Gayon and Dubourg (1894) have reported thefermentation products produced from alcohols andsugars by certain pentose-fermenting bacteria. In thislaboratory, Poe, Field, and Witt (1933) investigatedthe metabolic products produced by the Escherichiaand Aerobacter species in a medium containing D-sorbi-tol. Their investigation showed little difference in theamounts of products formed by the two genera ofbacteria.

* For reprints address the junior author, University ofColorado, Boulder, Colorado.

Hewitt and Steabben (1921), quoting an unpublishedwork of Harden, reported experiments with the fer-mentation of inositol. Meillere (1907) found that in-ositol under aerobic conditions is not attacked byE. coli or Salmonella typhosa. Weiss and Rice (1917)found that inositol reacted negatively when tested withthirty strains of E. coli. Six strains of A. aerogenes gaveuniformly good gas production in inositol medium,whereas two strains of E. acidi-lactici were tested andreacted negatively. Weldin and Levine (1923) foundthat inositol was usually not fermented by E. neapoli-tana but was frequently fermented by A. aerogenes,A. oxytocum, and A. chinense, and rarely by A. cloacaeand A. levans. Kumagawa (1922) found that the gasproductions brought about by species of Aerobacterin inositol medium ceased in twelve days. In two days,the presence of acetic acid and lactic acid was detected.In addition, he found succinic acid to be present.The object of the present research was to study

quantitatively the fermentation products produced byspecies of genus Echerichia and genus Aerobacter inmedia containing i-inositol.

MErTHODS AND PROCEDURE

A large number of cultures was used in this study.These organisms were members of the genera Eswhe-richia and Aerobacter, and were isolated from waters

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