REPLICA PLATING AND INDIRECT SELECTION OF BACTERIAL MUTANTS JOSHUA LEDERBERG AND ESTHER M. LEDERBERG Department of Genetics, College of Agriculture, University of Wiecomin. d!fadi8on, WiScOnSin Reprinted fmm JOUENAL OF BACTEEIOLOQT Vol. 63, No. 3, hfamh, 1362 Printed in U.S.A. REPLICA PIATING AUYD INDIRECT SELECTIOK OF B.iCTERIAL MUTANTS JOSHUA LE:nI~;RBE:ltG AXD b;STHER M. LIDERUERG Deptrrlment 0J" Geuetics ,I (`olleyc of Aqricrdlt~re, Cniucrsity of Wisconsin, ~lladison, W'isconsin Recrivcd for publication August, 31, 1951 Elective enrichment' is an indispensable technique in bacterial physiology and genetics (van Niel, 1949). Specifir biotypes are most readily isolated by the cs- tablishment of cultural conditions t,hut favor their growth or survival. It has heen repeatedly questioned, however, whether a selective environment, may I not ouly select but also direct) adaptive herit,ahle changes. In accord with similar discussious in evolutionary biology (Huxley, 1942), n-e may denote the concepts of spontaneous mubtion and natural select,ion in contr& to specific induction as "prenduptation" and "directed mutation", respectively. Many linrs of cyi- dence have been adduced in support of preadaptation in a variety of systems (Luria and Delbriick, 1943; Lea and Coulson, 1949; Burnet, 1929; Se\vcomt)e, 1949; I,ewis, 1934; Kristensen, 1934; n'ovick and Szilard: 1950; Ryan and Schneidcrj 1949; Demerec, 1948; Welsch, 1950; also reviewed: Brawl, 1947; Lucia, 1947; I,edertwg, 1948, 1949). This paper concerns an approach to this problem that makes use of a replica plating technique which facilitates the handling of large numbers of bacterial clones for classificaation on a variety of media. LILTHODS Replica plafiny. A frequentj chore in twcteriological work is the transfer of isolates from one suhstrate to other selective or indicator agar media. In plave of an inoculating needle, one might, imagine a devire consisting of many needle tips in fiwd array, so that one operation N-ould substitute for repeated transfers with a single needle. The requirements of this design arc met, by pile fabrics such as velvet, or velveteen. The pile provides space in a vertical plane for moisture that might ot,herwise cause lat,eral smearing of any impression. (=lwording to Dr. S. \Ywonti, in a private communication, dampened filter paper may t:c applicxt~le to some replicaation problems considered by him independently of the prcscut, work.) In our prwtice, twelve cm squares were cut from velveteen yardage, packed in large petri dishes, and sterilized in the aubclave. A square is plared, nap up, on a q%ndri(*al wood or vork support of nine cm diameter and held firmly in . place with a met,al flange or hoop pushed over the fabric and around the rim of the support). The agar plate carrying the initial colonies is invert,ed onto the I So. -173. This work 1~s IWPII support.~d 1)~ gr:lnts from ll>e Research Committcv. Gwdu- ate School. University ot' Wisconsin, with funds supplied t)y the Wisconsin Alumni Research Foutldntion. and from the Division of Research Gmnts and Frllowships, S:ltion:ll IIicro- I)ioIogiwl lnst itutr, Natiorml lnst itutes of Health, I:. S. Public IIr:~lth Sewire. 399 400 J0SHU.t LEDI*:RBERG AND E. M. LICDERBEHG [VOL. 63 fabric with slight digital pressure to transfer the groxth. The imprinted fabric then provides t)he pattern for transferring replica-inwula to subsectuent plates impressed in the same way. Replka plating is used to facilit,ate routine tests involvitlg repetitive inocula- tions of many isolates on different media. Such tests are frequently rcctuired in genet)ic work, but the method should be applicable to other routine practice. Traits which lend thcmsclves to classitiwtion by replica plating include anti- ~)iotic:-sensitivity spwtra, responses to bacteriophages (as in phagr typing), fermentation characters, nutritional requirements, or any characteristic for which a selective or indicatJor agar medium can be devised. AIn application of replica plating to the detection of auxoheterotrophic mutants is illustrated in 1%2] REPLICA PLr\TIh-G AiXD INDIRECT SELECTION -lo1 figure 1, which also demonstrates the precision of the replicas. However, more faithful reproductions than t,hose shown in the figure can be obtained with the use of dry, hard (2 or 2.5 per cent) agar. The t,ype of initial growth to be replicated may be varied according to specific needs. It may consist of surface colonies, localized growths from stab or spot inocula, or, as in the latter part of this paper, confluent growth from dense inocula. previously spread over the agar and incubated. Freshly seeded sites will yield replicas of rest,ricted inoculum size. A single initial plate may be used to imprint' more than one fabric if carryover from one replica plate to another vitiates serial transfer. A fabric square may be v-ashed, sterilized, and used repeakxlly. Replica plat)ings may he quantitatively variable and influenced by many physical factors in common Cth some conventional methods of rcpctitire inocu- lation. There is no practical limitation on the number of serial replicas available, except for the acacumulation of moistjure that may exude from the agar surfaces. The resolution depends on the texture of the agar, colonies, and fabric. I~nlcxs the initial colonies are very plump, the distortion in size and shape is minimal and usually less than illustrat)ed in figure I. A crude estimate v-as made of the eflkietwy of transfer from initial plates spread with measured numbers of Esch~richia coli cells. Approximately 10 t,o 30 per cent, of the initial cells wre transferred to the fabric, and an equal proportion again of these was found to be deposited on t,he replica plates. C(onnl occurrence oj pl2agr wsisfant ?rzManfs. Pread:ipti\e mutation as the lwis of bac4erial rcsistnnw to phages has been supported by two types of evidence. Tkiwt (1929) suweeded in isolating phage resistant mutants of Salmotlclla by observing the colonial morphology of the Ii and S phases. The other ej-idenw is biometric: T,uria and Iklbriic~k (1943) working I\-it11 E. coli, strain B ant1 phage `T-1 , she\\-ccl that the immbcrs of mtttants selected from parallel broth cultures followed a (#Ional rather than a random sampling distribution. This was xub- statitiatetl by more direct evidence of clonal ocwurence of the mutants. Se\\-- cornbe (1949) sprayed phage on films of growth WI agar to assay them for their couiit of rcsintant mutarlts. The cwmts \vrre greatly nugmcntcd by redisttil~uting the growth at the time the phage \\-as sprayed. The incw~ase n-as I-elked to result from the (preadaptive) occurrence of the mutants in coherent clonrs. On the undisturbed plates, t,he assay would give the count of clones; the rctlistri- bution I\-ould give the Ma1 count, of resistant cells. The replica plating method allows a more direct, demonstration of the c~lotial occurrence of the mutants: clones on an initial plate would be detected by the recurrence of resistSant, colonies at superimposable sites on serial replica-plates containing t,he phage. If t#he resistant (ells did not exist already in clones on the init,ial plate, t)hey should occur in only a random distribution in serial replicas from a ronfluent film of growth. For this t,est, a culture (W-l), derived from E. co& strain Ii-12, and the phage 40% JOSHUA LEDERBERG Ah-D E. M. LEDERBERG [VOL. G3 T-l I\-ere used. The culture is fully sensitive to the phage T-l, as n-r11 as to strep- tomycitt, and like most E. cnli strains gives rise to resistant mutants at rates of approximately 10-j and lo-`" per division, respectively. The media used included "Difco penassay" broth iii 5 or 10 ml volumes (re- fcrred to as "broth") and EMB lactose ngar ("plait1 agar"). The replicxs were made on EMB agar previously coated with (:a lo9 particles of T-l per plate ("phage agar"). In a typical experiment, a dense hrot,h culture was gro\vtt from a sittglr ~~~lotty on plain agar. One-tenth ml was spread on plain agar, attd the plate was incxt)atctl 3 to (i hours at 37 Cr. Serial replicxs then were transferred, as dewrit~ed previously, to two or more phage agar plates n-1tic.h t#hen were ittcwhated o~wttipltt. The plates were marked either with a glass marking petwil or, for greater preckion, by means of pills inserted into t,he velvet, which indented the agar. Figure 2 shows a typical result, except that a 0.01 ml inoculum MYIS usctl to rrstric$ the numtwr of rlottes. In several esperimettts, at, least half and often ttearly all of the resistatttj mutants on the replica-plat)es recurred at c*ottgructtt sites. The preoccurtwt(~e of the resistant, cells in coherent families or c~lottrs lvithitt the confluent film ott plain agar is inferred from t,his result. Intliwct sclcctio~l sf phagc resistant mutants. The hypothesis of prcadaptation would h(~ further strettgthened if adapted mutants csould be isolated in pure cultjure \vithout direct exposure of the hact,erin to the selective agettt. Replicaa platittg has made this possible. In the clsperiments of the previous se&Ott, the sites of preadapted mutants in the ittitial film of gro\vth on plain agar are disc~erttahle from tlif rrpliws. If the initial ittowla are made sufficktl; dilute, there may he ottly one or a fen clottes on a single plate. If t,he congruent' sites are chosen for the ittocvlum of a sewtttl broth tube, the mutants will he cottcaetttrated or enriched in Aout thr same proportiott as the cells per plate t,o the cells ittc4uded in the inoculum. -111 enrichment of a hundredfold was anticipated and wttfirmed for ewh stage and this sufic~etl for our experiments. After incubation, the enriched broth is treated itt the same way, cswpt that a more diluted inoculum is spread on plain agar to give agaitt hut otte 01' a fcv mutant clones per plate. After about, four stages of itidirwt srlwtiott, the re- sistatlt culottes appear as tliswctc colonies which c'att thctt he clt:trwterizrcl, purified, wtd maitttainctl by caonventional methods. The itttlirevt rclwtiott for phage resistance n-as applied in two itldrptttdcttt~ runs! both quite sucwsaful. The mut,ants showed the same indiff'ctwwe to the phagc as did mutatlts previously isolat,rd by dirert selection. Their stability was verified t)y makittg ten serial loopful to broth transfers, for a total of ahout 100 txwtcrial generations in eacsh series. The terminal cultures then vwc diluted and plated ott plain agar. A total of 836 and 417 colonies tested, respec.tiTrely, lucre all rcsktattt to the phage as determined by replira platings, and by a few tests of ww-sttwkittg cwlonies against, the phagc. It should 1~ reemphasized that the indirect wlcction line itself has ttot heen csposed to the phage at any time. Its hisby cwnsists of the trattsfrr cycle: 19521 REPLICA PLATING AND INDIRECT SELECTION 403 broth to plain agar to broth, with side transfers from agar to velvet discs used to imprint phage agar. Each broth was tested for any stray phage that might lead to fallacious conclusions with no indication of its presence. Replica plating Figure 2. Clonal occurrence of mutants resistant to phage T-l. A, Initial or replica plate on plain agar with diffuse, confluent growth, (semidiagrammatic). B, C, and D, Successive replicas from A to agar coated with phage, from tracing of a typical experiment. Superim- posable colonies of resistant cells are numbered. These are concluded to be derived from small clones of resistant mutants already present at corresponding sites on the plain agar plate, A. thus provides a technique for isolating resistant or otherwise adapted mutants without altering the media in which the bacteria are grown. Xtreptomycin resistance. In order to verify the general applicability of indirect selection, the procedure was also applied to streptomycin resistance. Instead of "phage agar", EMB agar containing 200 Fg of streptomycin per ml ("sm agar") was used for the replica plates. The only deviation of these experiments from 404 JOSHUA LEDERBERG AND E. M. LEDERBERG [VOL. 63 those with phage results from the extremely low rate of mutation to sm-resis- tance. In order to obtain any resistant mutants at all, the W-l culture was transferred several times in large volumes of broth. Concentrated inocula, about 3 X log celIs, were used on the initial plates. Only two or three resistants were seen in experiments with 20 or 30 initial plates. One mutant clone was found by recurrence on serial sm replicas, and its site on plain agar was used to initiate the selection. After six stages of enrichment, the sm-resistant was obtained in pure culture. Two hundred and thirty-nine colonies were tested after 100 addi- tional generations of growth in broth, and all retained resistance to sm. The sequence of transfers was identical with that exercised in indirect selection for phage resistance. The transfer operation itself could not have been responsible for resistance, for the sm selections remained sensitive to the phage and tie versa. The infrequency of sm-resistant mutants hinders the tests for clonal occurrence. The culture 58-278, derived from E. wli, strain K-12, has been found to exhibit a much higher rate for this mutation, about lo-' per division (H. P. Treffers, personal communication). Replicas of films of this culture on plain agar to sm agar plates repeatedly gave patterns similar to those illustrated in figure 1 for phage resistance. Indirect selection was aIso exerted successfully on this culture with results similar to those already described. We conclude that resistance to streptomycin, a,s to phage, is a spontaneous mutation that occurs independently of the presence of the selective agent. DISCUSSION Indirect selection is experimentally but not logically dependent on the clonal occurrence of the mutants. The latter had been established inferentially by Luria and Delbriick (1943) and by Newcombe (1949). It shows that the adapted cells are not randomly distributed in space. The success of indirect selection provides a sound basis for Ohis nonrandom clust'ering in hereditary transmission. In particular, neither the adaptive change nor its inheritance depends upon a specific environment, which is what we mean by spontaneous mutation or preadaptation. This demonstration does not conflict with reversible adaptive responses to a specific environment which disappear after some generations of growth in an indifferent medium. Directed, but nonheritable, responses have been clearly demonstrated in adaptive enzyme formation (Monod, 1947) and may be involved in the resistance phenomena investigated by Eagle (1951). However, no un- equivocal case of a mutation specifically direct'ed by and adapting cells to a chemical agent has yet been defended, despite numerous attempts of varying clarity (e.g., Barer, 1951). The concept of the "genotype as the norm of reaction" is pertinent to this discussion. The status of a microorganism's reaction as realized at any time, i.e., its phenotype, will reflect its immediate history, but its com- petence to react is an intrinsic quality subject for the most part only to sporadic, indeterminate mutations. Indirect selection and tests for clonal occurrence should be applicable to other 19521 REPLICA PLATING AND INDIRECT SELECTION 405 adaptive systems, but some difficulties may be anticipated. With low mutation rates, sufficient numbers of cells must be used t'o allow a reasonable number of mutants to appear. Since the proportion of mutants should also increase in time (Novick and Szilard, 1950), the serial transfer of large volumes of cultures is also indicated. A very high mutation rate may also cause difficulties if preexisting clones are outnumbered by new mutations during the growth of the initial plates. This can be compensated for again by serial transfer in broth to pre accumulate mutants and by restricting the inoculum size and time of incubation of the initial plates. The presumably indifferent "plain medium" may prove to be adverse to the mutants. For example, it is not likely that sm-dependent mutants will be detected as clones in plain agar films. However, any applications of replica plating that fail to demonstrate the clonal occurrence of a mutant type may be controlled by suitably designed reconstruction experiments. These would in- volve the addition of known numbers of directly selected mutants to the original cultures. If the intruded mutants are detected in clones, and new occurrences are not, this would support the conclusion that the latter is not of spontaneous origin. Some adaptations may be less amenable to these approaches, depending on the availability of suitable selective media for the replica plates. An example may be the development of lactose-positive papillae in cultures of E. coli "mu- tabile" on lactose-peptone agar. It would be necessary to devise a medium that would detect mutants in the replica inoculum without losing them in an ava- lanche of new mutations occurring on the indicator plate itself. The indirect selection procedure is paralleled by improvement methods which depend upon the performance of the kinships of a plant or animal rather than its own phenotype, as for example in the selection of roosters for egg production breeding stock (Lush, 1945). SUMMARY A method, replica plating, was developed to permit the copying of a pattern of microbial growth from one initial agar plate to a series of others. The method uses velveteen or other fabrics to make the transfer without disturbing spatial relationships. It may be useful in the detection of biochemical mutants, classifi- cation of fermentation reactions, determination of antibiotic sensitivity spectra, and other rout'ines requiring repetitive inoculation of several media. Replica plates were used in an approach to the problem of the preexistence of adaptive mutants prior to their selection by specific environments. Replicas to agar containing bacteriophage or streptomycin showed that mutants of Escherichia coli resistant to these agents existed in clones on the initial plates of indifferent agar medium. In addition, concentration or enrichment for such mutants was accomplished by taking inocula from bacterial films at sites demon- strated to contain mutants by replica plates. After several stages of enrichment, each type of resistant mutant was isolated in pure culture. The procedure at no time exposes the indirectly selected populations to the specific agent. 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