JOURNAL OF BACTERIOLOGY, Oct., 1966Copyright © 1966 American Society for Microbiology

Vol. 92, No. 4Printed in U.S.A.

Mutants of Escherichia coli Variably Resistant to

Bacteriophage Tl1GUY R. CARTA2 AND VERNON BRYSON

Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey

Received for publication 27 June 1966

ABSTRACT

CARTA, Guy R. (Rutgers, The State University, New Brunswick, N.J.), ANDVERNON BRYSON. Mutants of Escherichia coli variably resistant to bacteriophageTi. J. Bacteriol. 92:1055-1061. 1966.-Mutants resistant to bacteriophage Tiwere isolated from ultraviolet (UV)-irradiated cultures of Escherichia coli B/r, aUV-resistant variant. Bacterial populations derived from some of these mutantswere partially but not completely resistant to the bacteriophage. Such mutants,designated variably resistant (B/r/lv), could not be obtained from E. coli B.Phage-free mutant populations taken from different stages in growth consisted ofsignificantly different proportions of Tl-resistant and TI-sensitive cells. The growthstage-dependent range of variation exceeded 1,000-fold. In broth cultures, the highestproportion of resistant cells consistently appeared at mid-log phase, and the highestproportion of sensitive cells at lag and stationary phases. Comparable evidence forenvironmentally dependent changes in host-cell phenotype was obtained by effi-ciency of plating and cloning efficiency analysis tests. Micromanipulation showedthat, in clones growing in the presence of phage Ti, sensitive bacteria appeared withhigh frequency and underwent lysis.

Several investigators have isolated bacterialmutants that yield populations exhibiting sometype of intermediate resistance to phage. Thestudies include the work of Wahl and Blum-Emerique (10-13) and Jones, McDuff, and Wilson(8).

In a qualitative comparison of spontaneous andultraviolet-induced mutations to bacteriophageTi resistance in Escherichia coli B/r, Bryson andDavidson (2) described a type of bacterial mutantcharacterized by "partial" resistance. Suchmutants exhibit reduced growth rate in phage-containing broth, and reduced efficiency of plat-ing.

In the present study, we examined additionalproperties of the E. coli mutant phenotype. Canclones derived from single mutant bacteria giverise to sensitive descendants at a high enoughfrequency to account for the reduced growth ratein the presence of Ti? If so, what governs the

1 Based on a thesis submitted by the senior authorto Rutgers, The State University, in partial fulfillmentof requirements for the Ph.D. degree.

2Present address: Department of Biology, VirginiaPolytechnic Institute, Blacksburg, Va.

transition from resistance to sensitivity? Suchquestions are of interest because-although exten-sive knowledge exists concerning typical resist-ance, sensitivity, and lysogeny-little study hasbeen devoted to phenotypes that do not fallneatly into any of these three categories of hostcell-bacteriophage interaction.

MATERIALS AND METHODSBacteria. The organisms used were E. coli B, a

radiation-resistant mutant B/r (14), and two K-12strains: W3780 (Hfr2 met azi-r str-r) and W1177(F- thr leu mtlh ara- malh lac- galb xylb str-r T6-r;Bendigkeit, Kubitschek, and Hendrick, Bacteriol.Proc., p. 143, 1962).

Inoculum. Cultures were inoculated from 18-hr,aerated, Nutrient Broth (Difco) cultures.

Growth. Bacterial populations were grown to earlylogarithmic, mid-logarithmic, late logarithmic, andearly stationary phase by introducing 0.1 ml of theinoculum culture into 9.9 ml of Nutrient Broth andincubating the culture for appropriate periods.

Temperature and aeration. All liquid cultures weregrown at 37 C on a rotary shaker. Petri plates con-taining solid media were incubated at the same tem-perature.

Bacteriophages. Coliphages Ti and T5 were propa-

1055

on May 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CARTA AND BRYSON

gated on E. coli B/r and assayed by agar overlay orby direct spreading, by use of conventional methods(1).

Media. With a few exceptions, Nutrient Broth andNutrient Agar (Difco) were employed. For micro-manipulation studies, isolation droplets consistedeither of TI phage lysates containing 1010 to 3 X 1010plaque-forming units (PFU) per ml, or of DifcoPenassay Broth free of phage. For marker analysis ofbacteria, the following media were utilized: LevineEosin Methylene Blue Agar without lactose (BBL)supplemented with a specific sugar; Nutrient Agarsupplemented with 200 jg of streptomycin sulfate perml; Nutrient Agar containing 0.002 M sodium azide;and a modified form of minimal medium A agar (3).

Isolation of mutants. Mutants resistant, or variablyresistant, to TI were obtained by ultraviolet irradia-tion of 18-hr stationary-phase cultures diluted 1:10in Nutrient Broth, followed by incubation for 4 hrand dilution (1:10) in saline. Killing was 90 to 99%.A General Electric 15-w mercury vapor lamp wasused. Cultures were stirred magnetically during irra-diation, and precautions were taken to avoid photo-reactivation. Irradiated samples in 0.1-ml quantitieswere spread on Nutrient Agar plates and incubatedfor 2.5 hr to allow the expression of mutants. Plateswere then spread with 0.1 ml of Ti phage and incu-bated for 1 to 2 days. Resistant, or variably resistant,mutants were streaked four or five times, with inter-vening subculture, to eliminate bacteriophage.

Test of mutants. Milky saline suspensions of eachbacteriophage-free mutant were cross-streaked onNutrient Agar over a previously applied streak of Tibacteriophage. Resistant mutants, variably resistantmutants, and sensitive control bacteria could be dis-tinguished (Fig. 1) after 10 to 14 hr of incubation ofthe plates.

Micromanipulation. Methods used for pedigreeanalysis of single bacteria and progeny were based onthose of de Fonbrune (4). Bacteria were isolated inmicrodroplets of media contained within an oil cham-ber, by means of a Cailloux micromanipulator (C.M. Stoelting Co., Chicago, Ill.). Micropipettes weremade by hand, by use of a flame passed through ahypodermic needle.

Efficiency of plating. Bacteriophage TI was testedfor its ability to form plaques on a control strain(B/r) and on individual mutant strains. The ratio wasexpressed as efficiency of plating.

Determination of cloning efficiency. The ability ofbacteria to form colonies in the presence of bacterio-phage was determined by plating known numbers ofcells, sensitive and variably resistant to TI, on agarplates spread with an excess of Ti (109 to 3 X 109PFU per plate). After 24 hr of incubation, the per-centage of colonies formed in the presence of bacterio-phage and in its absence was determined.

Turbidimetric determination of lysis. Measurementof lysis was done turbidimetrically, with a Klett-Summerson colorimeter equipped with a blue filter.

Measurement ofbacteriophage production. Early logphase broth populations of the appropriate bacterialstrains were mixed with TI at a multiplicity of infec-

tion of approximately one. At various time intervalscommencing with zero-time, 0.1-ml samples werewithdrawn and diluted in chilled Nutrient Broth;the dilution tubes were maintained at 4 C. After thedesired number of samples had been withdrawn, theinitial 100-fold dilutions were turther diluted as neces-sary and assayed for infectious phage particles.

Adsorption. Two methods were used to determinebacteriophage adsorption. In both, broth cultureswere mixed with bacteria at a multiplicity of infectionof approximately 0.1 and incubated further with aera-tion. The first method allowed 8 min for adsorption;then the, mixture was poured into a chilled 125-mlflask. Next, an 8-ml sample of the chilled mixturewas centrifuged at 6,500 X g in a cold room (4 C).The supernatant fluid was assayed for infectious bac-teriophage particles, and the measurement of adsorbedphage was made by difference. The second methodwas a modified form of the Fredericq (6) chloroformtechnique, with materials equilibrated at 37 C. For theadsorption of phage, 6 min was allowed, followed byintroduction of 1.0 ml of chloroform into the 10-mlmixture. The tube contents were shaken vigorously20 to 30 times; then the chloroform was allowed tosettle. The aqueous phase was assayed for infectiousphage particles and measurement of adsorbed phagewas made by difference.

RESULTS

Preliminary characterization ofmutants. As hadbeen observed by Bryson and Davidson (2),mutants exhibiting an atypical resistance to bac-teriophage Ti may be isolated with little difficultyfrom ultraviolet-irradiated E. coli B/r cultures.The atypical strains variably resistant to Ti havenow been designated as B/r/lv, in contrast toresistant B/r/i and sensitive B/r. No mutantsubcultures lost the property of variable resistancefor which the mutants were initially isolated and,except for their anomalous resistance to Ti, allB/r/lv strains were found to be similar to theparent B/r strain. Thus, all were sensitive tostreptomycin and sodium azide, were able toferment lactose, arabinose, xylose, and galactose,and were unable to ferment maltose. None of25 B/r/lv strains was fully resistant to bacterio-phage T5, although 4 exhibited a cross-streakresponse to T5 which was similar to that of B/r/lvtested with Ti as shown in Fig. 1. Absence ofcross-resistance to T5 in any B/r/lv strain con-trasted with B/r/l types, of which an expectedlyhigh proportion showed resistance to this phage(5). Attempts to detect an ultraviolet-inducible orspontaneously inducible prophage in a B/r/lvculture were unsuccessful. Under conditions ofirradiation which resulted in the induction andeventual lysis of a prophage-carrying strain,W1177, no similar fate was observed for theidentically treated B/r/lv culture. Also, in no

1056 J. BACTERIOL.

on May 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

VARIABLE RESISTANCE TO BACTERIOPHAGE

FIG. 1. Portion of a petri dish containing NutrientAgar upon which different saline suspensions of Esch-erichia coli have been cross-streaked against phageTi. Top to bottom: sensitive (B/r), variably resistant(B/r/lv), and resistant (B/r/l). The bacterial streakswere made from right to left across a vertical phagestreak.

instance did plaques appear on Nutrient Agarplates seeded with TI-sensitive cells (B/r) andspread with a B/r/lv culture.

Effect of Ti on singly isolated B/r, B/r/l, andB/r/lv cells. In preliminary micromanipulationexperiments, 18 bacteria were singly isolated fromearly log and early stationary phase populationsof a model B/r/lv strain, and were allowed todivide once in Penassay Broth; one of thedaughters was then transferred to a Ti phagelysate. All results to be described were obtainedfrom experiments involving this model strain. Ithad a laboratory stock designation of W-RIM-3and had been given all tests described in the pre-vious section. In the presence of phage, all singlyisolated cells showed one of the following re-sponses: no division with eventual lysis; onedivision with eventual lysis of both daughter cells;formation of clones that lysed after 12 to 18 hr ofincubation. Where delayed lysis took place, itappeared that progeny susceptible to lysis arosethroughout the clonal histories. The 18 controlisolates in Penassay Broth divided regularly, andcultures derived from each of the developingclones gave a cross-streak response to Ti that wassimilar to the initial B/r/lv culture (Fig. 1).

In other investigations, single cells were isolatedfrom B/r/lv cultures in mid-log, and betweenlate-log and early-stationary phases. The cells

were placed into a phage Ti lysate. The progenyof these cells, if any, were separately isolated inthe phage lysate through five generations. Some ofthe descendants of the singly isolated cells were

sensitive to phage, whereas others appeared to beresistant (Fig. 2 and 3). Also, the number of phage-sensitive descendants reflected differences in thephage susceptibility of the mid-log, and late-logto early-stationary phase, populations.Table 1 gives the result of experiments in which

B/r/lv bacteria taken from various growth phaseswere placed singly into phage Ti medium andobserved periodically. By determining the num-ber of cells that formed clones (which eventuallylysed), and that did not, it was possible to deter-mine the proportion of resistant and sensitivebacteria in the cultures tested. The data indicatethat populations in the designated stages ofgrowth consist of different proportions of resist-ant and sensitive bacteria.For further studies, single bacteria were isolated

from B/r/lv populations in various phases ofgrowth. The bacteria were allowed to divide oncein the presence of phage Ti. One member of theresulting cell pair was washed by 16 serial transfersthrough Penassay Broth, and its progeny weresingly isolated through six generations. Under

CD1==c:1c ~ cI , IDC:

O, C.O c_, O= C=: c C,C=

/\ /\ ~/r '\/:,\ /\_ _\_

--

_/=

FIG 2 Effect of Ti on the progeny of si/gle cellsisolated from mid log phase (4.0 hr of incubationl)B/r/Jv cultures. Open figures representt cells that di-vided. Closed figures represenit cells that failed to divideand eventually lysed. All terminal cells formed cloniesthat eventually lysed.

1057VOL. 92, 1966

on May 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CARTA AND BRYSON

these conditions, the terminal cells consisted ofclones that were either normal or abnormal, theabnormal being characterized by chain formationand clumping. However, no infectious phage was

c= ~ ~~~~~~CZD C \

o\oo o \ / \ooo

= =

FIG./ wee\

-loo l( ofoin' C

C=I C=, C'C=Dooooo oooo:

FIG.3.Same a Fig. 2exceptthat sigle cels werislae CroB//vppltin nasag DfgotAewe lae-o Aneal-ttonr hs\(. roincubation).C

detectable upon assaying droplets containing 18normal and 36 abnormal clones from four experi-ments, with B/r as the indicator strain.

Controls for micromanipulation experimentsrequired observing the effect of Ti on singly iso-lated bacteria that were either sensitive or resis-tant to Ti: parental strain B/r versus conventionalB/r/l or B/r/1,5 mutants. In the presence ofphage, the sensitive cells failed to divide andeventually lysed, whereas resistant cells dividedregularly through five generations. Lysis of sensi-tive and variably resistant bacteria in the sensitivestate was observed microscopically to be similar:cells either faded out slowly or swelled and dis-appeared.

Cloning efficiency ofB/r/lv cells in the presenceof Ti. Figure 4 shows representative results ofexperiments designed to detect at various timesthe proportion of sensitive and resistant bacteriain growing B/r/lv broth cultures. As a control,sensitive B/r cells were treated in a similar man-

ner.There was a considerable change in the pro-

portion of resistant and sensitive cells in a grow-ing B/r/lv population. For corresponding periodsof time, the direction of this change, as deter-mined by percentage of colony formation in com-parison with bacteriophage-free controls, was inagreement with the changing percentage of cloneformation ascertained by micromanipulation(Table 1). In all experiments, the plots of per-centage of colony formation versus time were

similar, and the peak of resistance occurred con-

TABLE 1. Effect of Ti on single cells isolatedfrom B/r/lv populations in various phases ofgrowth

Single cells Cells forming Cumulative %Growth phase of B/r/lv populations placed in Cells not clones that cells forming

medium forming clones eventually lyse clones thatcontaining Ti eventually lyse

Early log (2.5 hr of incubation) 31 27 431 25 624 18 629 20 935 24 11

Total 150 114 36 24

Mid log (4 hr of incubation) 62 23 3932 15 1742 18 24

Total 136 56 80 59

Between late log and early stationary (7.5 hr 35 32 3of incubation) 36 34 2

51 48 3Total 122 114 8 7

Early stationary (9 hr of incubation) 50 50 024 24 0

Total 74 74 0 <1

1058 J. BACTERIOL.

on May 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

VARIABLE RESISTANCE TO BACTEIRIOPHAGE

0 1 2 3 4 5 6 7 8 9TIME IN HOURS

z0

I-20

z0-J0U

zLAJU

0.

10-2

FIG. 4. Viable count (0) and percentage of colonyformation in the presence of excess Ti (A) for aB/r/lv culture. Sampled at different stages of growth.

sistently during mid-log phase, with values rang-ing from 43 to 89 %. Also, there was aregular drop in percentage of colony formation0.5 to 1.5 hr after the start of incubation. Coloniesthat arose from B/r/lv cells spread with phageTi characteristically had within them areas oflysis. This independently indicated the unstablenature of B/r/lv phenotypes.

Plaque formation by Ti on B/r and B/r/lv cul-tures. Comparisons were made of the plaque-forming ability of equal volumes of the sameTi preparation plated on Nutrient Agar spreadwith bacteria from B/r and B/r/lv cultures thathad previously been incubated for various lengthsof time. These results (Fig. 5) further confirmthe change in Ti susceptibility of a growingB/r/lv population. The initially highly resistantB/r/lv culture, upon further incubation, becameprogressively more sensitive as indicated by theincrease in plaque count. Also, efficiency ofplating values showed a striking change, from0.05 at 4 hr to 0.93 at 7 hr, when the phage sus-ceptibility of the B/r/lv and B/r cultures wasnearly the same. Turbid plaques appearing onB/r/lv cultures were one-sixth to one-half thediameter of the clear plaques on the sensitive B/rstrain. The least turbid and largest plaques oc-curred on plates spread with bacteria from cul-tures previously incubated for 5.5 to 7.0 hr. Some

U)

a-JQ.

6 7TIME IN HOURS

1.00

0.90

0.80

0.70

0.60

0.50 C.0

0.40 i

0.30

0.20

0.100.00

FIG. 5. Plaque formation by equal volumes of thesame Ti preparation on B/r (0) and B/r/lv (A)cultures that had previously been incubated for variouslengths of time; (O) EOP (efficiency ofplating) is theratio ofplaques on B/r/lv to plaques on B/r.

of these plaques were picked and inoculated intoearly log phase B/r and B/r/lv cultures. Theformer cultures lysed, whereas the latter showedan irregular lysis typical for B/r/lv populationsinfected with B/r-propagated Ti (see below). Onthe basis of the cross-streak test, the B/r/lvstrain still showed a variable resistance to B/r/lv-propagated Ti. Evidently, there was no evidenceof host-induced modification.

Lytic effect of Ti on B/r, B/r/l, and B/r/lvcultures. When early log phase B/r, B/r/l, andB/r/lv populations were infected with Ti at amultiplicity of one, the turbidity of the B/r culturebegan to decrease within 20 min after phage addi-tion and that of the B/r/l culture continued toincrease linearly. However, the B/r/lv cultureshowed an initial steady rise in turbidity fromtime zero to 3 hr, followed by a divergence fromlinearity (indicating growth retardation) com-mencing between 3 and 4 hr. Control B/r/lvcultures grew normally when phage was omitted.It should be noted that the time intervals repre-sent total incubation periods of the cultures ofof 2.5 to 5.5 and 5.5 to 6.5 hr, respectively, thelater interval corresponding to the phenotypictransition phase of B/r/lv populations fromresistance to sensitivity (Fig. 4).Production of Ti by Blr and B/r/lv cultures.

The latent period of Ti production by B/r andB/r/lv cultures was 12 to 14 min (Fig. 6). The

1059VOL. 92, 1966

on May 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CARTA AND BRYSON

107 1, , , I II, I I

0 10 20 3061 2 3 4 5 6 7 8 9 10 12TIME IN MINUTES TIME IN HOURS

FIG. 6. Ti production by Blr (A), B/rl (O), andB/r/lv (0) populations infected during early logphase.

maximal phage titer in the B/r population was

reached 0.5 to 1 hr after infection; in the B/r/lvpopulation maximal titer was reached 10 hr afterinitial contact with phage. In the B/r/lv incuba-tion mixture, it appeared that the lytic cycle con-sisted of two major burst phases, one commencingafter the latent period and another at approxi-mately 3 hr of incubation (total incubation ofculture, 5.5 hr; see Fig. 4). For equal time inter-vals, the phage yields in both phases were lowerthan that resulting from the single burst period ofthe sensitive culture. This low but prolongedphage yield indicated both the phenotypic hetero-geneity of the B/r/lv population and the con-tinued descendance of sensitive cells frominitally resistant cells. Phage propagated on B/rand B/r/lv showed no apparent differences inplaque morphology when assayed on B/r.

Streaks from 30-hr incubation mixtures con-taining TI plus B/r/lv cells were made on Nu-trient Agar. All of 33 B/r/lv colonies thusisolated were sensitive to bacteriophage T5. Of35 typically Tl-resistant colonies, arising from thesame type of mixture incubated for 25 to 30 hr,30 colonies were resistant and 5 were sensitive toT5.

When Ti was propagated three consecutivetimes on early log phase B/r/lv populations, eachtime using the previous lysate, the following re-sults were obtained: each of the three consecu-tively infected cultures showed irregular lysis;each reached maximal phage titers of 4 X 1010 to5 X 10'°; on the basis of the cross-streak test, eachlysate versus B/r/lv gave the variable response.

Adsorption of Ti by B/r and B/r/lv cultures.By use of the procedure in which adsorption mix-tures were chilled prior to and during centrifuga-tion, no adsorption of Ti was observed for B/r/ior B/r/lv cultures. Under similar conditions, themaximal adsorption of phage by B/r cultures was95 %. With the modified Fredericq technique (6),however, B/r/i cultures showed no phage adsorp-tion, whereas B/r and B/r/lv cultures showedphage adsorption of 85 and 22%, respectively,both systems employing cells in the late-log phase(6.0 hr of incubation).

DISCUSSION

A B/r/lv cell, although genetically stable, maybe either phenotypically sensitive or resistant tothe typically virulent phage Ti. In our experi-ments, no evidence suggests that the variableresponse is attributable to a genetic heterogeneityof phage, host-modified phage, a lysogenic asso-ciation between phage and the B/r/lv cell, acarrier state, or genetic heterogeneity of thebacterial host.

Variable resistance and the "semi-resistance" ofWahl and Blum-Emerique (10-13) appear to bemanifestations of essentially similar host-phageinteractions. These investigators, employingShigella paradysenteriae and bacteriophage S13,proposed that a semiresistant population consistsof a constant proportion of cells in the receptiveand refractory states. They provisionally attributedto phage, and certain ions (calcium and phos-phate), the ability to alter the proportions of bothtypes of cells. However, their experiments showthat in peptone broth a 10-fold increase in thepercentage of refractory or resistant cells occursduring logarithmic growth of a semiresistant cul-ture when phage is omitted and calcium or phos-phate concentrations are not varied. These obser-vations are in agreement with results presentedhere: a variably resistant mutant population be-comes increasingly resistant during logarithmicgrowth.Our interpretation of plaque formation by Ti

on B/r/lv cultures is similar to the explanationgiven for plaques formed on semiresistant Shigellastrains (9); i.e., development of a plaque is initiatedby phage infection of a cell in the sensitive state,followed by release of phage progeny whichfurther infect other sensitive bacteria in the im-

1060 J. BACTERIOL.

on May 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

VARIABLE RESISTANCE TO BACTERIOPHAGE

mediate vicinity. Plaque size and turbidity wouldbe dependent on the proportion of sensitive andresistant cells in the area of lysis.With conventional methods of measurement by

difference, based on chilling and centrifugingadsorption mixtures, adsorption ofTi to a B/r/l vpopulation was not detectable. This result sug-gested that chilling of a B/r/lv-Tl mixturecaused an elution of weakly attached phage parti-cles. By employing the alternative chloroformtechnique, carried out at 37 C, a maximum phageadsorption of 22% was determined for a B/r/lvculture. It has been pointed out (1) that, fortypically sensitive strains, adsorption values below30% are not significant. Because of the unusualnature of the B/r/lv culture, however, the ob-served low value may indicate that in such apopulation the resistant-state cells do not adsorbphage, whereas sensitive state cells do (reversibly,prior to irreversible attachment). Evidence forsuch a two-step adsorption process has beendemonstrated for typically sensitive cells (7). Theview that resistant state cells do not adsorb phagewas further supported by results of single-celltechniques in which neither lysis nor phage couldbe detected in cultures derived from single B/r/lvbacteria that, after having divided once in thepresence of phage, were washed and isolated inphage-free medium. Two lines of evidence suggestthat B/r/lv cells in the sensitive state respond toTi much like typically sensitive bacteria. Individ-ual cell lysis of each type was visually similar,and the latent period of phage production forpopulations of either was approximately 13 min.The alternative responses to phage, exhibited

by B/r/lv bacteria, appear to be determined atthe adsorption stage of host-phage interaction. Ifsuch were the case, then a B/r/lv cell, dependingon its physiological state, would have a cell walleither conducive or nonconducive to phageattachment.

ACKNOWLEDGMENT

This investigation was supported by Public HealthService grants 5-T1-GM-507, from the Division ofGeneral Medical Sciences, and AI-2615, from theNational Institute of Allergy and Infectious Diseases.

LITERATURE CITED

1. ADAMS, M. H. 1959. Bacteriophages. IntersciencePublishers, Inc., New York.

2. BRYSON, V., AND H. DAVIDSON. 1951. Spontane-ous and ultra-violet-induced mutations to phagein Escherichia coli. Proc. Natl. Acad. Sci. U.S.37:784-791.

3. DAVIS, B. D., AND E. S. MINGIOLI. 1950. Mutantsof Escherichia coli requiring methionine orvitamin B12. J. Bacteriol. 60:17-28.

4. DE FONBRUNE, P. 1949. Technique de micromani-pulation, 1st ed. Masson et Cie, Paris.

5. DEMEREC, M., AND U. FANO. 1945. Bacterio-phage-resistant mutants in Escherichia coli.Genetics 30:119-136.

6. FRtDERICQ, P. 1952. Emploi du chloroforme pourmesurer le taux de fixation des enterobacterio-phages par les bacteries vivantes. Compt. Rend.146:327-329.

7. GAREN, A., AND T. T. PUCK. 1951. The first oftwo steps of the invasion of host cells by bac-terial viruses. II. J. Exptl. Med. 94:177-189.

8. JoNEs, L. M., C. R. McDUFF, AND J. B. WILSON.1962. Phenotypic alterations in the colonialmorphology of Brucella abortus due to a bac-teriophage carrier state. J. Bacteriol. 83:860-866.

9. WAHL, R. 1953. La semi-resistance aux bacterio-phage. Ann. Inst. Pasteur 84:51-59.

10. WAHL, R., AND L. BLUM-EMERIQUE. 1952. Lesbacteries semi-resistantes au bacteriophage. I.Techniques: Souches et phage utilises. Lyse surmilieu gelose. Ann. Inst. Pasteur 82:29-43.

11. WAHL, R., AND L. BLUM-EMERIQUE. 1952. Lesbacteries semi-resistantes au bacteriophage. II.etude quantitative de l'adsorption des phagessur les bacteries semi-resistantes et de l'infectionde des bacteries. Ann. Inst. Pasteur 82:44-49.

12. WAHL, R., AND L. BLUM-EMERIQUE. 1952. Lesbacteries semi-resistantes au bacteriophage.III. Etude quantitative de la production desphages par les bacteries semi-resistantes in-fectees. Ann. Inst. Pasteur 82:194-205.

13. WAHL, R., AND L. BLUM-EMERIQUE. 1952. Lesbacteries semi-resistantes au bacteriophage.IV. Influence de la concentration de certainsions sur le comportement des bacte'ries semi-resistantes vis-a-vis du phage. Signification deces bacteries. Ann. Inst. Pasteur 82:266-276.

14. WITKIN, E. M. 1946. Inherited differences in sensi-tivity to radiation in Escherichia coli. Proc.Natl. Acad. Sci. U.S. 32:59-68.

1061VOL. 92, 1966

on May 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from