Host-controlled variations in bacteriophages active against lactic streptococci

VIROLOGY

2,

261-271 (1956)

Host-Controlled

Variations in Bacteriophages Against Lactic Streptococci E. B.

Department

of Dairy

Industry,

Active

COLLINS

University

Accepted January

oj California,

Davis,

(‘alijornia

26, 1956

Certain bacteriophages were very active against some strains of Streptococcus cremoris, and restricted in activity against others. Drastic changes in activity were observed after one growth cycle. The results of adsorption of a restricted bacteriophage was either (1) no detected consequence, (2) bacterial death, or (3) bacterial death followed by bacteriophage reproduction. The occurrence of each of the last two possibilities was found to be influenced by the multiplicity of infection. Bacteriophage reproduction within t.he fruitful cells of a restrictive host produced progeny that were fully active on the cells of that host. Alteration of the bacteriophages appeared limited to changes in virulence for particular hosts, the alterations not. being accompanied by changes in adsorbability or changes in heat resist)ance. Both the strain of infecting bacteriophage and the particular host were important, factors in determining the specific virulence of the progeny. INTRODUCTION

Cross-infection has been used extensively as a technique for establishing similarities and differences among strains of lactic streptococci and the bacteriophages active against them. A study of similarities and differences in activity patterns resulted in the present investigation of host-controlled variations. Host-controlled variations are phenotypic, transient changes in bacteriophages produced by a single growth cycle on a new host (Luria and Human, 1952). Such variations have been reported for bacteriophages active against Escherichia coli and Shigella dysenteriae (Luria and Human, 1952; Bertani and Weigle, 1953), SalmoneEla typhi (Anderson and Felix, 1952), and Staphylococcus (Ralston and Krueger, 1952). The reports of Collins (1953) and Nelson (1954) suggest that host-controlled variations occurred in the bacteriophages active against Streptococcus cremoris, but t.he descriptions in the reports are inadequate for a definite conclusion. This paper describes host-controlled variat,ions in bacteriophages ac261

262

E.

B.

COLLINS

tive against 8. cremoris and shows that for the restricted bacteriophages the multiplicity of infection was important in determining the fate of adsorbed particles. MATERIALS

AND METHODS

Bacterial strains. Four strains of S. cremoris, isolated from four different commercial cultures used in making cottage cheese, were labeled Ml, M2, M3, and M4. Each strain was purified several times by colony isolation. None was found to carry a bacteriophage active against any of the other three strains. Bacterial inoculum. Unless indicated otherwise, young, actively growing bacteria were used for the studies that involved the use of adsorption mixtures. These young bacteria were prepared by inoculating bacteria into skimmilk and permitting them to grow at 32” for 2 hours just before they were used in an experiment. Cultures that had been incubated for 16 hours were used for determinations and experiments not requiring adsorption mixtures. Bacteriophage strains. Homologous strains of bacteriophage were isolated from vats of cottage cheese in which the commercial cultures (referred to above) had produced lactic acid slowly. The bacteriophages were purified several times by plaque isolation. The bacteriophage strains were labeled A(Ml), B(M2), C(M3), and D(M4), corresponding respectively to the four bacterial strains. The experiments reported in this paper involve bacteriophages that have been propagated on different hosts. Thus, the particular host that has been employed in the growth of a given phage preparation is identified in parentheses as a part of the designation of that bacteriophage preparation. Cross-activity. The cross-activities of bacteriophages were determined in litmus milk by a previously described method (Collins, 1955). Bacteriophage enumeration. Bacteriophages were enumerated either by the limiting active dilution method, as most probable numbers (Collins, 1951), or by plaque count (Potter and Nelson, 1952). RESULTS

Activity of the bacteriophages as determined with di$erent hosts. It was noticed from cross-activity determinations that bacteriophages A(M1) and B(M2) inhibited the growth of bacterial strains Ml, M2, and M3, but not M4; C(M3) and D(M4) inhibited M2, M3, and M4, but not Ml. Additional comparison showed that the plaque counts for each bact’erio-

HOST-CONTROLLED

VARIATIOSS

TABLE (:~IASGES

IS

Bacteriophage A (Ml)

3f312)

CN3)

THE

ACTIVITIES

IN

263

BACTERIOPHAGES

1

OF BACTERIOPHAGES DURIXG WITH DIFFERENT HOSTS

YLAQCE

FORMATION

Av most probable no.’ of particles/ml of four plaque suspensions Streplococcur actlve against culture: ctemeris strain used for plaque --m-mmII -x2 x3 formation Ml

m.2 M3 Ml M2 h’I3 hIZ M3

7.6 1.0 6.0 1.0 5.8 4.3

X 105” x 103 X lo3 x 10’ x 10” x 103 <0.33 <0.33

1 .o 1.0 6.6 6.9 1.0 2.6 6.X 1.0

x X x X X X X x

10’ 10’ 106 lo6 10’ IO6 106 105

7.2 1.0 1.0 1.6 I.‘4 1.0 1.0 1 .o

x 103 x 10’ x 10’ x 103 x 10’ x 107 x IO’ x 10’

(6 To facilitate comparison the av values for quadruplet suspensions were multiplied by the fact.or necessary t.o make the highest value 1.0 X 10’. * Plaque counts for bacteria-free filtrates did not substantiate this low value.

phage differed considerably, depending upon the host used for enumerat,ion. The plaque counts per ml on hosts Ml, M2, and M3, respectively, were: of a lysate of bacteriophage A(Ml), 2.0 X log, 3.0 X log, and 5.4 X 104; of a lysate of B(M2), 7.6 X 103, 1.3 X log, and 4.0 X lo*; and of a lysate of C(M3), <0.5, 2.2 X 106, and 2.2 X 10s. Ch,anges in activity during plaque formation with diferent hosts. Plaques were suspended in sterile skimmilk and titers were determined. The data in Table 1 shorn that the activity patterns of the bacteriophages did change during plaque formation on t,he different hosts. Propagation of bacteriophages A(M1) and B(M2) on a common host yielded progeny t#hat, were the same; B(M2) and C(M2) were different. Heat resistance after propagation with diferent hosts. The data in Fig. 1 show that changes in activiby patterns were not accompanied by changes in heat resistance or sensitivit,y. They substantiate t,he above indication (Table I) that bacteriophages A(M1) and B(M2) were the same, except, as modified by t’he hosts used for propagation. Bacteriophages C(M3) and D(M3) were different. Most of the work discussed in the following sections concerns phage B grown on various hosts. Eflect on plating eficiency of changes in the plaque-plating procedzlrc. Changes in the plaque-plating procedure were investigated to determine if t,hey would result in a larger production of plaques by B(M2) on MI. The following did not result in higher plaque counts: (1) substitution of

264

E.

B.

COLLINS

PLAQUESFORMEDON M2

PLAQUESFORMEDON M3

0 FIG. 1. Heat resistance of bacteriophages after propagation with different hosts. Lysates were diluted 1:lO in sterile skimmilk. Quantities of 11 ml of a diluted lysate were dispensed into screw-cap test tubes. One quantity served as a control. The others were placed in a water bath, thermostatically controlled at 65”. The tubes were permitted a “come-up” period of 5 minutes. Tap water was used for cooling the tubes after the desired exposure time. Plaque platings were made with Streptococcus cremoris, strain M2 or M3.

skimmilk for the calcium chloride solution ordinarily used as a constituent of the overlay; (2) incubation of plaque plates at 26” and 35”; (3) adjustments of the plaque-plating medium to pH values above and below pH 6.0; (4) use of Ml cultures of different ages as bacterial inoculum. Exclusion of two hypotheses. Plaque counts indicated that there were only about lo4 plaque-forming bacteriophage particles per ml in filtrates of B(M2) when Ml was used in the enumeration procedure, but there were about log infective particles when M2 was used. This difference was similar to that encountered by Luria (1945) in a study of host-range mutations of bacterial viruses; and similar hypotheses could be formulated, namely: (1) that among log particles of B(M2) there were about lo4 variant particles that had stemmed from mutations during the growth of normal particles on sensitive M2 bacteria, (2) that among log

HOST-CONTROLLED

VARIATIONS

IN

BACTERIOPHAGES

265

particles of B(M2) there were about lo4 variant particles that had stemmed from exceptional, abnormal M2 cells, or (3) that the population of particles in filtrate B(M2) was homogeneous-any one of the particles active against a small portion of exceptional, “active” Ml bacteria. The following results indicate that the first two hypotheses did not apply. 1. With a technique similar to that used by Luria, we tested a series of 32 lysates of bacteriophage B(M2), each started with a few M2 bacteria and a few normal bacteriophage particles (510 bacteria and 850 parUes per 0.5-ml sample). Following an incubation period of 6 hours the lysates were assayed on plates seeded with Ml. All of the plaque counts were between 5.0 X lo2 and 2.5 X 103; the average was 1.5 X lo3 per sample. The fluctuations in number of plaques produced on the resistant bacteria (Ml) can be accounted for by sampling error. A distribution with a variance much higher than the average (due to the occurrence of clones of mutant particles) should have resulted if those few particles in B(M2) lysat.es t,hat are active against Ml do in fact, stem from mutations during the growth of normal particles on sensitive M2 bacteria. 2. The hypothesis that, filtrate B(M2) contained a small number of variant particles (approximately one in lob) that were produced by abnormal M2 cells was eliminated by the following experiment. A sample t,hat contained 1.2 X lo8 M2 bacteria per ml was infected with B(M2) and assayed after an adsorption period of 15 minutes. The input bacteriophage assayed 9.4 X 10” and 1.0 X lo* particles per ml on plates seeded with Ml and M2, respectively-a ratio of 1: 106,000. The total infective centers were 7.0 X lo5 and 7.9 X 10’ per ml with Ml and M2, respectively. These values would require an unreasonably large fraction of the M2 cells t.o be abnormal (one in 110). The high frequency of plaques on the plates seeded with Ml was not’ related to the age of the M2 bacteria in the adsorption mixture, since a ratio of 1: 130 was the result) when older bacteria (incubated for 16 hours) were substituted for the young bacteria, The high frequency of plaques was explained, at least in part, by two facts: (1) Most of t’he plaques resulted from infected M2 cells (as opposed to free particles). If each plaque result’ed from a single infected cell, the frequency of particles active against Ml would be one in (110 X burst size). (2) The M2 cells occur in pairs and the input multiplicity of B(M2) was less than one particle per cell. The number of bacteriophage particles per plate (seeded with Ml) likely was increased by bacteriophage multiplication within some of t,he uninfected cells from tbe absorption mixture.

266

E.

B.

COLLINS

TABLE 2 OF BACTERIOPHAGES BY Slreplococcus cremoris, STRAIN Ml

ADSORPTION

Samples of Ml bacteria (about 1.0 X lo7 per ml) were irifected with bacteriophages. After allowing 15 minutes for adsorption at 32”, plaque counts were made immediately for total infective centers. Aliquots of the adsorption mixture?, were centrifuged and the supernatants assayed for unadsorbed bacteriophage. Two hours later the adsorption mixtures were plated for determination of progeny bacteriophage. All platings were made with M2. Bacteriophage B&W

Input bacteriophage/ml Total infective centers/ml Unadsorbed bacteriophage/ml Plaque count/ml after 2 hr Per cent adsorption Burst size

2.4 2.0 7.6 1.2

X x X x 68 137

BW) lo7 107 106 109

6.7 2.4 2.0 7.7

X X x x 70

BW3) lo7 10’ 107 105

6.2 1.9 1.6 1.2

X lo7 X.,10’ X 10’ x 106 74

Adsorption and lcilliv. In view of the probability that the population of particles in filtrate B(M2) was homogeneous, experiments were carried out to determine if the restricted bacteriophages, B(M2) and B(M3), differed from B(M1) in adsorption to Ml or in ability to kill Ml. The data in Table 2 show that the three bacteriophages were adsorbed equally well by Ml. However, plaque counts with M2 suggeted that only the unrestricted bacteriophage, B(Ml), multiplied after adsorption. The following data show that bacteriophages B(M2) and B(M3) did not kill Ml as effectively as did B(M1). Samples of Ml that contained 7.0 X 10’ bacteria per ml were infected with each of the bacteriophages (1.5 X log particles per ml). After 15 minutes for adsorption, the values for survival of bacteria were 81, 9, and 0 per cent with B(M2), B(M3), and B(Ml), respectively. Essentially all of the Ml bacteria were killed by the restricted bacteriophages when the Ml populations were 1.0 X lo’, 1.5 X 106, and 1.0 X lo6 bacteria per milliliter. Change from

restricted

to nonrestricted

activity

in

one growth

cycle.

The fact that essentially all of the Ml bacteria could be killed by the restricted bacteriophages made it possible to determine whether Ml changed the activities of restricted bacteriophages during one growth cycle, and also made it possible to study the latent periods. Experiments were similar

to those whose results are presented

in Table

2, except

that

input, multiplicities of bacteriophages were sufficiently high to permit the killing of very large fractions of the Ml bacteria, and the plaque platings were made on plates seeded with Ml instead of M2. The results

HOST-CONTROLLED

VARIATIONS

IS

TABLE IMULTIPLICATION

267

BACTERIOPHAGES

3

OF BACTERIOPHAGES ON Streptococcus cretaoris, IN ONE GROWTH CYCLE

Samples of Ml were for adsorption at 32”, surviving bacteria were were made immediately were made with Ml.

STRAIN Ml,

infected with bacteriophages. After allowing 20 minutes adsorption mixtures were diluted lo-fold. Platings for made immediately. Platings for numbers of bacteriophage and after different periods of incubation. Plaque platings

Incubation period (minutes)

20 30 40 50 60 70 Input bacteriophage/ml as determined with: Ml M2 Input bacteria/ml Surviving bact,eria/ml (20 min) Burst size/killed colony” Burst size/infective center at 20 min5

Plaaue count/ml after infection with bacterimhace:-

.._--~

B (Ml)

4.0 5.0 1.0 3.2 7.4 7.5

x 106 x 106 x 107 x 10’ x 107 x 107

3.0 x

106

5.2 X 1Oj 1.7 x 105 211 -

3.1 7.3 5.8 9.1 1.x 1.;

x 10’ x 10’ x 101 x 105 x 106 x 106

3.2 4.2 2.8 1.9 1 .o

x X x x x

10’ 10’ 105 106 IO’

1.4

x

107

1.3 1.6 7.1 2.1

x X x x

2.8 1.5 7.1 4.2

x x x x 14 312

IO” 108 105 103

2

58

STPlaque count, after 60 min f (input bact.eria - surviving h Plaque count after 60 min + plaque count after 20 min.

~~

B(M3)

B(M2)

103 lOa 105 10’

bact,eri:r).

in Table 3 show t,hat Ml did change the activities of B(M2) and B(Ms) during one growth cycle. The latent periods for bacteriophages B(M2) and B(M3) were the same as that for B(Ml)---about 50 minutes. Calculation of burst size per killed colony gave extremely low values for B(M2) and B(W). These low values indicated that reproduction of the restricted bacteriophages had been completed in only a portion of the killed bacteria, since reasonable burst sizes were obt,ained by calculaCng burst size per infective center at 20 minutes. Change ,from nonrestkfed to restricted aet~z~ity in one growth cycle. The following results show that during one growth cycle M2 lowered the activity (against Ml) of unrestricted B(M1). A sample of M2 (1.1 X IOx per ml) was infected with B(M1) and assayed after an incubation period of 1 hour. The input bacteriophage assayed 1.2 X lo6 and 2.0 X 106 particles per ml on plates seeded with Ml and M2, respectively. After 1

268

E.

B.

COLLINS

hour the total infective centers were 5.5 X 104 and 1.1 X 108 per ml with Ml and M2. In one growth cycle on M2 the bacteriophage had been modified so as to be restricted against Ml. Part of the plaques (on Ml) counted as infective centers undoubtedly resulted from infected M2 cells (as opposed to free particles), and the plates received some uninfected M2 bacteria from the adsorption mixture. These conditions account for the fact that the plaque count ratio was higher than 1: lo5 (on Ml and M2, respectively). Comparison of young and old bacteria. As can be seen in Table 3, plaque counts (with Ml) for Ml bacteria with preadsorbed, restricted bacteriophages were greater than the counts for input bacteriophage by factors of 11 and 24. These increases could not be the result of multiplication. There was a difference in age, and possibly physiological differences between the bacteria in the adsorption mixtures and those used for plating. But the results of the following experiment indicate that the difference in age did not cause the above differences in plaque count. Samples of young Ml bacteria and samples that had been incubated for 18 hours (old bacteria) were infected with B(M2). The bacterial inputs were 1.3 X lo8 and 1.6 X lo8 per ml for young and old bacteria, respectively; the bacteriophage input (as determined on M2) was 1.2 X log per ml. After 15 minutes at 32”, plaque counts were determined on plates seeded with Ml. For the adsorption mixtures that contained young and old bacteria, respectively, the counts were 9.9 X lo3 and 1.1 X lo4 plaques per ml. The difference between these counts is not sufficiently great to explain the increases shown in Table 3. Plate counts indicated that all of the bacteria survived the 15-minute adsorption period. Results with different multiplicities of infection. That all of the bacteria appeared to survive in the above experiment substantiated an earlier finding that high multiplicities of the restricted bacteriophages are required to kill appreciable numbers of Ml. Preadsorption (in the experiment covered in Table 3) increased the chances of multiple infection. Multiple infection thus might have made possible the high plaque counts for Ml bacteria with preadsorbed bacteriophage. If the numbers of Ml bacteria that are killed and the numbers that are fruitful are influenced by multiple infection with a restricted bacteriophage, the numbers of bacteria that survive should decrease rapidly and the plaque counts on plates seeded with Ml should increase more rapidly than expected when increasing numbers of a restricted bacteriophage are added to adsorption mixtures. These possibilities were found t,o occur wit.h bacteriophages B(M2) and B(M3) in the experiments whose

HOST-CONTROLLED

VARIATIONS

IN

269

BACTERIOPHAGES

results are presented in Table 4. The influence of multiple infection upon the fruitfulness of the Ml bacteria was particularly evident. The results in the table show that with the higher multiplicities of infection, 10 to 13 per cent of the Ml cells (plate count of input bacteria X 2) were fruitful. This eliminated an earlier possibility that only TABLE

4

ISFLITESCE OF MULTIPLE INFECTION WITH RESTRICTED BACTERIOPHAGES UPON THE SURVIVAL OF Streptococcus cremoris, STRAIS Ml, AND UPON THE PLAQUE COUNT OF ADSORPTION MIXTURES Samples

of Ml were adsorption

infected with bacteriophages. After allowing 15 min at 32”, platings for surviving bacteria and for plaque count were made immediately.

Bacteriophage Input/ml as deterrm$;d Strain

B(MI)

Multiplicity” of infection

Bacteria input/ml ..~

-~.-

H(M2)

1.1 2.8 5.6 1.7

x x X x

10’ 107 107 10”

1.1 1.1 1.1 8.5

x 10’ x 10’ x 10’ x 106

B (x3)

3.5 7.0 1.0 1.4 2.0 9.0

x 10” x 10’ x ,209 x 109 x 10” x 10’

7.1) 7.8 7.8 8.6 8.6 1.2

x x x X X X

106 106 106 lo6 lo6 107

15. 31. 45. 57. 81. 2.6

1.2 1.2 2.5 3.7 5.0

x x X x x

1.2 7.9 8.3 8.3 8.2

x x x x x

10’ 106 106 10” 106

3.5 5.3 10. 16. 21.

n It was that all of be in pairs. sumptions

108 10” 10H 10s 10”

0.5 0.0 1.8 T.0

Per tenth of bacteria that survived

100 6-l 19 50 (or greater) 43 22 11 3.5 0 50 (or greater! 39 30 5 0. 5 0

for

Plaque count/ml of adsorption mixture as detknin;d

3.0 x

103

4.3 6.1 5.8 1.5 1.7 6.2

x X X x x X

103 104 105 106 106 10”

2.5 3.2 1.3 2.2 1.2

x x x x x

10” IO’ 106 10” 106

assumed that there was 70 per cent adsorption in each mixture and the bacteria occurred in pairs. (Most of the Ml cells were observed to Some were single; a few’were in chains of four cells.) Wit,h these asthe formula for calculation was:

input ___~X 0.7 Bacteria input X 2 h With bacteriophage B(M1) it was assumed that all counts of input bacteria and of surviving bacteria represented growth from pairs of cells. With bacteriophages B(M2) and B(M3) it was assumed that all count’s of input bacteria represented pairs and that all counts of surviving bacteria represented single cells. In the latter case a survival of 100 per cent, would be recorded as 50 per cent. .Multiplicity

of infection

=

Bacteriophage

270

E.

B.

COLLINS

a very small fraction of the Ml bacteria were exceptional, and thus able to permit reproduction of a restricted bacteriophage. DISCUSSION

One growth cycle on a different host drastically changes the activity of certain bacteriophages. The transformation involves and appears limited to changes in virulence (changes that occur after adsorption in the process of bacteriophage reproduction) for various hosts. This transformation yields a bacteriophage that is considerably more active than was the original bacteriophage for the particular host used in propagation. In this respect the findings are similar to those of Ralston and Krueger (1952) and Bertani and Weigle (1953). They differ from the results of Luria and Human (1952), who found that passage of T2 on two B/4 mutant strains of E. coli resulted in production of bacteriophages that were not highly virulent for the B/4 mutants. Luria and Human (1952) in a study of host-controlled variations observed that the activities of restricted bacteriophages were influenced by the physiological conditions of the bacteria. This conclusion was based upon the fact that a restricted bacteriophage did not multiply in young, actively growing cells of E. coli, yet multiplied in an appreciable fraction of the cells that had been grown for 18 hours. For Streptococcus cremoris, strain Ml, no appreciable difference in plaque formation was found between young and the older bacteria infected with a restricted bacteriophage. Modification of a bacteriophage by growth in a host toward which it was restricted has been considered due to the acceptance of some particle of the restricted bacteriophage by some exceptional, “active” cell of the host (Bertani and Weigle, 1953; Luria, 1953). The results in this paper show that multiple infection with a bacteriophage of low virulence is an important factor influencing the numbers of X. cremoris, strain Ml, that are killed and, particularly, the numbers in which bacteriophage reproduction occurs. With high multiplicities of infection, instances were encountered in which 10 to 13 per cent of the restrictive cells were fruitful, whereas earlier results had indicated that the ratio of exceptional, “active” cells to normal cells was about one in 105. This influence of multiple infection upon fruitfulness cannot’ be attributed to qualitative differences in the ‘Lreceptors” on the bacterial surface. Interaction between particles of a restricted bacteriophage and the Ml cell apparently goes far enough to permit introduction of the material supplied by the bacteriophage. Acceptance (with subsequent production

HOST-COXTROLLED

VAItIATIONS

IN

R.4C!TEl~IOPH.4GES

271

of modified particles) or rejection (with or &hout’ cell death) seems influenced by the amount of material introduced within the cell, i.e., the number of infect,ing particles. Fruitfulness after multiple infection might, result either from a combination of a limiting essential (a mimke quantity possessed by each infecting particle), or from neutralization of a bacteriophage-growt’h-inhibiting mechanism. Each progeny particle would have a sufficient quantit’y of the limiting essential, or would be modified so as t,o be able to function in the presence of the nom ineffective inhibiting mechanism. Present data do not permit choosing bet,ween the above possibilities. In these host-controlled variations the specific virulenre of the progeny was influenced by the host and by the infecting bacteriophage. That different hosts produced different progenies is evident in Table 1 in that the activity pat,terns of B(Ml), B(M2), and B(W) were different. The influenre of the infecting bacteriophage in determining the activitv pattern of the progeny is apparent from t,he differences between B(M31 and C(M3) and between B(M2) and C(M2). REFEREKCES Ia:. H., md FELIX, A. (1952). Variations in Vi-phage II of Sal~onelln typhi. Suture 170, 492-493. HERTANI, G., and WEIGLE, J. J. (1953). Host controlled variations in bwterial viruses. J. Bwteriol. 66, 113-121. COLLIXS, Ii:. Is. (1951). Relation of different numbers of bacteriophage and b:tc*teria to population changes and acid production. J. Dairy Sci. 34, 894-904. COLLINS, I?. R. (1953). Influence of host on adaptat,ions of bacteriophage itct,ivc against lactic streptococci. J. Dairy Sci. 36, 563 (abstract). COLLINS, E. 13. (1955). Action of bacteriophage on mixed strain cultures. IV. 1)omination among strains of lactic streptococci. ;Ipp/. Microbid. 3, 1-11-144. I,VRIA, S. I<. (1945). Mutations of hactcrial viruses affecting their host range. Genetics 30, 81-99. I,URIA, S. I<. (1953) Host-induced modifications of virurrs. (‘old iipriuy Ha&u S~/ncposia Quant. Rid. 16, 237-243. IIv~~.4. S. I<., and Hunras, M. I,. (1952). A nonhereditary. host-induced variation of bact~erial viruses. J. Hacteriol. 64, 557-569. Xsr,sos. F. 15. (1954). The influence of host strain employed upon thr quantitatiw :tspect,s of lactic streptococcus bacteriophage activity. Hrrc(r~iol. /‘WC. 1964, 49 (abstract). POTTER, PG. N., and ~YELSON, F. 13. (1952). E:ffects of calcium on prolifer:tt,ion of lactic strept,ococcus bacteriophage. I. Studies on plaque formation with a modified plating technique. J. Bacterial. 64, 105-111. RALSTOX, I). J., and KRCEGER, A. I’. (1952). I’hage mulliplication on t.wo hosts. Isolation and activit,y of variants of staphylococcus phage I’, Prw. S’oc. &rpf/. Hid. Med. 80, 217-220. .INDERSOS,