JOURNAL OF VIROLOGY, Aug. 1976, p. 659-667 Copyright © 1976 American Society for Microbiology
Vol. 19, No. 2
Printed in U.S.A.
Characterization of Some Pneumococcal Bacteriophages RONALD D. PORTER AND WALTER R. GUILD* Biochemistry Department, Duke University, Durham, North Carolina 27710
Received for publication 8 March 1976
The growth of pneumococcal phages at high cell and phage densities is enhanced strongly by the substitution of potassium for sodium in the medium. Initial titers of 2 x 1010 to 4 x 1010 PFU/ml are readily obtained, and concentrated stocks are stable in a storage buffer described here. The mechanism of the cation effect is obscure. Phages cw3 and w8 each have linear double-stranded DNA of 33 x 106 daltons per particle, with an apparent guanine plus cytosine content of 47 to 49 mol%, as determined by buoyancy and melting temperature, but with an unusual absorbance spectrum. Efficiency of plating is high if sufficient time is allowed for a relatively slow adsorption, which differs severalfold in rate between the two phages. Morphologically, these and other pneumococcal phages are similar to coliphage lambda but with a longer tail and tail fiber. Upon UV inactivation, w3 and Cl8 have D37 values of 33 and 55 J/m2, respectively, and each shows multiplicity reactivation. A total of 13 ts mutants have been isolated from the two phages, representing only two complementation groups; complementation and recombination occur between co3 and co8 mutants. Both phages provoke high-titer antisera with extensive cross-reactivity against a number of newly isolated pneumococcal phages.
Well-characterized bacteriophages for pneu- blood was obtained from the Duke Hospital blood mococcus (Diplococcus pneumoniae) could offer bank. Bacteria and phage strains. D. pneumoniae Rxl, a number of genetic tools to complement those in all experiments unless otherwise specified, from transformation, until now the only means used is a of strain Rx (18). Phages w3 and cw8 were subline of studying gene transfer and recombination in obtained from Tiraby in 1973 and used for all of this species. Pneumococcal phages were first the experimentsG.described. isolated by Tiraby (personal communication, Phage storage and dilution buffer. TMF consists 1973; reference 20), and another such phage has of 0.01 M Tris, 0.001 M MgCl2, and 10-6 M FeSO4, been described by McDonnell et al. (13). In each adjusted with HCl to pH 7.4. This was used for case, a major barrier to full exploitation of the dilutions and routine storage of phage stocks at 4°C phage was that initial lysates showed low ti- (see Results). Media. In most of the work described here, standters, typically between 108 and 109 PFU/ml, and ard CAT medium or a modification of it, in which poor stability during storage. was substituted for NaCl, was used. Because We studied two of Tiraby's phages, w3 and KCl substitution is critical for obtaining high-titer w8, and found a strong cation effect on their this lysates (see Results), we use the terms Na-CAT and growth. This phenomenon, of potential interest K-CAT to denote which version is used. If not speciin itself, allows ready production of stable, fied, Na-CAT is implied as the normal growth mehigh-titer lysates and has facilitated a number dium for Rx strains. CAT consists of 1.0% Casitone of additional experiments described here, in- (Difco), 0.5% tryptone (Difco), 0.1% yeast extract cluding preliminary characterizations of the (Difco), and 0.5% NaCl or KCl; it was autoclaved and then supplemented with 10 ml of 20% glucose phage DNAs, UV inactivation and multiplicity and 30 ml of 0.5 M K2HPO4 per liter. reactivation of the phage, the isolation of ts growth. Routine growth of phage stocks mutant phages, and complementation and re- wasPhage in K-CAT (see Results). A starter culture done combination among the mutants. at 3 x 108 to 4 x 108 cells per ml was infected at a MATERIALS AND METHODS Reagents. Chloramphenicol was obtained from Sigma Chemical Co., hydroxylamine hydrochloride and polyethylene glycol 6000 (PEG) were from J. T. Baker Chemical Co., and Pronase (B grade) was from Calbiochem. Other compounds were reagent grade or were Difco products. Outdated human
multiplicity of infection (MOI) of about 3. After full lysis occurred, usually in 130 to 150 min, the culture was successively diluted 20-fold into more cells at the same density until the desired volume of lysate was obtained. This lysate typically contains 2 x 10'° to 4 x 1010 PFU/ml, whereas in Na-CAT or other media containing sodium ions the titer is 10 to 100 times lower (see Results). 659
660
PORTER AND GUILD
Phage purification. After clarification at low speed, the final lysate was concentrated 1,000-fold by the NaCI-PEG procedure of Yamamoto et al. (23), followed by centrifugation of the suspended pellet at 30,000 rpm and suspension in TMF. Recovery of PFU can be as high as 90% at this point, provided the PEG is added soon after lysis. For electron microscope examination, preparation of antibody, or DNA preparation, this stock was purified further on step gradients of CsCl in TMF, followed by dialysis against two changes of TMF, and storage at 4°C. We have not attempted to concentrate the phage in this step but only to remove debris. The titer usually drops about twofold at this step, and such stocks have ranged from 2 x 1012 to 8 x 1012 PFU/ml. Plaque assays. For routine titer, 0.1 ml of a phage dilution was preincubated for 30 min at 37°C with 0.9 ml of strain Rxl at about 108 cells per ml. Of this adsorption mix, 0.1 ml was added to 1.4 ml of CAT, mixed with 1.5 ml of top agar, and poured on plates. Top agar contains Na-CAT, 1.0% agar, 5 ,ug of chloramphenicol per ml, 2% blood, and 0.2% supplemental sterile yeast extract. Base plates (100-mm diameter) contain Na-CAT, 1.2% agar, and 10-2 M Tris (pH 7.5). This procedure puts about 107 cells into a 0.5% agar layer about 1 mm thick, comparable to the diameter of a typical plaque. With higher cell concentrations or thicker agar layers, visualization of plaques is poor. The chloramphenicol, suggested by T. A. Trautner, apparently slows the growth of the lawn more than that of the phage and gives larger plaques. This size still varies substantially, however, and plaque morphology mutants have not yet been observed. Omission of the preadsorption step reduces the number of plaques two- to fivefold. We find that Tris plus phosphate in the base plate gives better results than either buffer alone. Mutagenesis and selection of ts mutants. Phage at about 1010 PFU/ml in TMF were mixed with an equal volume of 0.8 M NH2OH-0.003 M EDTA (pH 6.0) (7), incubated at room temperature 30 h, diluted 100-fold into 5% acetone in CAT, and stored at 4°C. This stock showed a survival of 10% and was assayed, as follows, over a period of several weeks for mutants unable to produce plaques at 37°C. A culture of Rxl was infected at 30°C at low MOI. After 30 min it was divided into aliquots, each of which was diluted 10-fold so that, after 200 min at 30°C, 0.1 ml would yield about 200 plaques at 30°C from infective centers produced by adsorption of the progeny of the first burst. From these plaques, stabs were made with sterile toothpicks into duplicate fresh lawns of strain Rxl, one set of which was incubated at 30°C and the other at 37°C. After 12 to 15 h, apparent ts mutants were recovered from the 30°C plates and retested. To ensure isolation of independent mutations, no more than one mutant was kept from each initial subculture. Complementation spot tests. ts mutants were tested for possible complementation by spotting drops of a 5 x 107-PFU/ml stock of each mutant adjacent to each other on a freshly seeded lawn so that the drops overlapped slightly (3). After overnight incubation at 37°C, a clear zone where the drops had fused was taken as a positive indication of complementation.
J. VIROL.
Phage crosses. Phage crosses were done essentially by the method of Edgar and Lielausis (4). Cells (108/ml) in K-CAT were infected at an MOI of 3 to 4 with each of the two ts mutants to be tested. After 30 min at 30°C, the cultures were diluted 104 in K-CAT, and the incubation was continued at 30°C for another 175 min to allow complete lysis of all infected cells without readsorption of the progeny. The lysates were titered by the standard procedure, with parallel platings incubated overnight at 30 and at 370C. DNA preparation. A CsCl-purified phage stock was diluted to about 1012 PFU/ml with 1 x SSC (0.15 M NaCl-0.015 M Na3 citrate), made 0.5% (wt/vol) sodium dodecyl sulfate, and held at room temperature for 5 to 10 min to allow detergent lysis of the phage. This solution was then gently hand rolled for 10 min with double-distilled phenol saturated with 1 M Tris-0.1 M EDTA (pH 8). The tube was then chilled in ice and centrifuged for 5 min at 3,000 x g to separate the phases. The upper aqueous phase was removed with a sterile Pasteur pipette and again extracted with phenol. After the second extraction, the aqueous phase was removed, and the two phenol fractions were pooled and washed with 1 ml of SSC. After phase separation, the aqueous phase was added to the initial one and dialyzed against five changes of SSC for 48 h at 4°C in tubing boiled previously in SSC. UV inactivation. Phage stocks in TMF were irradiated at 254 nm, by swirling in a petri dish 37 cm from a germicidal lamp, where the dose rate was 5 J/m2 per s as determined with a Latarjet dose meter. Antibody preparation. Rabbit antibodies were prepared against w3 and c8. A 0.5-ml volume of Freund adjuvant was mixed with 0.5 ml of 0.15 M NaCl containing about 5 x 1011 PFU per rabbit. The rabbits were injected subcutaneously, and the adjuvant was omitted when booster injections were given. The rabbits were bled periodically through the marginal ear vein, and antiserum titers were determined by the method of Adams (1). Electron microscopy. Pelco grids (200 mesh) were coated with 0.25% Formvar and then carbon coated in a vacuum evaporator. Grids were glow discharged within 48 h of usage. A drop of phage solution containing approximately 1011 PFU/ml was placed on a Parafilm surface. The grid was floated on the phage for 15 s, on a drop of TMF for 30 s, and then on a drop of 2% aqueous uranyl acetate for the desired length of time, usually 1 to 3 min. The specimens were examined in a JEOL JEM-1OOC electron microscope.
RESULTS Initial efforts were directed at obtaining stable, high-titer stocks of one or more pneumococcal phages, a reproducible assay, and a better understanding of the growth cycle. High-titer lysates. In early experiments using Na-CAT medium with or without added blood, we found results comparable to those of others (13, 20), in that small-volume cultures would sometimes lyse readily and sometimes not, yielding 108 to a maximum of 2.5 x 109
VOL. 19, 1976
PFU/ml. Upon examination of the variables, it was discovered that omission of NaCl from the CAT gave severalfold higher yield, and addition of 0.5% KCl (0.085 M) in place of NaCl gave a further threefold increase (Table 1). The "salt-free" CAT was, in fact, 0.03 M in potassium from the K2HPO4 buffer, and further data (not shown) established that 0.10 to 0.12 M K+ is optimal. The depression of yield in Na-CAT, already 0.03 M K+, shows that sodium ion is deleterious to high yield. However, for plaque production on plates, Na-CAT in the top agar appears to be preferable (Table 1), and further tests with K-CAT in both basal and top agar confirmed that Na-CAT gives a larger mean plaque size, although the distribution still ranges from about 0.2 to 2 mm. Results for O3 phage are very similar to those for w8 with respect to Na and K, as illustrated in other experiments described below. Latent period and burst size. At low MOI and low cell density, the latent period of cA8 is about 55 min at 37°C in Na-CAT or in K-CAT, and the apparent burst size is 100 to 120 (Fig. 1). For w3, the latent period is near 60 min and the burst size is at least 200. These are minimum burst sizes because there is no certainty that all phage scored at early times had adsorbed prior to dilution. At 30°C, cw8 showed a latent period near 110 min, with the same burst size as at 37°C. When cells were infected with co3 at high multiplicity (10-min exposure at an MOI of 5) and then diluted 100-fold into either Na-CAT or K-CAT, it was found (data not shown) that (i) the results were independent of the ion in the adsorption medium, and (ii) there was only a small effect of MOI on burst size, more pronounced in Na-CAT than in K-CAT, but not nearly as great as that implied by titers of 2 x 109 PFU/ml from cells infected at 3 x 108 to 4 x 108/ml (Table 1). This suggested strongly that it is a combination of high cell density and multiple infection that is sensitive to the presence of Na or K in liquid medium. We therefore followed culture turbidity and TABLE 1. Dependence of titer of w8 on NaCI or KCl added to CAT medium during growth and plating Salt added to growth mediuma Salt added to top agar plating mediuma
0.5% NaCl
None 0.5% KCl
661
PHAGES OF PNEUMOCOCCUS
0.5%
NaCl
2.5 x 109 1.6 x 109 1.9 x 109
None
7.5 x 109 5.7 x 109 6.4 x 109
0.5%
KCI
2.2 x 1010 1.9 x 1010 1.9 x 1010
a The phosphate buffer adds 0.03 M potassium to each medium. Basal agar on the plates was made with Na-CAT.
0060.120
180
40 I0
E~~~~TM 3
(
i.
.
10
60 120 180 TIME (min.) FIG. 1. Single-step growth curves at low cell density. Strain Rxl at 3 x 108 to 4 x 108 cells per ml was infected at an MOI of about 0.003 at 30 or 37°C and after 5 min was diluted 103 times in fresh medium at the same temperature. At intervals, samples were removed and assayed for PFU. Symbols: *, w8 in KCAT at 37°C; *, co8 in Na-CAT at 3 7°C; 0, c3 in KCAT at 37°C; A, w8 in K-CAT at 30°C. 0
phage production simultaneously for such conditions in the two media (Fig. 2). In K-CAT there is always a substantial increase in turbidity before massive lysis, which sometimes starts at 110 min and is usually complete by 150 min, although it is somewhat delayed in this experiment. In Na-CAT the turbidity rise is much less, and the fall is less sharp. The latent periods for phage production are significantly longer than those at low cell density. Furthermore, in this experiment the phage titer in NaCAT fell at late times to near 2 x 108, whereas it continued to rise in K-CAT. Comparison with other data, such as those in Table 1, shows that such a fall does not always occur in Na-CAT, but titers in the range 108 to 2 x 108 are not uncommon (13, 20). The longer latent period may reflect exhaustion of the medium, in that an uninfected culture at this concentration commonly reaches stationary phase within 90 min in either Na-CAT or K-CAT (data not shown). Adsorption. By titration of supernatants after various times of adsorption, it was found that adsorption rate constants were about 5 x 10-10 for w3 and 10-10 ml/cell per min for w8, both lower than those for most coliphages (1),
662
J. VIROL.
PORTER AND GUILD
and were the same in Na-CAT as in K-CAT (data not shown). The experiments in Table 2, showing increasing number of plaques as time is allowed for adsorption, confirm the faster adsorption of co3, give rate constants about the same as those determined by the usual method, and also show that 20% or more of the phage can adsorb on the plate. Eff'iciency of plating. Comparison of the 5x 10'0
1 10
20-
40
0
80
120
160
200
TIME AFTER INFECTION (min.)
FIG. 2. Growth of &3 at high cell density in KCAT and Na-CAT. Circles, Turbidity of culture infected at zero time at an MOI of3; squares, PFU per milliliter; open symbols, in K-CAT; closed symbols, in Na-CAT. The initial cell concentration was about 4 x 108 cells per ml (2 x 108 colony-forming unitsl ml). Turbidity was measured in 18-mm tubes in a Coleman Nepho-Colorimeter at 600 nm. TABLE 2. Plaques as function of time of preadsorption of &3 and w8a Adsorption time (min)
Plaques observed w3
(O)b
Vol. 19, No. 2
Printed in U.S.A.
Characterization of Some Pneumococcal Bacteriophages RONALD D. PORTER AND WALTER R. GUILD* Biochemistry Department, Duke University, Durham, North Carolina 27710
Received for publication 8 March 1976
The growth of pneumococcal phages at high cell and phage densities is enhanced strongly by the substitution of potassium for sodium in the medium. Initial titers of 2 x 1010 to 4 x 1010 PFU/ml are readily obtained, and concentrated stocks are stable in a storage buffer described here. The mechanism of the cation effect is obscure. Phages cw3 and w8 each have linear double-stranded DNA of 33 x 106 daltons per particle, with an apparent guanine plus cytosine content of 47 to 49 mol%, as determined by buoyancy and melting temperature, but with an unusual absorbance spectrum. Efficiency of plating is high if sufficient time is allowed for a relatively slow adsorption, which differs severalfold in rate between the two phages. Morphologically, these and other pneumococcal phages are similar to coliphage lambda but with a longer tail and tail fiber. Upon UV inactivation, w3 and Cl8 have D37 values of 33 and 55 J/m2, respectively, and each shows multiplicity reactivation. A total of 13 ts mutants have been isolated from the two phages, representing only two complementation groups; complementation and recombination occur between co3 and co8 mutants. Both phages provoke high-titer antisera with extensive cross-reactivity against a number of newly isolated pneumococcal phages.
Well-characterized bacteriophages for pneu- blood was obtained from the Duke Hospital blood mococcus (Diplococcus pneumoniae) could offer bank. Bacteria and phage strains. D. pneumoniae Rxl, a number of genetic tools to complement those in all experiments unless otherwise specified, from transformation, until now the only means used is a of strain Rx (18). Phages w3 and cw8 were subline of studying gene transfer and recombination in obtained from Tiraby in 1973 and used for all of this species. Pneumococcal phages were first the experimentsG.described. isolated by Tiraby (personal communication, Phage storage and dilution buffer. TMF consists 1973; reference 20), and another such phage has of 0.01 M Tris, 0.001 M MgCl2, and 10-6 M FeSO4, been described by McDonnell et al. (13). In each adjusted with HCl to pH 7.4. This was used for case, a major barrier to full exploitation of the dilutions and routine storage of phage stocks at 4°C phage was that initial lysates showed low ti- (see Results). Media. In most of the work described here, standters, typically between 108 and 109 PFU/ml, and ard CAT medium or a modification of it, in which poor stability during storage. was substituted for NaCl, was used. Because We studied two of Tiraby's phages, w3 and KCl substitution is critical for obtaining high-titer w8, and found a strong cation effect on their this lysates (see Results), we use the terms Na-CAT and growth. This phenomenon, of potential interest K-CAT to denote which version is used. If not speciin itself, allows ready production of stable, fied, Na-CAT is implied as the normal growth mehigh-titer lysates and has facilitated a number dium for Rx strains. CAT consists of 1.0% Casitone of additional experiments described here, in- (Difco), 0.5% tryptone (Difco), 0.1% yeast extract cluding preliminary characterizations of the (Difco), and 0.5% NaCl or KCl; it was autoclaved and then supplemented with 10 ml of 20% glucose phage DNAs, UV inactivation and multiplicity and 30 ml of 0.5 M K2HPO4 per liter. reactivation of the phage, the isolation of ts growth. Routine growth of phage stocks mutant phages, and complementation and re- wasPhage in K-CAT (see Results). A starter culture done combination among the mutants. at 3 x 108 to 4 x 108 cells per ml was infected at a MATERIALS AND METHODS Reagents. Chloramphenicol was obtained from Sigma Chemical Co., hydroxylamine hydrochloride and polyethylene glycol 6000 (PEG) were from J. T. Baker Chemical Co., and Pronase (B grade) was from Calbiochem. Other compounds were reagent grade or were Difco products. Outdated human
multiplicity of infection (MOI) of about 3. After full lysis occurred, usually in 130 to 150 min, the culture was successively diluted 20-fold into more cells at the same density until the desired volume of lysate was obtained. This lysate typically contains 2 x 10'° to 4 x 1010 PFU/ml, whereas in Na-CAT or other media containing sodium ions the titer is 10 to 100 times lower (see Results). 659
660
PORTER AND GUILD
Phage purification. After clarification at low speed, the final lysate was concentrated 1,000-fold by the NaCI-PEG procedure of Yamamoto et al. (23), followed by centrifugation of the suspended pellet at 30,000 rpm and suspension in TMF. Recovery of PFU can be as high as 90% at this point, provided the PEG is added soon after lysis. For electron microscope examination, preparation of antibody, or DNA preparation, this stock was purified further on step gradients of CsCl in TMF, followed by dialysis against two changes of TMF, and storage at 4°C. We have not attempted to concentrate the phage in this step but only to remove debris. The titer usually drops about twofold at this step, and such stocks have ranged from 2 x 1012 to 8 x 1012 PFU/ml. Plaque assays. For routine titer, 0.1 ml of a phage dilution was preincubated for 30 min at 37°C with 0.9 ml of strain Rxl at about 108 cells per ml. Of this adsorption mix, 0.1 ml was added to 1.4 ml of CAT, mixed with 1.5 ml of top agar, and poured on plates. Top agar contains Na-CAT, 1.0% agar, 5 ,ug of chloramphenicol per ml, 2% blood, and 0.2% supplemental sterile yeast extract. Base plates (100-mm diameter) contain Na-CAT, 1.2% agar, and 10-2 M Tris (pH 7.5). This procedure puts about 107 cells into a 0.5% agar layer about 1 mm thick, comparable to the diameter of a typical plaque. With higher cell concentrations or thicker agar layers, visualization of plaques is poor. The chloramphenicol, suggested by T. A. Trautner, apparently slows the growth of the lawn more than that of the phage and gives larger plaques. This size still varies substantially, however, and plaque morphology mutants have not yet been observed. Omission of the preadsorption step reduces the number of plaques two- to fivefold. We find that Tris plus phosphate in the base plate gives better results than either buffer alone. Mutagenesis and selection of ts mutants. Phage at about 1010 PFU/ml in TMF were mixed with an equal volume of 0.8 M NH2OH-0.003 M EDTA (pH 6.0) (7), incubated at room temperature 30 h, diluted 100-fold into 5% acetone in CAT, and stored at 4°C. This stock showed a survival of 10% and was assayed, as follows, over a period of several weeks for mutants unable to produce plaques at 37°C. A culture of Rxl was infected at 30°C at low MOI. After 30 min it was divided into aliquots, each of which was diluted 10-fold so that, after 200 min at 30°C, 0.1 ml would yield about 200 plaques at 30°C from infective centers produced by adsorption of the progeny of the first burst. From these plaques, stabs were made with sterile toothpicks into duplicate fresh lawns of strain Rxl, one set of which was incubated at 30°C and the other at 37°C. After 12 to 15 h, apparent ts mutants were recovered from the 30°C plates and retested. To ensure isolation of independent mutations, no more than one mutant was kept from each initial subculture. Complementation spot tests. ts mutants were tested for possible complementation by spotting drops of a 5 x 107-PFU/ml stock of each mutant adjacent to each other on a freshly seeded lawn so that the drops overlapped slightly (3). After overnight incubation at 37°C, a clear zone where the drops had fused was taken as a positive indication of complementation.
J. VIROL.
Phage crosses. Phage crosses were done essentially by the method of Edgar and Lielausis (4). Cells (108/ml) in K-CAT were infected at an MOI of 3 to 4 with each of the two ts mutants to be tested. After 30 min at 30°C, the cultures were diluted 104 in K-CAT, and the incubation was continued at 30°C for another 175 min to allow complete lysis of all infected cells without readsorption of the progeny. The lysates were titered by the standard procedure, with parallel platings incubated overnight at 30 and at 370C. DNA preparation. A CsCl-purified phage stock was diluted to about 1012 PFU/ml with 1 x SSC (0.15 M NaCl-0.015 M Na3 citrate), made 0.5% (wt/vol) sodium dodecyl sulfate, and held at room temperature for 5 to 10 min to allow detergent lysis of the phage. This solution was then gently hand rolled for 10 min with double-distilled phenol saturated with 1 M Tris-0.1 M EDTA (pH 8). The tube was then chilled in ice and centrifuged for 5 min at 3,000 x g to separate the phases. The upper aqueous phase was removed with a sterile Pasteur pipette and again extracted with phenol. After the second extraction, the aqueous phase was removed, and the two phenol fractions were pooled and washed with 1 ml of SSC. After phase separation, the aqueous phase was added to the initial one and dialyzed against five changes of SSC for 48 h at 4°C in tubing boiled previously in SSC. UV inactivation. Phage stocks in TMF were irradiated at 254 nm, by swirling in a petri dish 37 cm from a germicidal lamp, where the dose rate was 5 J/m2 per s as determined with a Latarjet dose meter. Antibody preparation. Rabbit antibodies were prepared against w3 and c8. A 0.5-ml volume of Freund adjuvant was mixed with 0.5 ml of 0.15 M NaCl containing about 5 x 1011 PFU per rabbit. The rabbits were injected subcutaneously, and the adjuvant was omitted when booster injections were given. The rabbits were bled periodically through the marginal ear vein, and antiserum titers were determined by the method of Adams (1). Electron microscopy. Pelco grids (200 mesh) were coated with 0.25% Formvar and then carbon coated in a vacuum evaporator. Grids were glow discharged within 48 h of usage. A drop of phage solution containing approximately 1011 PFU/ml was placed on a Parafilm surface. The grid was floated on the phage for 15 s, on a drop of TMF for 30 s, and then on a drop of 2% aqueous uranyl acetate for the desired length of time, usually 1 to 3 min. The specimens were examined in a JEOL JEM-1OOC electron microscope.
RESULTS Initial efforts were directed at obtaining stable, high-titer stocks of one or more pneumococcal phages, a reproducible assay, and a better understanding of the growth cycle. High-titer lysates. In early experiments using Na-CAT medium with or without added blood, we found results comparable to those of others (13, 20), in that small-volume cultures would sometimes lyse readily and sometimes not, yielding 108 to a maximum of 2.5 x 109
VOL. 19, 1976
PFU/ml. Upon examination of the variables, it was discovered that omission of NaCl from the CAT gave severalfold higher yield, and addition of 0.5% KCl (0.085 M) in place of NaCl gave a further threefold increase (Table 1). The "salt-free" CAT was, in fact, 0.03 M in potassium from the K2HPO4 buffer, and further data (not shown) established that 0.10 to 0.12 M K+ is optimal. The depression of yield in Na-CAT, already 0.03 M K+, shows that sodium ion is deleterious to high yield. However, for plaque production on plates, Na-CAT in the top agar appears to be preferable (Table 1), and further tests with K-CAT in both basal and top agar confirmed that Na-CAT gives a larger mean plaque size, although the distribution still ranges from about 0.2 to 2 mm. Results for O3 phage are very similar to those for w8 with respect to Na and K, as illustrated in other experiments described below. Latent period and burst size. At low MOI and low cell density, the latent period of cA8 is about 55 min at 37°C in Na-CAT or in K-CAT, and the apparent burst size is 100 to 120 (Fig. 1). For w3, the latent period is near 60 min and the burst size is at least 200. These are minimum burst sizes because there is no certainty that all phage scored at early times had adsorbed prior to dilution. At 30°C, cw8 showed a latent period near 110 min, with the same burst size as at 37°C. When cells were infected with co3 at high multiplicity (10-min exposure at an MOI of 5) and then diluted 100-fold into either Na-CAT or K-CAT, it was found (data not shown) that (i) the results were independent of the ion in the adsorption medium, and (ii) there was only a small effect of MOI on burst size, more pronounced in Na-CAT than in K-CAT, but not nearly as great as that implied by titers of 2 x 109 PFU/ml from cells infected at 3 x 108 to 4 x 108/ml (Table 1). This suggested strongly that it is a combination of high cell density and multiple infection that is sensitive to the presence of Na or K in liquid medium. We therefore followed culture turbidity and TABLE 1. Dependence of titer of w8 on NaCI or KCl added to CAT medium during growth and plating Salt added to growth mediuma Salt added to top agar plating mediuma
0.5% NaCl
None 0.5% KCl
661
PHAGES OF PNEUMOCOCCUS
0.5%
NaCl
2.5 x 109 1.6 x 109 1.9 x 109
None
7.5 x 109 5.7 x 109 6.4 x 109
0.5%
KCI
2.2 x 1010 1.9 x 1010 1.9 x 1010
a The phosphate buffer adds 0.03 M potassium to each medium. Basal agar on the plates was made with Na-CAT.
0060.120
180
40 I0
E~~~~TM 3
(
i.
.
10
60 120 180 TIME (min.) FIG. 1. Single-step growth curves at low cell density. Strain Rxl at 3 x 108 to 4 x 108 cells per ml was infected at an MOI of about 0.003 at 30 or 37°C and after 5 min was diluted 103 times in fresh medium at the same temperature. At intervals, samples were removed and assayed for PFU. Symbols: *, w8 in KCAT at 37°C; *, co8 in Na-CAT at 3 7°C; 0, c3 in KCAT at 37°C; A, w8 in K-CAT at 30°C. 0
phage production simultaneously for such conditions in the two media (Fig. 2). In K-CAT there is always a substantial increase in turbidity before massive lysis, which sometimes starts at 110 min and is usually complete by 150 min, although it is somewhat delayed in this experiment. In Na-CAT the turbidity rise is much less, and the fall is less sharp. The latent periods for phage production are significantly longer than those at low cell density. Furthermore, in this experiment the phage titer in NaCAT fell at late times to near 2 x 108, whereas it continued to rise in K-CAT. Comparison with other data, such as those in Table 1, shows that such a fall does not always occur in Na-CAT, but titers in the range 108 to 2 x 108 are not uncommon (13, 20). The longer latent period may reflect exhaustion of the medium, in that an uninfected culture at this concentration commonly reaches stationary phase within 90 min in either Na-CAT or K-CAT (data not shown). Adsorption. By titration of supernatants after various times of adsorption, it was found that adsorption rate constants were about 5 x 10-10 for w3 and 10-10 ml/cell per min for w8, both lower than those for most coliphages (1),
662
J. VIROL.
PORTER AND GUILD
and were the same in Na-CAT as in K-CAT (data not shown). The experiments in Table 2, showing increasing number of plaques as time is allowed for adsorption, confirm the faster adsorption of co3, give rate constants about the same as those determined by the usual method, and also show that 20% or more of the phage can adsorb on the plate. Eff'iciency of plating. Comparison of the 5x 10'0
1 10
20-
40
0
80
120
160
200
TIME AFTER INFECTION (min.)
FIG. 2. Growth of &3 at high cell density in KCAT and Na-CAT. Circles, Turbidity of culture infected at zero time at an MOI of3; squares, PFU per milliliter; open symbols, in K-CAT; closed symbols, in Na-CAT. The initial cell concentration was about 4 x 108 cells per ml (2 x 108 colony-forming unitsl ml). Turbidity was measured in 18-mm tubes in a Coleman Nepho-Colorimeter at 600 nm. TABLE 2. Plaques as function of time of preadsorption of &3 and w8a Adsorption time (min)
Plaques observed w3
(O)b
