and Fong (1937), in their work with Staphylococcus aureus, found that increasing

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1 INFLUENCE OF ph ON PROLIFERATION OF LACTIC STREPTOCOCCUS BACTERIOPHAGE1' 2 W.. W. OVERCAST,' F. E. NELSON, AND C. E. PARMELEE Dairy Industry Section, Iowa Agricultural Experiment Station, Ames, Iowa Received for publication October 16, 1950 Variations in the production of lactic acid by bacteria can be of major importance in the dairy industry, especially in the making of cheddar and cottage cheese. One of the principal causes for failure of organisms to produce acid is bacteriophage. This study was undertaken to determine the influence of the reaction of the medium on the proliferation of lactic streptococcus bacteriophage. Nelson et al. (1939) found that a reaction approximating ph 6.8 was most favorable for the lysis of sensitive strains of Streptococcus lactis. Turner (1948) found the reaction of the medium an important factor in plaque formation by the lactic streptococcus bacteriophage, ph 6.0 to 6.2 being optimum for most strains. Cherry and Watson (1949) suggested that ph 5.0 was about the critical level below which lysis could not occur for S. lactis Gold and Watson (1950) found that the ph of the medium was the limiting factor in the lysis of the cells of Clostridium madisonii by the homologous bacteriophage. Krueger and Fong (1937), in their work with Staphylococcus aureus, found that increasing the hydrogen ion concentration prolonged the lag phase of the organism without a corresponding lengthening of the lag phase of bacteriophage production; increasing the hydroxyl ion concentration caused no pronounced change in the curves of either organism growth or bacteriophage proliferation. EXPERIMENTAL METHODS Litmus milk was used as the basal medium for all determinations at ph 5.2 or above. Broth containing 400 ml of filtered V-8 juice,4 10 g peptonized milk, 10 g proteose peptone, and 600 ml of distilled water was used as the basal medium at all ph levels below 5.2. The substratum was sterilized in 150-ml quantities in 6-oz screw-cap bottles. The reaction of the medium was lowered by the addition of 10 per cent lactic acid or raised by the addition of N NaOH. Two samples were run as a single experiment; the first sample contained the organism and the second contained the organism plus the homologous bacteriophage. In each case the ph of the samples was maintained by periodic additions of N NaOH. The samples were incubated at C for the duration of the trial. At various intervals an aliquot was withdrawn aseptically andused forthe determina- 1 Journal paper No. J-1846 of the Iowa Agricultural Experiment Station, Project This investigation was supported in part by a research grant from the National Institute of Health, U.S. Public Health Service. 3Present address: Department of Dairying, University of Tennessee, Knoxville, Tennessee. 4A combination of eight vegetable juices made by the Campbell Soup Company, Camden, New Jersey. 87

2 88 W. W. OVERCAST, F. E. NELSON, AND C. E. PARMELEE [vol. 61 tion of ph, organism numbers, and bacteriophage titer. All ph measurements were made using either a Beckman or a Leeds and Northrup glass electrode potentiometer. Organism numbers were determined by the standard plate count method (American Public Health Association, 1948) on tryptone glucose extract milk agar with incubation at 32 C. Bacteriophage numbers were determined by the three-tube limiting dilution technique with fortified" litmus milk as the substratum; bacteriophage titer was expressed as the most probable number of particles per ml, based upon significant retardation of acid production, reduction, or coagulation of litmus milk. TIME (HOURS) TIME (HOURS) Figure 1. Population changes at different ph levels for the combination of Streptococcus cremoris 573 and bacteriophage F55. RES3ULTS Streptococcus cremoris 673 and bacteriophage F55 combination. Typical population changes for bacteriophage strain F55 and S. cremoris 573 at various ph levels are shown in figure 1. This bacteriophage strain proliferated over a ph range from 5.2 to 7.6. When the ph approached the limiting values for bacteriophage proliferation, a marked increase in the lag phase and a reduction in the rate of proliferation occurred. Although strain F55 showed considerable increase in numbers at ph 5.2, neither organism growth nor bacteriophage proliferation occurred in 20 hours at ph 5.0. Although there was very little difference in the growth of the organism at ph 7.5 and ph 7.6, bacteriophage proliferation was delayed considerably at ph 7.6, 5 Ten per cent filtered V-8 juice was added.

3 1951] PROLIFERATION OF LACTIC STREPTOCOCCUS BACTERIOPHAGE 89 but at ph 7.5 proliferation was retarded only slightly as compared to that at ph 6.5. Other trials at ph 7.6 using longer incubation periods indicated that bacteriophage proliferation occurred but only at a very slow rate. Acid production by the host organism was so rapid at ph 7.6 that a constant ph was difficult to maintain after 12 to 14 hours' incubation. There was a definite increase in organism numbers at ph 8.0, but the bacteriophage failed to multiply at this ph level in 18 hours. At ph 6.5 mas lysis occurred in about 6 hours. Other trials indicated that mass lysis also would occur at ph 5.4 in 14 to 16 hours. Figure S. Population changes at different ph levels for the combination of Streptococcus lactis W2 and bacteriophage E24. Streptococcus ljcti8 W2 and bacteriophage F24 combination. Figure 2 shows typical population changes for the W2-F24 combination. Bacteriophage proliferation occurred over the ph range from 4.8 to 9.4, essentially the entire range of organism growth. The bacteriophage proliferation rate during the logarithmic phase was much the same from ph 5.0 to ph 8.3, although considerable differences in the length of the lag phase were encountered. The organism failed to grow during a 24-hour incubation period at ph 4.7, and the bacteriophage also failed to multiply. The organism showed retarded growth at ph 4.8, and bacteriophage nuimbers increased only after an extended lag phase. The orgaism was slow in getting started to grow at ph 5.0, but bacteriophage proliferation occurred at a high rate after organism growth was proceeding fairly rapidly.

4 ^f% W. W. OVERCAST, F. E. NELSON, AND C. E. PARMELEE [VOL. 61 The lag phase for both organism growth and bacteriophage proliferation became longer as the reaction was raised from ph 7.6 to 9.4. At ph levels of 9.1 and 9.4 the lag phase of bacteriophage proliferation was extended by approximately 12 hours and the rate of proliferation, after initiation, was somewhat retarded. At ph levels of 9.1 and 9.4 a decrease in organism numbers, followed by a slight increase, usually occurred. Bacteriophage multiplication occurred during this increase. In one trial at ph 9.4 there was virtually no increase in organism numbers, as deterrmined by the plate count; yet the final bacteriophage titer was fairly high. s 1--~~~~~~~~~~~~~~~-,S. 3 x 8..P H O O 9 H 5. si z7o lactis 712andbacipH6a5 0 0 w5~~~~~~~~~~~~~~c ove a iep ag.iniainnohr phal weeta ms.iswt h W2-2 -a cobnto occu aslwa H. n s iha H.ne TIME (HOURS) TIME (HOURS) necessary~ Figure 3. for Population mslyitoocraph76asopretoph ~~ changes P at different levelsi for the combination 6.5. of Streptococcus lactis 712 and bacteriophage F56. Mass lysis of the organisms occurred during a 12- to 14-hour incubation period over a wide ph range. Indications in other trials were that mass lysis with the W2-F24 combination may occur as low as ph 4.9 and as high as ph 8.0 under proper conditions, although the data presented for the typical trials do not demonstrate this. From figure 2 it wil be noted that 5 to 6 additional hours were necessary for mass lysis to occur at ph 7.6, as compared to ph 6.5. Streptococcus lactis 712 and bacteriophage F56 combination. Typical population changes for the 712-F56 combination at various ph levels are shown in figure 3. S. lactis 712 differed from other test cultures in several respects. It was not so sensitive to the acid reactions as were the other organisms used in this study. Colonies on TGEM agar were several times larger than colonies, from other strains. In order to promote proliferation of F56 in V-8 broth, 0.1 per cent of

5 1951] PROLIFERATION OF LACTIC STREPTOCOCCUS BACTERIOPHAGE 91 CaCl2 was added to the medium. Collins et al. (1950) reported similar results for this strain of bacteriophage in a defined medium. Bacteriophage strain F56 was more sensitive to both acid and alkaline reactions than was the host organism. Little, if any, proliferation of bacteriophage occurred below ph 5.0, despite the comparatively good growth of the host organism at lower ph levels. Retarded growth of the organism was evident at ph 8.6, but the bacteriophage failed to proliferate at this ph level. Proliferation of the bacteriophage was retarded considerably at ph levels of 8.3 and 8.4, whereas there was considerable growth of the organism at these ph levels. 9 I. I, I.I I..-PH w -I H ~~~~~ ph cn4 0~~~~~~~~~~~~~~~ ~~~H 9.4 Maslssocre oe Hrnefo 5. to 6..5Ol P 0 ~~~~~~~~0 Hutr 51221udr lae o6hur tp.,a copre to ph 6.,bt a nl lghl e layed ~ 5H 6..5 ph strain~~~~~~~~~~~~~~~~~~ F4poieae9ve Hrnefo 52t.,a4yicldt rsne levls f52,.6 9., ad 94 re f iterst Afer actriphagpolfeato had ben iniiated it prceede at a rairte The orgnism ofthis cobinatio wa 2oesniiet teakln ecin infigure 4. Phopuato chaxtnges latgifrnphalevel baterohae for mbintionlficaetiocnuatp lemesorfh.2,1 and91 ad94ae F4lieato bacteriophage fitret fe occurred Mass lysis as occurrted,i deulteriochngedsb poverde a ath ph platercun,n trange from51toe.5onyclue121ndr t ph levelsfothcmbnin of8.6 incfigurred4 ashow rndpaov;cu etermnded e lg phases intbacteophae m pliationd atvp

6 92 W. W. OVERCAST, F. E. NELSON, AND C. E. PARMELEE [VOL. 61 likewise there was no increase in bacteriophage numbers. However, when there was multiplication of the organism at these high ph levels, bacteriophage proliferation occurred after an extended lag period, indicating that failure of the organism to multiply prevented bacteriophage proliferation. Mass lysis occurred at ph 5.4, and there was an indication that mass lysis also occurred at ph 8.6 between 18 and 22 hours. This was not the typical abrupt and complete lysis that occurs at lower ph levels but a more gradual type, not nearly so complete on a percentage basis. Preliminary trials with this organism-bacteriophage combination seemed to indicate that the low ph actually inhibits the bacteriophage proliferation within V * -r V 1C 1 t *. U- V TIME (HOURS) TIME (HOURS) Figure 5. Population changes at different ph levels for the combination of Streptococcue cremoris 122,1 and bacteriophage F43. the cell, rather than at some other phase of the cycle. When the organism and the bacteriophage were incubated together at 32 C at ph 6.5 for 3 hours and then the ph was lowered, by the addition of lactic acid, to the limiting level for the bacteriophage, proliferation ceased. Streptococcus cremoris 122, 1 and bacteriophage F43 combination. Figure 5 shows typical population changes at various ph levels for the S. cremoris 122,1 and bacteriophage F43 combination. When V-8 broth was used as the substratum, added calcium was necessary for good proliferation of F43. Both S. cremoris 122, 1 and bacteriophage F43 were sensitive to the alkaline ph levels. Although the organism grew well at ph 4.8, proliferation of bacteriophage could not be demonstrated. At ph 5.0 the organism increased rapidly, bacteriophage pro-

7 1951] PROLIFERATION OF LACTIC STREPTOCOCCUS BACTERIOPHAGE 93 liferation occurred at a very rapid rate after a slight lag, and mass lysis occurred after a delay of 4 to 6 hours as compared to ph 6.5. Bacteriophage proliferation occurred at ph levels of 8.4 and 8.6 after prolonged lag phases. In this reaction range, the number of bacteria declined for the first 16 to 18 hours, indicating that the alkaline reaction was inhibitory to the organisms. After the early decline, slight growth took place; this apparently conditioned the cells to support bacteriophage multiplication. At ph 8.0 considerable bacteriophage increase was noted while the organism population decreased. Evidently the susceptible cells were in the right physiological state for bacteriophage adsorption and multiplication, but detectable bacterial multiplication did not occur before mass lysis began. DISCUSSION The optimum ph for lactic streptococcus bacteriophage proliferation was slightly on the acid side of neutrality, at about ph 6.5. Considerable proliferation occurred over a ph range of 5.4 to 7.5 for all five strains and extended beyond this range for some strains. At ph 5.4 there was very little difference in the rate of bacteriophage proliferation as compared to ph 6.5 for these strains; the lag phases were increased slightly but high final bacteriophage titers were obtained with all strains. At ph 7.5 proliferation of F55 was retarded somewhat, as was evident by the increased lag phase. Wide variations existed among the strains in their ability to proliferate under alkaline conditions. Although F55 increased very little at ph 7.6, and then only after an extended lag phase, F24 caused mass lysis of the susceptible cells at this level. Strains F56 and F43 were able to proliferate at ph 8.4 and ph 8.6, respectively. F24 and F4 did not show the sensitivity to alkaline reactions that the other strains did, both multiplying at ph 9.4, although only after a prolonged lag phase. Proliferation of F4 at ph 9.1 and ph 9.4 depended upon growth of the organism (as determined by the plate count), which occurred in some instances and not in others. The reasons for the variability were not apparent from the data available. Bacteriophage proliferation generally was dependent upon growth of the organisms. However, on one occasion with the 122,1-F43 combination at ph 8.0, proliferation apparently occurred without organism increase, which, of course, may not be synonymous with organism growth. The organisms evidently were in the right physiological state for bacteriophage proliferation. Considerable variation exists in the ability of the different bacteriophage strains to proliferate over the entire range in which host organism multiplication occurs. Strain F24 proliferated over the entire ph range of organism growth. F56 failed to increase below ph 5.0 or above ph 8.4, whereas its host organism, 712, grew rapidly at ph 4.7 and also above ph 8.4. Organisms H1,1 and 573 failed to exhibit definite increases in number of organisms at ph 5.0; but H1,1 did, upon occasion, show very slight increase at ph 9.1 and ph 9.4, and bacteriophage proliferation took place when this increase occurred. When mass lysis occurred at the higher ph levels, it was not the typical

8 94 W. W. OVERCAST, F. E. NELSON, AND C. E. PARMELEE [VOL. 61 abrupt lysing of the cells but rather a gradual process taking place over several hours. Cherry and Watson (1949) stated that ph 5.0 was near the critical level below which lysis does not occur. The data in this study indicate that mass lysis for the W2-F24 and 122,1-F43 combinations occurred at ph 5.0. Lysis of the W2 organism occurred at ph levels as low as 4.8. The results obtained with the W2-F24 combination are in direct contrast to those obtained with combination 573-F55, with which neither bacteriophage nor organism increased in numbers at ph 5.0 and both were retarded considerably at ph 7.6. Mass lysis for the 573-F55 combination occurred at ph 5.4 but not at ph levels as high as 7.5. Because F24 proliferated over the entire range of organism growth (ph 4.8 to ph 9.4), it is quite probable that this represents approximately the extremes at which lactic streptococcus bacteriophage can multiply, since these ph levels are about the limits of growth for the host organism. F55 could represent the other extreme, since its active proliferation was confined to such a narrow ph range (5.2 to 7.6). The probability of obtaining other strains vastly different in their response to ph seems small. These data offer an explanation for the rather rapid suspension of acid production sometimes encountered in lactic cultures, or the products made by their use, when the culture employed for inoculum appeared normal. If mass lysis of the sensitive strain of bacteria did not occur before the ph of the culture dropped below the limiting level for bacteriophage proliferation, mass lysis would be prevented and acid production would continue essentially unchanged until the lowest ph level characteristic for that particular culture had been reached. This situation would be most likely to arise if the level of the bacteriophage contamination was very low, such as might be the case when air was the source. Because of the exceedingly rapid rate of bacteriophage proliferation, particles might be present in very considerable numbers after the incubation of such a culture, even though the original contamination had been at a very low level. When such a culture, containing large numbers of bacteriophage particles but not obviously abnormal, was inoculated into milk at normal ph, the bacteriophage particles would proliferate rapidly and mass lysis, accompanied by cessation of acid production, would occur early in the incubation period. The basis for the recommendation to use an abnormally heavy inoculum when bacteriophage troubles are encountered probably is that the ph then drops more rapidly and the limiting reaction for bacteriophage proliferation has a greater chance to be reached before mass lysis occurs. SUMMARY Five strains of lactic streptococcus bacteriophage and the homologous organisms were studied over a wide ph range to determine the influence of reaction on bacteriophage proliferation. The maximum and minimum ph levels at which bacteriophage proliferation occurred varied for each strain studied. Bacteriophage proliferation usually failed to occur at ph levels at which the growth of the host organism was inhibited considerably. Two strains of bacteriophage proliferated over the entire ph range of organism growth, but three strains did not.

9 1951] PROLIFERATION OF LACTIC STREPTOCOCCUS BACTERIOPHAGE 95 REFERENCES American Public Health Association 1948 Standard methods for the examination of dairy products. 9th ed. Am. Pub. Health Assoc., New York, N. Y. CHERRY, W. B., AND WATSON, D. W The Streptococcus lactis host-virus system. I. Factors influencing quantitative measurement of the virus. J. Bact., 58, COLLINS, E. B., NELSON, F. E., AND PARMELEE, C. E Relation of calcium and other constituents of a defined medium to proliferation of lactic streptococcus bacteriophage. J. Bact., 60, GOLD, W., AND WATSON, D. W Studies on the bacteriophage infection cycle. II. Phage infection and lysis of Clostridium madisonii, a function of ph. J. Bact., 59, KRUEGER, A. P., AND FONG, J The relationship between bacterial growth and phage production. J. Gen. Physiol., 21, NELSON, F. E., HARRIMAN, L. A., AND HAMMER, B. W Slow acid production by butter cultures. Iowa Agr. Expt. Sta., Research Bull TURNER, G. E Dynamics of the Streptococcus lactis bacteriophage relationship. Unpublished Ph.D. thesis, Iowa State College Library, Ames, Iowa. Downloaded from on January 11, 2019 by guest