MOISTURE REQUIREMENTS OF BACTERIA

Transcription

1 II. MOISTURE REQUIREMENTS OF BACTERIA INFLUENCE OF TEMPERATURE, ph, AND MALATE CONCENTRATION ON REQUIREMENTS OF Aerobacter aerogenes' R. J. WODZINSKI2 AND W. C. FRAZIER Department of Bacteriology, University of Wisconsin, Madison, Wisconsin Received for publication July 8, 1960 Scott (1957) defines water activity or a,., as a fundamental property of aqueous solutions that is equal to p/po, where p is the vapor pressure of the solution and po is the vapor pressure of the solvent. The a,, also is numerically equal to the corresponding relative humidity expressed as percentage divided by 100. Mossel and van Kuijk (1955) expressed availability of water in terms of the equilibrium relative humidity (h) which is numerically equal to a,,,. Wodzinski and Frazier (1960) noted that as the a. was lowered from a favorable level the apparent lag phase and the generation time of Pseudomonas fluorescens were progressively lengthened at any one temperature until growth was prevented. Also, as the temperature was lowered below the optimal temperature for rate of growth, 30 C, the lower limit of a, for growth was raised progressively. The apparent lag phase and the generation time were also proportionally longer at low values of a,, than would normally be expected for the drop in temperature. When the ph of the medium was made unfavorable to the organism, the tolerance of P. fluorescens to low a,,, was less than within the optimal ph range. They also noted that if both the ph and the temperature were made unfavorable, the lower limit of a, for growth was higher than if only one condition was made adverse. The apparent lag phase and the generation time were lengthened I This investigation was supported in part by a grant from the Research Committee of the Graduate School with funds provided by the Wisconsin Alumni Research Foundation. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 Present address: Division of Microbiology, The Squibb Institute for Medical Research, New Brunswick, N. J. by the combination of these two factors to a greater extent than if only one factor was made adverse. Christian (1955) showed that the range of a,,, for growth was appreciably smaller for Salmonella oranienburg in a chemically defined glucose-salts medium than in a complex medium and the lower limit of growth was 0.97 a.. The addition of five amino acids and eight vitamins to the defined medium permitted growth down to 0.95 a,,,. It may be, however, as Scott (1957) states, that the limiting a,,, for growth is substantially independent of nutrient supply provided certain minimal requirements are satisfied. The purpose of the present work was to determine whether Aerobacter aerogenes has the same water requirements as P. fluorescens or if it differed in its water requirements. The effects of absence or presence of malate on the a,,, requirements of A. aerogenes were observed incidentally. MATERIALS AND METHODS A strain of A. aerogenes from the stock cultures of the department was used throughout. Repeated tests on this strain had shown it to have the characteristics of A. aerogenes described in Bergey's Manual of Determinative Bacteriology (Breed, Murray, and Smith, 1957). Precautions were taken to keep the inoculum the same size and age (10 hr) for all experiments so that the original inoculum was about 100,000 bacteria per ml of medium. The basal medium for the propagation of the inoculum, for the stock cultures, and for the experiments contained, per liter, 5 g tryptone (Difco) and 3 g yeast extract (Difco). The approximate lowest and highest ph values necessary for the initiation of growth of the organism under approximately optimal conditions were determined by the use of appropriate buffers that varied by 0.1 intervals of ph. The ph read- 353

2 354 WODZINSKI AND FRAZIER [vol. 81 ings were made with a Beckman H-2 meter. It was found that the lower limit of ph at 30 C, as controlled by a 0.05 molal malic acid buffer, for the initiation of growth was near 3.9. The highest ph for the initiation of growth, as controlled by a Seitz filtered carbonate-bicarbonate buffer was found to be 8.4 at 30 C. The a.. was controlled by the addition of KCI to the basal medium. The methods used to determine and to control the a,,, of the medium employed were the same as those given by Wodzinski and Frazier (1960). Incubation was at 15, 20, 25, 30, and 37 C. The time of incubation for all experiments was 430 hr. A Bausch and Lomb Spectronic 20 was used for optical density measurements for all experiments at a wavelength of 610 m,u. Five replicates at each temperature, at each a,,,, and at each ph were made. The increase of optical density from the zero hour values of the five replicates were averaged at different time intervals. Two plus the logarithms of these averages were plotted against time in hours. A correlation of two plus log optical density with log2 plate count was made. Peptone water (Straka and Stokes, 1957) was used for the dilution blanks. Eight replicate plates were prepared at each time of sampling to minimize the effects of errors inherent in plating. The medium used for plate counting was yeast glucose agar (Difco). Growth curves measuring the response of A. aerogenes were made at intervals of 0.05 a.. Some of these values have been omitted from the tables to conserve space. The average generation times were calculated from the linear portion of the growth curves from the log2 values. The apparent lag times (Hinshelwood, 1946) were calculated by determining the time which elapsed after inoculation to a point on the inoculum baseline where the extrapolated linear portion of an average growth curve cut the baseline. A statistical analysis was performed on the data for each environmental condition. Comparisons between conditions were made by Duncan's (1955) multiple range test after the data were transformed to logarithms. The data were transformed to logarithms since the effects observed were multiplicative. When the data were transformed, the effects were additive and the data were in appropriate form for Duncan's multiple range tests. Since the error mean square was calculated from one set of data, it was advisable to use the 0.01 level of probability in testing for significance instead of the customary 0.05 level of probability. When the 0.01 level is used to test for significance it enhances the possibility of making type II errors by claiming no significance, when significance actually exists. RESULTS Effect of a. on apparent lag phase at ph 7.0, 3.9, and 8.4. In Tables 1, 3, 5, and 7 it is shown that as the a, of the medium was lowered by the addition of KCI, the apparent lag phase of A. aerogenes was progressively lengthened at any one temperature until a level of a, was reached where no growth was observed for the duration of an experiment. Effect of a, on generation time during logarithmic phase at ph 7.0, 3.9, and 8.4. Tables 2, 4, 6, and 8 indicate that as the a,,, was lowered by the addition of KCl, the generation time in the logarithmic phase of growth was progressively increased at any one temperature until a level of a,,, was reached where no growth was observed for the duration of an experiment. Effect of temperature on growth at various levels of aw at ph 7.0, 3.9, and 8.4. It is evident from a comparison of the generation times in Tables 2, 4, 6, and 8 that as the temperature was lowered from the optimum for generation time (37 C), the tolerance of the organism to low a,,, became progressively less. Tables 1, 3, 5, and 7 indicate that the apparent lag phase was increased beyond what would normally take place if the tempera- TABLE 1 Effect of temperature and of a,, on apparent lag phase of Aerobacter aerogenes at ph 7.0 aw, * NG NG NG NG NG NG 234

3 1961] MOISTURE REQUIREMENTS OF A. AEROGENES 355 TABLE 2 Effect of temperature and of a., on generation time of Aerobacter aerogenes at ph 7.0 atw * NG NG NG NG NG NG 11 TABLE 3 Effect of temperature and of a, on apparent lag phase of Aerobacter aerogenes at ph 8.4 aw s * X 20 X 5 Z X Z NG 92 W 58 Z 24 Z 23 Z NG 174 W 100 W 45 W 76 Z *Control, no solute added. NG W = No growth after 430 hr. = No growth at ph 7.0. X = Significantly longer apparent lag than at ph 7.0 (0.01 level of P). Z = Significantly shorter apparent lag than at ph 7.0 (0.01 level of P). ture was lowered and the a,, was not lowered. Tables 2, 4, 6, and 8, show that the generation time was also progressively longer at low values of a,, than would normally be expected for the lowered temperature. Effect of ph on growth at various levels of a,. Comparison of the apparent lag times and the generation times, at the various ph values at any one temperature in Tables 1 to 8, shows the effect of high and low ph on growth. When the ph was raised to 8.4, which is near the maximal ph permitting the initiation of growth, the tolerance of the organism to low a,, was greater than at 7.0. When the ph was made more acid, and lowered to 3.9, which is near the lower limit TABLE 4 Effect of temperature and of a,, on generation time of Aerobacter aerogenes at ph 8.4 a 15is * X XO.54 X X 2.0 X X X X NG 6.3 W 4.8 Z X NG 14 W 10 W 8.2 W 6.7 X W = No growth at ph 7.0. X = Significantly longer generation time than at ph 7.0 (0.01 level of P). Z = Significantly shorter generation time than at ph 7.0 (0.01 level of P). TABLE 5 Effect of temperature and of a., on apparent lag phase of Aerobacter aerogenes at ph 7.0 with 0.05 molal malic acid buffer aw s * 17 3 X 2.8 Z 13 X X Z 22 X NG 88 W 54 X 37 X 36 X NG NG 168 W 190 W 182 W = Growth in presence of. malate not in absence of malate. X = Significantly shorter apparent lag in presence of malate (0.01 level of P). Z = Significantly longer apparent lag in presence of malate (0.01 level of P).

4 356 WODZINSKI AND FRAZIER [VOL. 81 TABLE 6 Effect of temperature and of a. on generation time of Aerobacter aerogenes at ph 7.0 with 0.05 molal malic acid buffer aw 15i * X Z NG 3.9 W 3.1 X 3.5 X NG NG 8.6 W 12 W 12 Z W = Growth in presence of malate not in absence. X = Significantly shorter generation time in presence of malate (0.01 level of P). Z = Significantly longer generation time in presence of malate (0.01 level of P). TABLE 7 Effect of temperature and of a. on apparent lag phase of Aerobacter aerogenes at ph 3.9 with 0.05 molal malic acid buffer ata * NGW 32Z 29Z 1OZ 1OZ NGW NGW NGW NGW 56Z NGW NGW NGW NGW NGW W = Growth at ph 7.0, not at ph 3.9. Z = Significantly longer apparent lag at ph 3.9 (0.01 level of P). of ph permitting the initiation of growth, the tolerance of the organism to low a, was less than at ph 7.0 or 8.4. In general, the apparent lag phase at ph 8.4 was shorter at the lowest a,, level permitting growth than at ph 7.0. At all temperatures except 15 C the lowest aw, level permitting growth at ph 7.0 was longer than at 8.4. The apparent lag TABLE 8 Effect of temperature and of a,, on generation time of Aerobacter aerogenes at ph 3.9 with 0.05 molal malic acid buffer aw * NGW 4.3 Z 3.7 Z 2.2 Z 3.0 Z NGW NGW NGW NGW 87 Z NGW NGW NGW NGW NGW MG = No growth after 430 hr. W = Growth at ph 7.0, not at ph 3.9. Z = Significantly longer generation time at ph 3.9 (0.01 level of P). phase at ph 3.9 was significantly longer than at ph 7.0 at all a,, levels where growth occurred. Where a significant difference did occur, the generation time in the logarithmic phase of growth was usually longer at acid and alkaline ph values than within the optimal ph range. Effect of combined ph and temperature on growth at various levels of a.. Comparison of Table 5 with 7 and Table 6 with 8 indicated that as the temperature was lowered and the ph was made acid the organism required a much higher level of a,, for growth. The organism needed more water under these conditions, than if only one environmental condition was made inhibitory. Both the apparent lag phase and the generation time were increased to a greater extent than if only one factor was varied: When the ph was made more alkaline (Tables 1 and 2 versus 3 and 4), and the temperature was lowered, the organism required less available water than at a neutral ph value at the same temperatures. The organism needed less water, under these conditions, than if only one of the environmental conditions was made adverse. Also, the apparent lag phase was decreased at ph 8.4 to a greater extent than if only one factor was varied. The generation times were not similarly affected by the two factors. Effect of malate on growth at various levels of a, at ph 7.0. Comparison of the apparent lag times (Tables 1 and 5) and the generation times (Tables 2 and 6), in the presence and absence of malate, showed the effect of malate on growth.

5 1961] MOISTURE REQUIREMENTS OF A. AEROGENES 357 In the presence of malate the tolerance of the organism to low a, was greater than in the absence of malate. Both the apparent lag phase and the generation time were longer in the malatedeficient medium than in the malate medium. DISCUSSION Results with different solutes such as 1 molal sucrose plus KCl, and a salts mixture of NaCl, KCl, and Na2SO4, although not reported here, indicated that the availability of water in the solution or substrate was most accurately described by the a, of the medium. Osmotic pressure was not used to describe the status of water in the substrate since it does not adequately explain the response of microorganisms to a gelled particle. Loeb (1922) noted that the osmotic pressure of a solution of gelatin was negligible when compared with the same weight concentration of gelled gelatin particles. It is true, however, that the a. of a medium may be calculated from the molal osmotic coefficient by a derivation of the Gibbs Duhem equation (Scott, 1957), if the solute is not a hydrophilic colloid. That the results have not been caused by salt tolerant mutants is indicated by the following: (i) the results are similar regardless of the solute(s) employed, including sucrose; (ii) it seems extremely unlikely that mutants would be active at near limiting au, levels which caused increasingly longer lag times and generation times as the a. was lowered; and (iii) Scott (1957) and others have reported that at no time were there any indications that more rapidly growing mutants had appeared in any of the cultures of low aw or that there was any appreciable difference in the water requirements of different strains of species. The effects of a,,, at levels between and were not studied extensively since preliminary investigations at the optimal temperature and ph for A. aerogenes indicated that there was no difference between these intermediate levels and responses noted at a.. It should be noted, however, that differences probably do exist at these intermediate a. levels under adverse conditions of ph and temperature. If the results obtained with A. aerogenes are compared with the similar study of Wodzinski and Frazier (1960) with Pseudomonas fluorescens, it is evident that these two organisms differ in their available water requirements. A. aerogenes was able to grow at a lower a,,, than P. fluorescens under optimal conditions, versus A much more striking effect on the apparent lag phase was observed with A. aerogenes as the a. became limiting. The generation times also became much longer as the a,,, became limiting. The different effect observed on the apparent lag phase of the two organisms might be explained by the rapid autolysis the strain of P. fluorescens employed undergoes if growth does not take place in a relatively short time. The rate of autolysis was more rapid at high temperatures than at low temperatures. This phenomenon was not observed with the strain of A. aerogenes employed. The striking effect of a,,, on the apparent lag phase of microorganisms has also been reported by Sakaguchi (1959) who used Pediococcus soyae while studying the effects of osmotic pressure on growth. It is apparent with P. fluorescenrs and A. aerogenes that the available water requirements of both organisms are different under different environmental conditions. The tolerance of both organisms to low a,,, was greatest under optimal conditions and least under adverse environmental conditions. The apparent lag phase and the generation time were shortest under optimal conditions and longest under adverse environmental conditions. The effects observed with both organisms were more pronounced if more than one condition was made adverse. Therefore, the moisture requirements of both organisms varied with the temperature and ph and with A. aerogenes in the presence or absence of malate. The application to preservation of food and to cultivation of bacteria in the laboratory is obvious. SUMMARY The effect that solute concentration, expressed as water activity or as available water (a,), has on the apparent lag phase and the logarithmic phase of growth of Aerobacter aerogenes in a tryptone, yeast extract broth was studied. The effect of water activity was studied at different temperatures and ph values, and in the presence and absence of malate. The a,, of the medium was adjusted by the use of KCl. It was found that as the a,,, was lowered from a favorable level by the addition of KCl, the apparent lag phase and the generation time

6 358 WODZINSKI AND FRAZIER (VOL. 81 were progressively lengthened, at any one temperature, until growth was prevented. As the temperature was decreased below the optimal for rate of growth (37 C) the lower limit of a,, for growth was raised progressively. The apparent lag phase and the generation time were also proportionally longer at low values of a,t than would normally be expected for the drop in temperature. When the ph of the medium was acid, the tolerance of the organism to low a,, was less than within the optimal ph range. The apparent lag phase and the generation time were longer at the adverse ph values than within the optimal ph range. When the ph of the medium was alkaline, the tolerance of the organism to low a,,, was greater than at ph 7.0. The apparent lag phase was shorter at the alkaline ph than at 7.0 at low a,,, levels. GCenerally, the generation time was longer at the alkaline ph. When both the ph and the temperature were made unfavorable the lower limit of a,, for growth was higher than if only one condition was made adverse. The apparent lag phase and the generation time were lengthened by the combination of these two factors to a greater extent than if only one factor was made adverse. In the presence of malate, A. aerogenes needed less water for growth than in its absence. REFERENCES BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH 1957 Bergey's manual of determinative bacteriology, 7th ed. The Williams & Wilkins Co., Baltimore. CHRISTIAN, J. H. B The influence of nutrition on the water relations of Salmonella oranienburg. Australian J. Biol. Sci., 8, DUNCAN, D. B Multiple range and multiple F tests. Biometrics, 11, HINSHELWOOD, C. N The chemical kinetics of the bacterial cell, p. 55. The Clarendon Press, Oxford. LOEB, J On the influence of aggregates on the membrane potentials and the osmotic pressure of protein solutions. J. Gen. Physiol., 4, MOSSEL, D. A. A., AND H. J. L. VAN KUIJK 1955 A new and simple technique for the direct determination of the equilibrium relative humidity of foods. Food Research, 20, SAKAGUCHI, K Growth of Pediococcus soyae nov. sp., in highly concentrated solutions of inorganic salts and sugars. Rep. Noda Inst. Sci. Research (Japan), 3, SCOTT, W. J Water relations of food spoilage microorganisms. Advances in Food Research, 7, STRAKA, R. P., AND J. L. STOKES 1957 Rapid destruction of bacteria in commonly used diluents and its elimination. Appl. Microbiol., 5, WODZINSKI, R. J., AND W. C. FRAZIER 1960 Moisture requirements of bacteria. I. Influence of temperature and ph on requirements of Pseudomonas fluorescens. J. Bacteriol., 79,