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The response of bacterial spores to vacuum treatments. II. Germination and viability studies
CRYOBIOLOGY
13, 71-79
(19%)
The Response of Bacterial II. Germination G. J. SOPER,
Spores to Vacuum and Viability
J. M. WHISTLER,*
AND
Treatments.
Studies D. J. G. DAVIES
School of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom
The change from the dormant spore to the vegetative cell involves sequential processes that have been subdivided into three main classes, activation, germination and outgrowth, and we have attempted to determine any changes that might be induced in one or more of these processes by the vacuum treatment, since these would not necessarily be apparent from viability data alone. Previous work in this department (13) has shown that the heat resistance of vacuum-treated spores varies with the aqueous vapor pressure to which they are reequilibrated, a peak sensitivity being shown at 2 x IOe3 Torr and a peak resistance at 10-22 Torr. It was also shown that at all water levels heat sensitivity was increased in the presence of oxygen. Since the dehydration treatments we are studying often involve exposure of the spores to temperatures above ambient for periods up to 24 hr, it was considered that any damage induced by these treatments would also be more apparent in the presence of oxygen and that the modification of this damage by water would be more noticeable at reequilibration aqueous vapor pressures of 2 x 1O-3 and 10 Torr. We report here the influence of the oxygen exposure and rehydration treatments on the activation, germination, outgrowth and overall viability of Bacillus megaterium spores subjected to different vacuum-dehydration conditions.
In the first paper of this series (16) we presented details of the design and characterization of the instrumented vacuum apparatus being used in our studies of dehydration-induced changes in microorganisms. Physical measurements made during use of the apparatus were shown to be reproducible, and it was found that under the most severe experimental conditions studied, i.e., 24-hr treatment at 65°C there was no significant loss of bacterial spores from the samples under test. We now report on our studies to determine whether changes in the biological characteristics of the spore are induced by the various dehydration treatments. The primary and most direct method of assessing biological integrity is to measure viability. In this context the viability of a spore is defined as its ability to germinate and reproduce to the point of microscopic detection as a colony when inoculated onto solid nutrient medium. While this criterion is by no means a complete measure of spore integrity, it is nevertheless an extremely sensitive assay requiring a fully operative replicating system, since, for example, if only a small number of divisions were possible in the vegetative cells formed after germination, the spore would be scored as nonviable. Received January 15, 1975. 1 Present address: School of Pharmacy, Leicester Polytechnic, Leicester, U.K.
City
of
71 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserve.i.
SOPER, WHISTLER
72 MATERIALS
AND METHODS
Test organism. Spores of Bacillus megaterium ATCC 8245 were used throughout this work. The spores were obtained after g-day incubation of the organism on potato extract agar medium and were washed by repetitive centrifugation and resuspension in sterile glass-distilled water. A standard spore suspension of known total and viable count was prepared and stored at 0-4°C. As a spore sample, 0.06 ml of this suspension was used. Counting medium. This consisted of 2% peptone (Oxoid), 0.5% sodium chloride (Analar grade) and 1.5% agar (Oxoid) in glass-distilled water, adjusted with 1~ sodium hydroxide to give a pH, after autoclaving, of 7.2-7.4. Growth medium. The preparation and composition of the liquid growth medium was similar to that of the counting medium except that the agar was omitted. Viable counts. Samples of spore suspensions were diluted serially, when necessary, with sterile distilled water to give a suspension containing 2 X lo*-5 X lo2 viable spores ml-l. Three-tenths milliliter of this suspension was plated by a surfacespread technique onto each of five plates of counting medium. Preliminary experiments had verified that, under all experimental conditions tested, maximum values were obtained when colony counts were performed on the plates after a 48-hr incubation at 35°C. Tot& counts. The total number of spores was estimated by the method of Cook and Lund (2), using a suitable dilution of the spore suspension and a platelet counting chamber of a nominal O.Ol-mm depth. Optical density measurements. These were carried out at 25°C in l-cm silica cuvettes using an S.P.1800 recording spectrophotometer ( Unicam Ltd. ) . Vacuum treatment of spore samples. The theoretical and practical considerations involved in the design of the instrumented vacuum Iine used for treating
AND DAVIES
spore samples was presented in detail in the accompanying paper ( 16), and data for temperature and weight changes induced by the treatments may be obtained from it. The methods described there for vacuum dehydration, reequilibration to defined aqueous vapor pressures and exposure of samples to controlled gaseous environments have been utilised in this work. RESULTS
Viability
of the Standard Spore Suspension
Values for the mean number of viable spores ml-l and for the mean total number of spores ml-; determined from quintuplicate 0.06-ml samples of the untreated standard spore suspension were 4.55 x lOlo and 7.52 x lOlo, respectively, giving a mean viability of 60.5%. No significant change in these values was detected during storage of the suspension at 0.4”C over a 2-yr period. The Effect of Drying Temperature and Reequilibration to 2 x 1C3 and 10 TOW Aqueous Vapor Pressure The drying temperatures investigated were 0, 10, 15, 25, 35, 50, 60 and 65°C. Ten 0.06-ml samples of standard spore suspension were dried for 6 hr at each temperature. At the end of the drying treatment half of the samples were sealed in anoxia. The remaining samples were reequilibrated to an aqueous vapor pressure of 2 X 10e3 or 10 Torr prior to sealing in anoxia. ImmediateIy after sealing the sample vessels were opened ‘and the contained spore samples resuspended in 6 ml of sterile water. Total and viable counts were performed on each resuspended sample. Statistical analyses performed on the total counts confirmed our previous findings that there is no significant loss of spores from the samples during vacuum dehydration or reequilibration treatments. However, similar analyses performed on
GERMINATION
AND
VIABILITY
the viable-count data indicated that the drying treatments had a significant effect on spore viability. A curvilinear relationship exists between percentage viability and drying temperature (Fig. l), with a minimum viability in the region of 15°C. At temperatures above 35°C a significant increase in viability occurs with increase in drying temperature suggesting that a phenomenon analogous to activation is occuring under these conditions. At all drying temperatures, vapor rehydration of dried spores, as occurs during reequilibration to 2 x lOA or 10 Torr aqueous vapor pressure, had no detectable effect on spore viability as can also be seen in Fig. 1. We have previously demonstrated that vacuum-dried spores have maximum heat sensitivity when reequilibrated to 2 x 10e3 Torr (13), and it was anticipated that a sensitizing effect on spore viability may also be observed if dried spores were vapor rehydrated to this aqueous vapor pressure prior to reconstitution. Data obtained with samples dried for 6 hr at 0, 15, 25 and 65°C prior to reequilibration to 2 x 1O-3 Torr showed that reequilibration to this level resulted in no significant
I
T
OF DRIED
73
changes in viability and did not modify the curvilinear relationship between percentage viability and drying temperature (Fig. 1). The Efect
of Oxygen
Survival of lyophilized vegetative bacteria is lower if oxygen is admitted to samples after drying than if the samples are maintained in anoxic conditions (3, 5). It was therefore considered that any damage induced in spores by vacuum dehydration might also be more apparent in the presence of oxygen. Samples were dried for 6 hr at 0, 15, 25 and 65°C. After drying, half of the samples were sealed in anoxia, and dry oxygen was admitted to the remainder prior to sealing. In a repeat experiment, samples were reequilibrated to 10 Torr aqueous vapor pressure before sealing in either anoxia or oxygen. Viability data obtained from the resuspended samples are recorded in Table 1. Statistical analyses of these data indicated that admission of oxygen to samples after drying does not result in loss of spores from the sample. In addition, they confirmed the previous observation that reequilibration to 10 Torr aqueous vapor pressure has no effect on the viability of dried spores and also showed that there is no interaction between vapor rehydration to this level and the presence of oxygen. Furthermore, the data showed that in contrast to vegetative bacteria (5) the influence of oxygen on the viability of dried spores is negligible under the conditions investigated. Effect of Drying
FIG. 1. Mean percentage viability as a function of drying temperature for Bacillus megatetium spores, vacuum dried for 6 hr and sealed in anoxia: Unequilibrated ( l ), reequilibrated to 2 X lo4 Torr aqueous vapor pressure ( A), reequilibrated to 10 Torr aqueous vapor pressure (0).
SPORES
Time
It appeared likely that the extent of the “activation” phenomenon illustrated in Fig. 1 would be dependent upon the amount of water removed from the spore and would therefore be a function of the drying time as well as of the drying temperature. To verify this, samples were dried at 15, 25, 35 and 65°C for periods of 6, 12, 18 and 24 hr and were sealed in
7-t
SOPER,
WHISTLER
AND
DAVIES
anoxia. As in previous experiments, no significant decrease was observed in the total number of spores in the samples. The relationship between percentage viability and drying time at each temperature is shown in Fig. 2. At a temperature of 65°C maximum viability is observed after 12-hr drying, further exposure resulting in a decrease in viable count. This maximum (71.170) might have been reached at 25 and 35°C if sufficient exposure had been allowed, but within the experimental limit of 24-hr drying it is not achieved. At a temperature of 15°C the decrease in viability observed after 6-hr drying is maintained during the increased drying times. Control experiments in which samples were exposed to the drying temperatures for periods up to 24 hr, without dehydration, produced no change in spore viability, indicating that the spores did not exhibit true heat activation. It is interesting that attempts to activate the spores by chemical
6 DRYING
12 TIME
IS (hours)
FIG. 2. Mean percentage viability as a function of drying time for Bacillus megaterium spores, vacuum dried at different temperatures and sealed in anoxia: 15°C (A), 25°C (X), 35°C (a), 65°C (0).
GERMINATION
AND VIABILITY
OF DRIED
SPORES
75
methods, e.g., incubation with L-alanine and n-glucose were also unsuccessful. Time for Outgrowth Resultant Vegetative
and Growth Cells
Rate of
It is possible that the vacuum and reequilibration treatments could induce changes in spore integrity that would not necessarily be manifest as differences in spore viability. It was hoped to gain evidence of such changes by determining the time for outgrowth of spores subjected to such treatments and the growth rate of the resultant vegetative cells. The spore suspension under investigation was diluted in pre-warmed growth medium to give a culture having an optical density of 0.02 at a wavelength of 420 nm. The culture was incubated at 35°C and its growth followed by optical density measurements at 420 nm. Preliminary experiments had demonstrated that optical density changes were reproducible and could be directly correlated with changes in the number of viable organisms in the suspension, The log optical density/incubation time relationship showed an initial lag during which the optical density remained constant, followed by a logarithmic region where optical density increases with time at a constant rate (Fig. 3). The time to reach the end of the initial lag period is a measure of the time for commencement of outgrowth (t,), and the slope of the logarithmic region represents the growth rate constant (k) for the resultant vegetative cells. The values of t, (215 I+ 5 min) and k (2.69 X 10e2-t 6.12 X W4 mm-l) were unaffected by the drying or reequilibration of the spore samples. Kinetics of Germination
During germination the optical density of a spore suspension decreases by up to 60% and optical density measurements can provide a convenient method for studying the kinetics of germination (4, 18). Under the conditions used in the
INCUBATION
TIME
(minx)
FIG. 3. Plot of optical density at 420 nm on a log scale against incubation time at 35°C in growth medium for Bacillus megaterium spores, vacuum dried for 6 hr at different temDeratur& EdCsTle;l in anoxia: Undried ( l ), 35°C ( /J ),, 0
growth experiments above, the absorbance of the growth medium and the low initial optical density of the suspension prevent the expected decreases in optical density from being detected. Preliminary investigations into the effects of dehydration on the kinetics of germination have therefore been carried out using the technique of McCormick (9). The spore sample under test was diluted in tris(hydroxymethyl) aminomethane (Tris) buffer, pH 8.5, to give a suspension with an initial optical density at 625 nm of about 0.6. At zero time, Galanine was added to give a fina concentration of 1 mmole ml-l and decreases in optical density, after incubation at 25°C were measured at 625 nm. The optical density/incubation-time pIots for undried spores and for spores dried for 6 hr at 35 and 65°C are given in Fig. 4. In each case the initial germination lag is approximately 6 min, and the subsequent rate of optical density
SOPER, WHISTLER
76
INCUBATION
TIME
(mins.)
J?IG.4. Plot of optical density at 625 nm against incubation time at 25”C, for Bacihs megaterium spores, vacuum dried for 6 hr at different temperatures and resuspended in Tris buffer, pH 8.5 containing 1 mmole/ml L-alanine: Undried ( l ), 55-Z (01, 65°C (0).
decrease appears of the function spores.
is constant. Vacuum dehydration to affect the final optical density suspension, this value being a of the total number of germinated DISCUSSION
Under all experimental conditions investigated the differences between the total number of spores in treated and untreated samples have been shown to be less than So/o and within the limits of error associated with normal sampling. In contrast, significant differences have been observed between numbers of viable spores and, thus, percentage viability in treated and untreated samples. When samples are exposed for 6 hr to drying temperatures between 0 and 65°C spore viability is seen to vary with drying temperature in a complex manner. Of particular interest is the decrease in viability that occurs between 10 and 20°C and the marked increases in viability that are observed above
AND DAVIES
35°C. At a drying temperature of 0°C and over the range 2535”C, the viability of the dried samples is indistinguishable from that of undried standard spore suspensions. Interpretation of these data is complicated by the multiplicity of factors that could theoretically be operative under any experimental condition. Any change in viability observed after a defined drying treatment may be the result of prolonged holding at the drying temperature, the removal of biologically essential water, or damage to spore contents induced by vacuum per se, or it may be due to a combination of one or more of these factors. Heat activation of spores is time and temperature dependent, shorter times being required at high temperatures (SO-SO’C) than at low temperatures (2040°C) ( 1). Consequently, if the observed changes were entirely the result of heat activation due to prolonged exposure to the drying temperature, ‘a direct relationship would be expected to exist between viability and drying temperature. This is not observed in practice (Fig. 1). Furthermore, the increased viability observed after drying at 65°C would be expected to occur also in spores dried at lower temperatures and subsequently heated at 65°C. Extensive studies on the kinetics of heat resistance of dried Bacillus megaterium spores have demonstrated that the log survivor/heating time curves for these spores exhibit a shoulder in the region of high-surviving fraction but have consistently failed to show any increases in viability in this region ( 14). A further possibility is that the observed viability differences may be a function either of the number of sites from which water is removed during dehydration or of the methods of spore water-removal. However, as no relationship between viability and any function of the drying process such as sample temperature changes or drying rates could be found,
GERMINATION
AND
VIABILITY
this would stem unlikely, and an alternative interpretation must be found. To explain our findings we would propose that during vacuum dehydration two processes are operative, one ‘inactivating” and the other “activating.” At the lowest temperature (0°C ), a 6-hr exposure induces neither inactivation nor activation with the result that the viability of the dried spores is indistinguishable from that of uutreated spores. In the region lo-20°C the loss of viability observed is the result of inactivating processes induced by the 6-hr vacuum treatment. The proposed activation processes are assumed to be either nonoperative or insignificant under these conditions. The inactivating processes also occur during drying at high temperatures, but at these temperatures the 6-hr treatment also induces a process analogous to heat activation, and thus the observed spores viability is a summation of the effects of the inactivating and activating processes. Over the range of drying temperature 25-35”C, activation completely compensates for inactivation, with the result that the viability of the dried spores is unchanged from that of untreated spores. After 6-hr drying at 65°C the induced activation is in excess of any inactivation that occurs, ,and a significant increase in spore viability is observed. Since the balance between activation and inactivation is altered by changes in drying temperature, it would be expected that this balance would also be affected by extension of the drying time at any particular temperature. Data obtained when the period of drying was increased from 6 to 24 hr (Fig. 2) would further suggest that only a limited amount of activation can take place. At drying temperatures of 15, 25 and 35°C this is still sufficient to nullify or even overcompensate for the effects of increased inactivation. This is not the case at 65°C where the amount of inactivation induced by drying periods in excess of 12 hr exceeds the amount of
OF DRIED
SPORES
77
activation and a resultant decrease in spore viability is observed. Previous workers have shown that water must be present for heat activation to occur and that, consequently, it is not possible to heat-activate lyophilized spores ( 11). The activation processes that are proposed to occur during vacuum treatments are almost certainly different from those of true heat-activation. Evidence for this was obtained when spore suspensions were exposed to time and temperature conditions identical to those existing during the vacuum treatments with no significant change in spore viability being observed. These results show that the Bacillus megaterium spores used in this work are not activated to germinate under conditions that have been shown to effectively heat activate other strains of B. megaterium ( 1, 11). Similarly, no change in viability was observed after incubation of the test spores with n-glucose or r.-alanine. Again the incubation conditions used have been shown to induce up to 100% activation in spores of a number of Bacillus species, including B. megaterium (6, 8, 17), and it must be concluded that the test organism cannot be activated by n-glucose or L-alanine. The increases in viability observed during vacuum treatments must therefore be attributed to an activation process that does not resemble conventional heat- or chemical-induced activation. Previous reports have suggested that vapor rehydration, either on its own or as a pretreatment to Iiquid resuspension, is necessary for optimum recovery of freezedried microorganisms. Support for this suggestion has been found with yeasts (10, 12), but the data obtained with bacteria are conflicting. Slow addition of water has been shown to be beneficial to the survival of Vibrio sp., but had no effect on the viability of Staphylococcus sp. Streptococcus sp. or Enterobacteriaceae (7). The data reported here indicate that dried Bacillus megaterium spores also do not re-
78
SOPER,
WHISTLER
quire vapor rehydration for optimum recovery. It is also apparent that vapor rehydration of samples to 2 x 10W3-10 Torr prior to liquid resuspension has no significant effect on spore viability and does not influence the relationship between viability and drying temperature (Fig. 1). Thus, although it is likely that both “activation” and “inactivation” processes in some way involve the removal of water from the spore, as this occurs during vacuum treatment, we cannot conclude that these processes are reversible by water or affected by the method of rehydration. Admission of oxygen to the spores after drying or reequilibration also had no effect on spore viability under the conditions investigated. These findings are in contrast to those reported with lyophilised vegetative bacteria where a marked reduction in survival occurs if oxygen is admitted to the cells after drying (3, 5). Oxygen has aIso been shown to enhance thermal damage in bacterial spores, but if one considers the long exposure times that are required, even at high temperatures, before oxygen sensitisation of spores becomes apparent, i.e., 212 hr at lOO”C, 42 hr at 120°C (15), the results are hardly surprising. The data suggest, however, that the proposed “inactivation” and “activation” processes are probably oxygen independent. Spore viability as measured by colony counts assesses the final outcome of the sequential processes of activation, germination and outgrowth, and consequentIy results obtained could mask any changes that may be induced in these component processes by vacuum treatments. At present such changes have not been successfully demonstrated experimentally. We have, in fact, shown that the time for outgrowth of the spores and the growth rate of the subsequent vegetative cells are unaltered by the drying conditions. Furthermore, preliminary experiments on the kinetics of
AND
DAVIES
spore germination (Fig. 3) would suggest that the “activation” process induced by vacuum dehydration does not accelerate either the commencement or the rate of germination, as might be expected, but rather renders spores that have been shown to remain consistently dormant susceptible to the normal germination conditions. SUMMARY
The viability of Bacillus megaterium spores has been determined after exposure to vacuum dehydration at temperatures between 0 and 65”C, for periods up to 24 hr. A curvilinear relationship has been demonstrated between viability and drying temperature, with minimum viability occuring around 15°C and increases in viability being shown above 35°C. In contrast to vegetative bacteria, reequilibration of the dried spores to 2 x 1O-3 or 10 Torr aqueous vapor pressure, and/or subsequent exposure to oxygen had no effect on viability. Dehydration, rehydration and oxygen treatments had no effect on the time for outgrowth of the spores or on the growth rate of the resultant vegetative celIs. Physical loss of spores from samples was not demonstrated during any of these treatments. Evidence has been presented for a novel type of spore activation, which occurs during vacuum dehydration at high temperatures, to an extent that is dependent upon drying time. The mechanism of this activation is unlike that of conventional heat or chemical activation but is oxygen independent and unaffected by reequilibration to 2 x 10e3 or 10 Torr. REFERENCES 1. Busta, F. F., and Ordal, J. J. Heat activation kinetics of endospores of Bacillus subtilis. J. Food Sci. 29, 345-353 ( 1964). 2. Cook, A. M., and Lund, B. M. Total counts of bacterial spores using counting slides. J. Gem Microbial. 29, 97-104 ( 1962). 3. Fry, R. M. Freezing and drying of bacteria. In “Cryobiology” (Meryman, H. T., Ed.), p. 665, Academic Press, New York, 1966.
GERMINATION
AND VIABILITY
4. Gould, G. W., and Hitchins, A. D. Sensitisation of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulphide bonds. J. Gen. Microbial. 33, 413423 ( 1963). 5. Heckly, R. J. Preservation of bacteria by lyophilisation. Advan. Appl. Microbial. 3, 1 (1961). 6. Hyatt, M. T., and Levinson, H. S. Effects of sugars and other carbon compounds on germination and post-germinative developspores. j. ment of Bacillus megaterium BacterioZ.
88, 1403-1415
12.
13. 14. 15.
( 1964).
7. Leach, R. H., and Scott, W. J. The influence of rehydration on the viability of dried micro-organisms. J. Gen. Microbial. 21, 295-307. 8. Levinson, H. S., and Hyatt, M. T. Nitrogenous compounds in germination and post germinative development of Bacillus megaterium spores. J. Bacterial. 83, 1224-1230 ( 1962). 9. McCormick, N. G. Kinetics of spore germination. I. Bacterial. 89, 1180-1185 (1962). 10. Mitchell, J. H., Jr., and Enright, J. J. Effect of low moisture levels on the thermostability of active dry yeast. Food Technol. 11, 359-362 (1951). 11. Powell, J. F., and Hunter, J. R. Spore germinaiton in the genus Bacillus: Modification of germination requirements as a result of
16.
17.
18.
OF DRIED
SPORES
79
pre-heating. 1. Gen. Microbial. 13, 59-67 (1955). Sant, R. K., and Peterson, W. H. Factors affecting the loss of nitrogen and fermenting power of rehydrated active dry yeast. Food Technol. 12, 359-362 ( 1958). Soper, C. J. MSc. thesis. University of Bath, 1966. Soper, C. J. Ph.D. thesis. University of Bath, 1970. Soper, C. J., and Davies, D. J. G. The effect of high vacuum drying on the heat response of Bacillus megaterium spores. In “Spore Research 1971” (A. N. Barker, G. W. Gould and J. Wolf, Eds.), pp. 275-288. Academic Press, London, 1971. Soper, C. J., and Davies, D. J. G. The response of bacterial spores to vacuum treatments. I. Design and characterisation of the vacuum apparatus. Cryobiology 13, 6170 (1975). Thorley, C. H., and Wolf, J. Some germination factors of mesophilic spore formers. In “Spores II” (Halvorson, H. O., Ed.) p.1. Burgess Publishing, Minneapolis, 1961. Vary, J. C., and McCormick, N. G. Kinetics of germination of aerobic Bacillus spores. In “Spores III” (L. L. Campbell and H. 0. Halvorson, Eds.), p. 188. American Society of Microbiologists, Ann Arbor, MI, 1965.
13, 71-79
(19%)
The Response of Bacterial II. Germination G. J. SOPER,
Spores to Vacuum and Viability
J. M. WHISTLER,*
AND
Treatments.
Studies D. J. G. DAVIES
School of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom
The change from the dormant spore to the vegetative cell involves sequential processes that have been subdivided into three main classes, activation, germination and outgrowth, and we have attempted to determine any changes that might be induced in one or more of these processes by the vacuum treatment, since these would not necessarily be apparent from viability data alone. Previous work in this department (13) has shown that the heat resistance of vacuum-treated spores varies with the aqueous vapor pressure to which they are reequilibrated, a peak sensitivity being shown at 2 x IOe3 Torr and a peak resistance at 10-22 Torr. It was also shown that at all water levels heat sensitivity was increased in the presence of oxygen. Since the dehydration treatments we are studying often involve exposure of the spores to temperatures above ambient for periods up to 24 hr, it was considered that any damage induced by these treatments would also be more apparent in the presence of oxygen and that the modification of this damage by water would be more noticeable at reequilibration aqueous vapor pressures of 2 x 1O-3 and 10 Torr. We report here the influence of the oxygen exposure and rehydration treatments on the activation, germination, outgrowth and overall viability of Bacillus megaterium spores subjected to different vacuum-dehydration conditions.
In the first paper of this series (16) we presented details of the design and characterization of the instrumented vacuum apparatus being used in our studies of dehydration-induced changes in microorganisms. Physical measurements made during use of the apparatus were shown to be reproducible, and it was found that under the most severe experimental conditions studied, i.e., 24-hr treatment at 65°C there was no significant loss of bacterial spores from the samples under test. We now report on our studies to determine whether changes in the biological characteristics of the spore are induced by the various dehydration treatments. The primary and most direct method of assessing biological integrity is to measure viability. In this context the viability of a spore is defined as its ability to germinate and reproduce to the point of microscopic detection as a colony when inoculated onto solid nutrient medium. While this criterion is by no means a complete measure of spore integrity, it is nevertheless an extremely sensitive assay requiring a fully operative replicating system, since, for example, if only a small number of divisions were possible in the vegetative cells formed after germination, the spore would be scored as nonviable. Received January 15, 1975. 1 Present address: School of Pharmacy, Leicester Polytechnic, Leicester, U.K.
City
of
71 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserve.i.
SOPER, WHISTLER
72 MATERIALS
AND METHODS
Test organism. Spores of Bacillus megaterium ATCC 8245 were used throughout this work. The spores were obtained after g-day incubation of the organism on potato extract agar medium and were washed by repetitive centrifugation and resuspension in sterile glass-distilled water. A standard spore suspension of known total and viable count was prepared and stored at 0-4°C. As a spore sample, 0.06 ml of this suspension was used. Counting medium. This consisted of 2% peptone (Oxoid), 0.5% sodium chloride (Analar grade) and 1.5% agar (Oxoid) in glass-distilled water, adjusted with 1~ sodium hydroxide to give a pH, after autoclaving, of 7.2-7.4. Growth medium. The preparation and composition of the liquid growth medium was similar to that of the counting medium except that the agar was omitted. Viable counts. Samples of spore suspensions were diluted serially, when necessary, with sterile distilled water to give a suspension containing 2 X lo*-5 X lo2 viable spores ml-l. Three-tenths milliliter of this suspension was plated by a surfacespread technique onto each of five plates of counting medium. Preliminary experiments had verified that, under all experimental conditions tested, maximum values were obtained when colony counts were performed on the plates after a 48-hr incubation at 35°C. Tot& counts. The total number of spores was estimated by the method of Cook and Lund (2), using a suitable dilution of the spore suspension and a platelet counting chamber of a nominal O.Ol-mm depth. Optical density measurements. These were carried out at 25°C in l-cm silica cuvettes using an S.P.1800 recording spectrophotometer ( Unicam Ltd. ) . Vacuum treatment of spore samples. The theoretical and practical considerations involved in the design of the instrumented vacuum Iine used for treating
AND DAVIES
spore samples was presented in detail in the accompanying paper ( 16), and data for temperature and weight changes induced by the treatments may be obtained from it. The methods described there for vacuum dehydration, reequilibration to defined aqueous vapor pressures and exposure of samples to controlled gaseous environments have been utilised in this work. RESULTS
Viability
of the Standard Spore Suspension
Values for the mean number of viable spores ml-l and for the mean total number of spores ml-; determined from quintuplicate 0.06-ml samples of the untreated standard spore suspension were 4.55 x lOlo and 7.52 x lOlo, respectively, giving a mean viability of 60.5%. No significant change in these values was detected during storage of the suspension at 0.4”C over a 2-yr period. The Effect of Drying Temperature and Reequilibration to 2 x 1C3 and 10 TOW Aqueous Vapor Pressure The drying temperatures investigated were 0, 10, 15, 25, 35, 50, 60 and 65°C. Ten 0.06-ml samples of standard spore suspension were dried for 6 hr at each temperature. At the end of the drying treatment half of the samples were sealed in anoxia. The remaining samples were reequilibrated to an aqueous vapor pressure of 2 X 10e3 or 10 Torr prior to sealing in anoxia. ImmediateIy after sealing the sample vessels were opened ‘and the contained spore samples resuspended in 6 ml of sterile water. Total and viable counts were performed on each resuspended sample. Statistical analyses performed on the total counts confirmed our previous findings that there is no significant loss of spores from the samples during vacuum dehydration or reequilibration treatments. However, similar analyses performed on
GERMINATION
AND
VIABILITY
the viable-count data indicated that the drying treatments had a significant effect on spore viability. A curvilinear relationship exists between percentage viability and drying temperature (Fig. l), with a minimum viability in the region of 15°C. At temperatures above 35°C a significant increase in viability occurs with increase in drying temperature suggesting that a phenomenon analogous to activation is occuring under these conditions. At all drying temperatures, vapor rehydration of dried spores, as occurs during reequilibration to 2 x lOA or 10 Torr aqueous vapor pressure, had no detectable effect on spore viability as can also be seen in Fig. 1. We have previously demonstrated that vacuum-dried spores have maximum heat sensitivity when reequilibrated to 2 x 10e3 Torr (13), and it was anticipated that a sensitizing effect on spore viability may also be observed if dried spores were vapor rehydrated to this aqueous vapor pressure prior to reconstitution. Data obtained with samples dried for 6 hr at 0, 15, 25 and 65°C prior to reequilibration to 2 x 1O-3 Torr showed that reequilibration to this level resulted in no significant
I
T
OF DRIED
73
changes in viability and did not modify the curvilinear relationship between percentage viability and drying temperature (Fig. 1). The Efect
of Oxygen
Survival of lyophilized vegetative bacteria is lower if oxygen is admitted to samples after drying than if the samples are maintained in anoxic conditions (3, 5). It was therefore considered that any damage induced in spores by vacuum dehydration might also be more apparent in the presence of oxygen. Samples were dried for 6 hr at 0, 15, 25 and 65°C. After drying, half of the samples were sealed in anoxia, and dry oxygen was admitted to the remainder prior to sealing. In a repeat experiment, samples were reequilibrated to 10 Torr aqueous vapor pressure before sealing in either anoxia or oxygen. Viability data obtained from the resuspended samples are recorded in Table 1. Statistical analyses of these data indicated that admission of oxygen to samples after drying does not result in loss of spores from the sample. In addition, they confirmed the previous observation that reequilibration to 10 Torr aqueous vapor pressure has no effect on the viability of dried spores and also showed that there is no interaction between vapor rehydration to this level and the presence of oxygen. Furthermore, the data showed that in contrast to vegetative bacteria (5) the influence of oxygen on the viability of dried spores is negligible under the conditions investigated. Effect of Drying
FIG. 1. Mean percentage viability as a function of drying temperature for Bacillus megatetium spores, vacuum dried for 6 hr and sealed in anoxia: Unequilibrated ( l ), reequilibrated to 2 X lo4 Torr aqueous vapor pressure ( A), reequilibrated to 10 Torr aqueous vapor pressure (0).
SPORES
Time
It appeared likely that the extent of the “activation” phenomenon illustrated in Fig. 1 would be dependent upon the amount of water removed from the spore and would therefore be a function of the drying time as well as of the drying temperature. To verify this, samples were dried at 15, 25, 35 and 65°C for periods of 6, 12, 18 and 24 hr and were sealed in
7-t
SOPER,
WHISTLER
AND
DAVIES
anoxia. As in previous experiments, no significant decrease was observed in the total number of spores in the samples. The relationship between percentage viability and drying time at each temperature is shown in Fig. 2. At a temperature of 65°C maximum viability is observed after 12-hr drying, further exposure resulting in a decrease in viable count. This maximum (71.170) might have been reached at 25 and 35°C if sufficient exposure had been allowed, but within the experimental limit of 24-hr drying it is not achieved. At a temperature of 15°C the decrease in viability observed after 6-hr drying is maintained during the increased drying times. Control experiments in which samples were exposed to the drying temperatures for periods up to 24 hr, without dehydration, produced no change in spore viability, indicating that the spores did not exhibit true heat activation. It is interesting that attempts to activate the spores by chemical
6 DRYING
12 TIME
IS (hours)
FIG. 2. Mean percentage viability as a function of drying time for Bacillus megaterium spores, vacuum dried at different temperatures and sealed in anoxia: 15°C (A), 25°C (X), 35°C (a), 65°C (0).
GERMINATION
AND VIABILITY
OF DRIED
SPORES
75
methods, e.g., incubation with L-alanine and n-glucose were also unsuccessful. Time for Outgrowth Resultant Vegetative
and Growth Cells
Rate of
It is possible that the vacuum and reequilibration treatments could induce changes in spore integrity that would not necessarily be manifest as differences in spore viability. It was hoped to gain evidence of such changes by determining the time for outgrowth of spores subjected to such treatments and the growth rate of the resultant vegetative cells. The spore suspension under investigation was diluted in pre-warmed growth medium to give a culture having an optical density of 0.02 at a wavelength of 420 nm. The culture was incubated at 35°C and its growth followed by optical density measurements at 420 nm. Preliminary experiments had demonstrated that optical density changes were reproducible and could be directly correlated with changes in the number of viable organisms in the suspension, The log optical density/incubation time relationship showed an initial lag during which the optical density remained constant, followed by a logarithmic region where optical density increases with time at a constant rate (Fig. 3). The time to reach the end of the initial lag period is a measure of the time for commencement of outgrowth (t,), and the slope of the logarithmic region represents the growth rate constant (k) for the resultant vegetative cells. The values of t, (215 I+ 5 min) and k (2.69 X 10e2-t 6.12 X W4 mm-l) were unaffected by the drying or reequilibration of the spore samples. Kinetics of Germination
During germination the optical density of a spore suspension decreases by up to 60% and optical density measurements can provide a convenient method for studying the kinetics of germination (4, 18). Under the conditions used in the
INCUBATION
TIME
(minx)
FIG. 3. Plot of optical density at 420 nm on a log scale against incubation time at 35°C in growth medium for Bacillus megaterium spores, vacuum dried for 6 hr at different temDeratur& EdCsTle;l in anoxia: Undried ( l ), 35°C ( /J ),, 0
growth experiments above, the absorbance of the growth medium and the low initial optical density of the suspension prevent the expected decreases in optical density from being detected. Preliminary investigations into the effects of dehydration on the kinetics of germination have therefore been carried out using the technique of McCormick (9). The spore sample under test was diluted in tris(hydroxymethyl) aminomethane (Tris) buffer, pH 8.5, to give a suspension with an initial optical density at 625 nm of about 0.6. At zero time, Galanine was added to give a fina concentration of 1 mmole ml-l and decreases in optical density, after incubation at 25°C were measured at 625 nm. The optical density/incubation-time pIots for undried spores and for spores dried for 6 hr at 35 and 65°C are given in Fig. 4. In each case the initial germination lag is approximately 6 min, and the subsequent rate of optical density
SOPER, WHISTLER
76
INCUBATION
TIME
(mins.)
J?IG.4. Plot of optical density at 625 nm against incubation time at 25”C, for Bacihs megaterium spores, vacuum dried for 6 hr at different temperatures and resuspended in Tris buffer, pH 8.5 containing 1 mmole/ml L-alanine: Undried ( l ), 55-Z (01, 65°C (0).
decrease appears of the function spores.
is constant. Vacuum dehydration to affect the final optical density suspension, this value being a of the total number of germinated DISCUSSION
Under all experimental conditions investigated the differences between the total number of spores in treated and untreated samples have been shown to be less than So/o and within the limits of error associated with normal sampling. In contrast, significant differences have been observed between numbers of viable spores and, thus, percentage viability in treated and untreated samples. When samples are exposed for 6 hr to drying temperatures between 0 and 65°C spore viability is seen to vary with drying temperature in a complex manner. Of particular interest is the decrease in viability that occurs between 10 and 20°C and the marked increases in viability that are observed above
AND DAVIES
35°C. At a drying temperature of 0°C and over the range 2535”C, the viability of the dried samples is indistinguishable from that of undried standard spore suspensions. Interpretation of these data is complicated by the multiplicity of factors that could theoretically be operative under any experimental condition. Any change in viability observed after a defined drying treatment may be the result of prolonged holding at the drying temperature, the removal of biologically essential water, or damage to spore contents induced by vacuum per se, or it may be due to a combination of one or more of these factors. Heat activation of spores is time and temperature dependent, shorter times being required at high temperatures (SO-SO’C) than at low temperatures (2040°C) ( 1). Consequently, if the observed changes were entirely the result of heat activation due to prolonged exposure to the drying temperature, ‘a direct relationship would be expected to exist between viability and drying temperature. This is not observed in practice (Fig. 1). Furthermore, the increased viability observed after drying at 65°C would be expected to occur also in spores dried at lower temperatures and subsequently heated at 65°C. Extensive studies on the kinetics of heat resistance of dried Bacillus megaterium spores have demonstrated that the log survivor/heating time curves for these spores exhibit a shoulder in the region of high-surviving fraction but have consistently failed to show any increases in viability in this region ( 14). A further possibility is that the observed viability differences may be a function either of the number of sites from which water is removed during dehydration or of the methods of spore water-removal. However, as no relationship between viability and any function of the drying process such as sample temperature changes or drying rates could be found,
GERMINATION
AND
VIABILITY
this would stem unlikely, and an alternative interpretation must be found. To explain our findings we would propose that during vacuum dehydration two processes are operative, one ‘inactivating” and the other “activating.” At the lowest temperature (0°C ), a 6-hr exposure induces neither inactivation nor activation with the result that the viability of the dried spores is indistinguishable from that of uutreated spores. In the region lo-20°C the loss of viability observed is the result of inactivating processes induced by the 6-hr vacuum treatment. The proposed activation processes are assumed to be either nonoperative or insignificant under these conditions. The inactivating processes also occur during drying at high temperatures, but at these temperatures the 6-hr treatment also induces a process analogous to heat activation, and thus the observed spores viability is a summation of the effects of the inactivating and activating processes. Over the range of drying temperature 25-35”C, activation completely compensates for inactivation, with the result that the viability of the dried spores is unchanged from that of untreated spores. After 6-hr drying at 65°C the induced activation is in excess of any inactivation that occurs, ,and a significant increase in spore viability is observed. Since the balance between activation and inactivation is altered by changes in drying temperature, it would be expected that this balance would also be affected by extension of the drying time at any particular temperature. Data obtained when the period of drying was increased from 6 to 24 hr (Fig. 2) would further suggest that only a limited amount of activation can take place. At drying temperatures of 15, 25 and 35°C this is still sufficient to nullify or even overcompensate for the effects of increased inactivation. This is not the case at 65°C where the amount of inactivation induced by drying periods in excess of 12 hr exceeds the amount of
OF DRIED
SPORES
77
activation and a resultant decrease in spore viability is observed. Previous workers have shown that water must be present for heat activation to occur and that, consequently, it is not possible to heat-activate lyophilized spores ( 11). The activation processes that are proposed to occur during vacuum treatments are almost certainly different from those of true heat-activation. Evidence for this was obtained when spore suspensions were exposed to time and temperature conditions identical to those existing during the vacuum treatments with no significant change in spore viability being observed. These results show that the Bacillus megaterium spores used in this work are not activated to germinate under conditions that have been shown to effectively heat activate other strains of B. megaterium ( 1, 11). Similarly, no change in viability was observed after incubation of the test spores with n-glucose or r.-alanine. Again the incubation conditions used have been shown to induce up to 100% activation in spores of a number of Bacillus species, including B. megaterium (6, 8, 17), and it must be concluded that the test organism cannot be activated by n-glucose or L-alanine. The increases in viability observed during vacuum treatments must therefore be attributed to an activation process that does not resemble conventional heat- or chemical-induced activation. Previous reports have suggested that vapor rehydration, either on its own or as a pretreatment to Iiquid resuspension, is necessary for optimum recovery of freezedried microorganisms. Support for this suggestion has been found with yeasts (10, 12), but the data obtained with bacteria are conflicting. Slow addition of water has been shown to be beneficial to the survival of Vibrio sp., but had no effect on the viability of Staphylococcus sp. Streptococcus sp. or Enterobacteriaceae (7). The data reported here indicate that dried Bacillus megaterium spores also do not re-
78
SOPER,
WHISTLER
quire vapor rehydration for optimum recovery. It is also apparent that vapor rehydration of samples to 2 x 10W3-10 Torr prior to liquid resuspension has no significant effect on spore viability and does not influence the relationship between viability and drying temperature (Fig. 1). Thus, although it is likely that both “activation” and “inactivation” processes in some way involve the removal of water from the spore, as this occurs during vacuum treatment, we cannot conclude that these processes are reversible by water or affected by the method of rehydration. Admission of oxygen to the spores after drying or reequilibration also had no effect on spore viability under the conditions investigated. These findings are in contrast to those reported with lyophilised vegetative bacteria where a marked reduction in survival occurs if oxygen is admitted to the cells after drying (3, 5). Oxygen has aIso been shown to enhance thermal damage in bacterial spores, but if one considers the long exposure times that are required, even at high temperatures, before oxygen sensitisation of spores becomes apparent, i.e., 212 hr at lOO”C, 42 hr at 120°C (15), the results are hardly surprising. The data suggest, however, that the proposed “inactivation” and “activation” processes are probably oxygen independent. Spore viability as measured by colony counts assesses the final outcome of the sequential processes of activation, germination and outgrowth, and consequentIy results obtained could mask any changes that may be induced in these component processes by vacuum treatments. At present such changes have not been successfully demonstrated experimentally. We have, in fact, shown that the time for outgrowth of the spores and the growth rate of the subsequent vegetative cells are unaltered by the drying conditions. Furthermore, preliminary experiments on the kinetics of
AND
DAVIES
spore germination (Fig. 3) would suggest that the “activation” process induced by vacuum dehydration does not accelerate either the commencement or the rate of germination, as might be expected, but rather renders spores that have been shown to remain consistently dormant susceptible to the normal germination conditions. SUMMARY
The viability of Bacillus megaterium spores has been determined after exposure to vacuum dehydration at temperatures between 0 and 65”C, for periods up to 24 hr. A curvilinear relationship has been demonstrated between viability and drying temperature, with minimum viability occuring around 15°C and increases in viability being shown above 35°C. In contrast to vegetative bacteria, reequilibration of the dried spores to 2 x 1O-3 or 10 Torr aqueous vapor pressure, and/or subsequent exposure to oxygen had no effect on viability. Dehydration, rehydration and oxygen treatments had no effect on the time for outgrowth of the spores or on the growth rate of the resultant vegetative celIs. Physical loss of spores from samples was not demonstrated during any of these treatments. Evidence has been presented for a novel type of spore activation, which occurs during vacuum dehydration at high temperatures, to an extent that is dependent upon drying time. The mechanism of this activation is unlike that of conventional heat or chemical activation but is oxygen independent and unaffected by reequilibration to 2 x 10e3 or 10 Torr. REFERENCES 1. Busta, F. F., and Ordal, J. J. Heat activation kinetics of endospores of Bacillus subtilis. J. Food Sci. 29, 345-353 ( 1964). 2. Cook, A. M., and Lund, B. M. Total counts of bacterial spores using counting slides. J. Gem Microbial. 29, 97-104 ( 1962). 3. Fry, R. M. Freezing and drying of bacteria. In “Cryobiology” (Meryman, H. T., Ed.), p. 665, Academic Press, New York, 1966.
GERMINATION
AND VIABILITY
4. Gould, G. W., and Hitchins, A. D. Sensitisation of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulphide bonds. J. Gen. Microbial. 33, 413423 ( 1963). 5. Heckly, R. J. Preservation of bacteria by lyophilisation. Advan. Appl. Microbial. 3, 1 (1961). 6. Hyatt, M. T., and Levinson, H. S. Effects of sugars and other carbon compounds on germination and post-germinative developspores. j. ment of Bacillus megaterium BacterioZ.
88, 1403-1415
12.
13. 14. 15.
( 1964).
7. Leach, R. H., and Scott, W. J. The influence of rehydration on the viability of dried micro-organisms. J. Gen. Microbial. 21, 295-307. 8. Levinson, H. S., and Hyatt, M. T. Nitrogenous compounds in germination and post germinative development of Bacillus megaterium spores. J. Bacterial. 83, 1224-1230 ( 1962). 9. McCormick, N. G. Kinetics of spore germination. I. Bacterial. 89, 1180-1185 (1962). 10. Mitchell, J. H., Jr., and Enright, J. J. Effect of low moisture levels on the thermostability of active dry yeast. Food Technol. 11, 359-362 (1951). 11. Powell, J. F., and Hunter, J. R. Spore germinaiton in the genus Bacillus: Modification of germination requirements as a result of
16.
17.
18.
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SPORES
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pre-heating. 1. Gen. Microbial. 13, 59-67 (1955). Sant, R. K., and Peterson, W. H. Factors affecting the loss of nitrogen and fermenting power of rehydrated active dry yeast. Food Technol. 12, 359-362 ( 1958). Soper, C. J. MSc. thesis. University of Bath, 1966. Soper, C. J. Ph.D. thesis. University of Bath, 1970. Soper, C. J., and Davies, D. J. G. The effect of high vacuum drying on the heat response of Bacillus megaterium spores. In “Spore Research 1971” (A. N. Barker, G. W. Gould and J. Wolf, Eds.), pp. 275-288. Academic Press, London, 1971. Soper, C. J., and Davies, D. J. G. The response of bacterial spores to vacuum treatments. I. Design and characterisation of the vacuum apparatus. Cryobiology 13, 6170 (1975). Thorley, C. H., and Wolf, J. Some germination factors of mesophilic spore formers. In “Spores II” (Halvorson, H. O., Ed.) p.1. Burgess Publishing, Minneapolis, 1961. Vary, J. C., and McCormick, N. G. Kinetics of germination of aerobic Bacillus spores. In “Spores III” (L. L. Campbell and H. 0. Halvorson, Eds.), p. 188. American Society of Microbiologists, Ann Arbor, MI, 1965.