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The response of bacterial spores to vacuum treatments. I. Design and characterization of the vacuum apparatus
CRYOBIOLOGY
13, 61-70 (1976)
The Response of Bacterial Spores to Vacuum Treatments. I. Design and Characterization of the Vacuum Apparatus G. J. SOPER School of Pharmacy
AND
and Pharmacology,
Freeze-drying including secondary drying has been used for many years as a routine method for preserving microorganisms. The techniques have also been extensively used as pretreatments in studies on the response of bacteria to adverse physical and chemical agents. During these studies it has often been suggested that damage is induced in microorganisms by the dehydration and rehydration treatments per se. Unfortunately the treatments employed have been many and varied, and in the majority of instances the design and physical characteristics of the apparatus used have only been briefly documented. As a consequence, comparison of published data is rarely possible, and attempts to reproduce reported results have invariably proved difficult. We have commenced a detailed investigation of the changes induced in bacteria and bacterial spores by vacuum dehydration and subsequent rehydration. For these studies it has been necessary to develop an apparatus that would enable microorganisms to be (a) dehydrated at defined pressures between atmospheric and lOmE Torr, under controlled thermal conditions; (b) reequihbrated to defined aqueous vapor pressures in the absence of permanent gases; and (c) exposed to controlled gaseous environments. The equipment needed to be such that a number of samples could be treated Received January 15, 1975.
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
D. J. G. DAVIES University
of Bath,
Bath, United
Kingdom
together under identical conditions and individual samples removed, if required, at intervals during the treatments. Furthermore, for the apparatus to be of use for biological studies it is essential that there should be no physical loss of organisms from samples during the treatments and that each treatment should be completely characterized in terms of physical parameters, such as pressure, operating temperatures and sample weight-loss, to ensure reproducibility. The theoretical and practical considerations involved in the design of an “ideal” freeze-drying and secondary drying apparatus have been extensively detailed by Rowe (9) and Meryman (5). To attempt to build laboratory-scale equipment complying with these optimal requirements would not have been practicable. The alternative was to construct an ,apparatus that would fulfil the requirements listed above, and then to characterise it fully, using both physical and biological parameters. In this communication we give details of the design and characterisation of the vacuum apparatus that is being used in our studies. Bacterial spores have been subjected to different treatments with this apparatus with careful monitoring of pressure, temperature and weight changes throughout the whole procedure and we include data on these physical effects induced by the treatments. Some biological effects concerned with changes in the viability and germination characteristics of
SOPER AND DAVIJZS
FIG. 1. The vacuum apparatus.
the spores are given in the second paper of this series. MATERIALS
AND METHODS
Test Organism A standard suspension of Bacillus megaterium ATCC 8245 spores in sterile water, containing approximately 8 x lOlo viable spores ml-1 was used as the biological test system to assess the suitability of the vacuum apparatus. The preparation of the spore suspension and the techniques used to determine the total and viable counts of spore samples are described in the accompanying paper (10). Vacuum Apparatus This was constructed in 1.5mm wall thickness Pyrex glass and is illustrated in Fig. 1. The main section of the apparatus was 150 cm long and 3.5cm id. and was evacuated by a Speedivac G.M.2 glass mercury diffusion pump (Edwards High Vacuum Ltd.) backed by a GDRl twostage rotary vacuum pump ( A.E.I. Ltd.). The vacuum line was closed at each end by lo-mm stopcocks (A and J).
Twelve sample-vessels were attached to the conical manifold ( M ), which was of lo-cm diameter, 5-cm height, by means of BlO cones mounted radially. The manifold could be isolated from the vacuum line by the lo-mm stopcock (H) . The sample vessels were constructed from 7.5-mm id. Pyrex glass tubing. One end was sealed by fusion of the glass to give a closed flat-bottomed tube 12 cm long. The sample under test was measured into it, and the open end was fused onto a B.10 socket. The vessel was then constricted to 2-mm i.d., for a length of 5 mm, at a distance of 8 cm from the base. The environment surrounding the sample vessels in position on the manifold was controlled by a constant-temperature bath. The temperature of the bath was designated the “drying temperature.” A solid carbon dioxide/acetone cold-trap at -78°C (G) and a liquid nitrogen coldtrap at -196°C (T) were incorporated into the vacuum line at distances of 100 and 150 cm, respectively, from the manifold. Pressure measurement. Pressure was monitored continuously by means of a Pirani gauge (Edwards High Vacuum Ltd.) and a Bayard Alpert ionisation gauge
CHARACTERISTICS
OF THE VACUUM
( U.H.V. Ltd.). Pirani gauge-heads were mounted on either side of cold trap G, and a third Pirani gauge-head together with the ionisation gauge-head was mounted directly above the manifold outlet. A McLeod gauge and a simple mercury manometer were incorporated via lo-mm ports fitted with 2-mm stopcocks (C, D.). Temperature measurement. This was by means of copper-constantan thermocouples manufactured from 0.4-mm plasticcoated wire sheathed in fiber glass. One thermocouple was located in the centre of the samples with the leads from it being led out through the waII of the sample vessel below the constriction. A second thermocouple recorded the temperature of the bath surrounding the sample vessels. Measurement of sample weight changes. An electronic Micro-Force Balance Mark IIB (C. I. Electronics) was remotely mounted in a Pyrex glass vacuum-bottle attached to the vacuum line at joint K. This enabled sample weight-changes in the range of 1 pg-100 mg to be recorded automatically and continuously during any treatment. The balance pans were constructed of aluminium foil and were manufactured to the same internal dimensions as the sample vessels. Reequilibration apparatus. This enabled samples, after dehydration, to be exposed to water vapor over ice or water at known temperatures in the absence of T.I\BL,E 1 COMPOSITION OF CONST.%NT TEMPERATURE AND CORRESPONDING I~EEQUILIBRATION AQUEOUS VAPOR PRESSURES Constant
temperature
composition
BATHS
bath
Solid carbon dioxide/acetone Melting Z-chloroethanol Melting chlorobenzene Ammonium nitrate/ice Water
-78 -G9 -45 -16 10
5 x lo-’ 2 x 10-s 5 x IO--* 1 10
APPARATUS
63
permanent gases. The apparatus consisted of a double vapor trap of IO-mm i.d. Pyrex glass fitted with a 2-mm L-type three-way stopcock (E). Sterile glass-distilled water was placed in Q and degassed under vacuum over into arm R. Maintaining the degassed water at a defined temperature under vacuum produced the required reequilibration aqueous vapor pressure in the system. The composition of the constant-temperature baths and the corresponding reequilibration aqueous vapor pressures are recorded in Table 1. Exposure to controlled gaseous environments. Gases could be introduced into the apparatus via a O-250 ml min-l Rotameter flow tube and a fine needle-valve (Edwards High Vacuum Ltd. ). Gases were admitted to the sample vessels at a flow rate of 200 ml min-l and were dried by passage through cold-trap G. Operation
of the Apparatus
Dehydration. With cold-trap G in position and all stopcocks cIosed except that to the pumping system (A), the pressure in the apparatus was reduced to 1O-2 Torr, by use of the rotary vacuum pump. The sample vessel containing the thermocouple and 11 other sample vessels, each containing 0.06-ml volumes of standard spore suspension were attached to the manifold. An additional 0.06-ml sample was placed on the Micro-force balance. Constant-temperature baths were placed around the sample vessels and the vacuum bottle. After 5 min, thermocouple readings showed that the samples had equilibrated to the drying temperature, and the stopcocks to the manifold and the balance were opened. Drying was timed from this point. Reduction of pressure in the sample vessels caused degassing and snap freezing of the samples within 1 min of opening the stopcocks. When the pressure in the system had again reached 10s2 Torr, the liquid nitrogen cold-trap (T) and the mercury diffusion pump were brought into
SOPER
AND
DAVIES
decrease in pressure resulted in a rapid decrease in sample temperature (A-B). Subsequent degassing of the sample is shown by a slower, and irregular cooling rate (B-C). After degassing the sample underwent classical “pseudo-freezing,” as described by Luyet (2). The unfrozen material cooled rapidly until the initial invasion of ice occurred (C-D). At this FIG. 2. Schematic diagram of the sample temstage, the latent heat of fusion that was perature/drying-time profile obtained during generated was not dissipated as rapidly as vacuum drying of Bacillus megaterium spores it was formed and a sharp rise in temperaat 65°C. ture to the freezing point resulted (D-E). operation. Dehydration was continued at The temperature of the frozen sample then reduced pressures below 10e5 Torr for de- followed a typical cooling curve to its minimum value (E-F). fined periods up to 24 hr. Reequilibration and exposure to conPrimary drying (the removal of water by trolled gaseous environments. Reequilibrasublimation) commenced when ice was tion when required was carried out as folfirst formed and continued until the temlows. At the end of the drying period coldperature of the sample was at equilibrium trap G was removed and, when it had with that of the surrounding bath. The reached room temperature, the pumping sample temperature therefore increased system was isolated. The stopcock to the during the primary stage and the sample requilibration apparatus was opened, and temperature/drying-time profile took the the samples were left to reequilibrate with form of a classical warming curve (F-G). the water vapor at the defined aqueous During the final stage of the profile, the vapor pressure for 1 hr. sample temperature remained constant at To obtain samples under anoxic con- the chosen drying temperature. This repreditions, the sample vessels were sealed sented isothermal desorption or secondary after the dehydration treatment or after drying. reequilibration, by fusion of the glass at Five sample temperature/drying-time the constriction. On occasions, dry gases profiles were recorded at each drying temwere admitted to the samples, immediately perature, and the mean values for measureafter drying or reequilibration, prior to ments from these profiles are given in sealing. Table 2. For each measurement the five individual values obtained at a particular RESULTS AND DISCUSSION drying temperature were statistically inPhysical Measurements During Dehydradistinguishable at the 5% probability level. tion The time from opening the samples to The drying temperatures investigated the vacuum line until they reequilibrated were 0, 15, 25, 35, 50 and 65°C. Samples to the temperature of the bath, tA - tG, i.e., were dried for 24 hr at each temperature. primary drying, was dependent upon the Sample temperature measurements. The drying temperature, a longer time being sample temperature/drying-time profiles required at low temperatures than at high recorded at all drying temperatures can be temperatures. A similar dependence on represented by the schematic diagram drying temperature was seen in the time shown in Fig. 2. required to reach the minimum temperaWhen the sample vessels were first opened to the vacuum line (A), the initial ture ( tA - tF) and in the value of the
CHARACTERISTICS
OF THE TABLE
ME,ISUREMENTS
FROM SAMPLE megaterium
Bacillus
VACUUM 2
TEMPERATURE/DRYINGTIME SPORE SUSPENSION DRIED
PROFILES OBTAINED WITH AT DIFFERENT TEMPERATURES Drying 15
0
Minimum temperature of sample prior to freezing (D) Time for sample to freeze (tn - tD) Minimum tempersture of frozen sample (F) Time for frozen Time for drying
sample to reach minimum temperature (tA - TV) sample to m-equilibrate to the temperature (tr - to)
(“Cl (min) (“C)
-14
1.0
-27
10
5.5
(min)
35
18.5
-25.5 3.5 12
OF
(“C)
-13
1.03
-30
SAMPLES
35
-11.8
1.0
(min)
minimum sample temperatures ( F). In contrast, the sample temperature prior to freezing (D) was -13 * 1.2”C regardless of drying temperatures, showing that in all cases the spores underwent 1214°C of supercooling. At all temperatures except 65°C the time taken to achieve this minimum ( tA - tn) was 0.98 * 0.05 min. At 65°C the time was shorter, 0.78 min, due to evaporative drying during cooling. Sample weight changes. Sample weight/ drying-time profiles obtained at all drying temperatures were similar in shape and show the expected decrease in sample weight with increase in drying time. A condensed version of a typical profile obtained at a drying temperature of 35°C is shown in Fig. 3. For ease of comparison the sample weight/drying-time profiles were treated according to the method of Suzuki et al. (11) and expressed in the form of drying rate/drying-time curves (Fig. 4). Replicate curves obtained at each drying temperature were superimposable. Each curve exhibited an initial steady drying rate during the 5-min period in which the sample equilibrated to the drying temperature. Since the samples were in air at this stage it was expected that water loss would be by simple evaporation and would result in an activation energy for the water removal process comparable to the heat of vaporisation of pure water (1.05 x lo4 cal mole-l). The experimental value for activation energy
temperature 25
-13
0.93 -33.5
65
APPARATUS
50
65
-11.8 0.92
- 12.5
-23
-21.5
0.78
2.25
1.5
1.08
9.5
6.5
5.25
calculated from the initial drying rates (Table 3). 1.107 x lo4 cal moIe-I, confirmed this. When samples were opened to the vacuum line, the pressure changes that resulted in degassing also produced faster,
I
-2
cn
I
I
I
0
2
4
, IO
I 8
I 12
(fni,*)
I 14
(mind
I 04mg I> 23.5
23 DRYING
24
(hrr)
TlME
FIG. 3. Condensed portions of a sample weight/ drying-time profile obtained with a sample of Bacillus megaterium spores during vacuum drying at 35°C.
66
SOPER
AND
DRYING
FIG. 4. Drying dried at different 65°C (A).
rate/drying-time temperatures.
TIME
(hours)
curves for samples of Bacillus 0°C (+), 15°C (A), 25°C (
drying rates until a maximum rate was reached. These fast drying rates (Table 3) were maintained relatively unchanged for a defined period, after which they decreased rapidly until they again became constant. The duration of the fast drying rate gives an indication of the extent of primary drying. A clear or defined division between the primary drying stage and the secondary drying stage cannot be obtained, since a certain amount of secondary drying must inevitably occur, once primary drying has commenced. This is particularly illustrated at a drying tem-
irregular
TBBLE
DAVIES
3
FROM DRYING RATE/DRYING TIME PROFILES FOR Bacillus megaterium SNORES
l
megaterium spores vacuum ), 35°C (X), 50°C (O),
perature of 0°C where the end of the fast drying-rate period cannot be defined. The final drying rate at high drying temperatures was so slow that its precise measurement was not possible. However, the observation that there was always a positive drying rate and that the sample was still losing weight after 24-hr drying at 65°C casts doubt on the validity of the techniques used by previous workers whereby the residual moisture content of a sample was calculated with reference to the weight after a 3-hr drying at 60°C under a vacuum of either 10-l or 1O-5 Torr, this being considered as the weight of a completely dry sample (6, 7, 11).
MEASUREMENTS DRIED Drying
temperature (“C) 0 15 25 35 50 65
AT DIFFERENT
TEMPERATURES
Drying
rate (mg min-1)
During initial equilibration
During fast dyqzte
Not measurable 9.59 x 10-z 1.91 x 10-l 4.0 x 10-l 9.52 x 10-1 1.88
1.41 2.05 2.35 2.59 3.43 4.16
Physical tion
Measurements
During
Rehydra-
After dehydration at different temperatures for defined periods, samples were sequentially exposed to reequilibration aqueous vapor pressure of 5 X W4, 2 X 1O-s, 5 x 10-2, 1 and 10 torr for 1-hr periods prior to sealing in anoxia or in oxygen. Sample temperature measurements. Reequilibration of spore samples and sub-
CHARACTERISTICS
OF THE VACUUM
6o:y9 m
(0) (b)
(cl
r--i
I
I
I
0
20
40
RE-EQUILIBRATION
TIME
I
(mins)
FIG. 5. Sample weight/reequilibration-time profiles obtained with samples of Bacillus megutetium spores vacuum dried at 25°C and reequilibrated to different aqueous vapor pressures. (a) 2 X 10-’ Torr; (b) 5 X lo-’ Torr; (c) 1 Torr; (d) 10 Torr.
sequent admission of dry oxygen to the sample vessels prior to sealing had no measurable effect on sample temperature. Sample weight changes. Typical sample profiles for weight/reequilibration-time those samples obtained after 6-hr drying at 25°C are illustrated in Fig. 5. Under all conditions, no change in sample weight was observed after reequilibration to 5 X lo-* Torr. With other high reequilibration aqueous vapor pressures a rapid initial increase in sample weight occurred, followed by a period of reduced weight increase up to a maximum sample weight. The maximum value (Table 4) was obtained after the I-hr equilibration period, indicating that reequilibration was complete. These weight increases were unaffected by the addition of dry gases after reequilibration. Provided that removal of water has taken place to a point below the aqueous vapor
67
pressure of the reequilibration bath it would be expected that, after sufficient time, samples would reequilibrate to the same level and would attain the same weight. The weight of water taken up by the spores should therefore be a function of spore “dryness” and consequently of the degree of drying. In fact, the experimental results show that for each reequilibration level the increase in sample weight at the end of rehydration is constant and independent of the drying conditions. The rehydration process therefore results in a specific weight of water being taken up by the spore sample rather than the sample attaining a defined weight level. To explain these findings, we would suggest that water is being removed from specific sites within the spore and that the sites are of two types: Those at which dehydration is reversible sand those at which dehydration is irreversible. If the number of specific sites at which dehydration is reversible is a function of the aqueous vapor pressure of the surroundings, then, during reequilibration, water would only be taken up at these “rehydratable” sites, and thus the increase in weight of the spores would be a function only of the reequilibration aqueous vapor pressure and would be independent of the drying conditions. Furthermore, the number of sites at which dehydration is reversible and thus the weight of water taken up by the spores would be expected to increase with increase in reequilibration aqueous vapor pressure. The observation that the time taken for the sample to attain constant weight is independent of the reequilibration aqueous vapor pressure, and of the drying conditions, wouId further suggest that the available sites in the spore are readily accessible to the rehydrating water vapor and that ease of rehydration is more likely to be a function of the molecular configuration of the sites rather than of their position within the spore.
(d) 60
APPARATUS
SOPER AND DAVIES
68
TABLE
4
WEIQHT INCREBSES AFTER REEQUILIBRATION TO DEFINED AQUEOUS V.~POR PRESSURES FOR SAMPLES OF Bacillus megaterium SPORE SUSPENSION DRIED UNDER DIFFERENT CONDITIONS Drying temperature (“C)
Drying time (hr)
2 XTor
5 xTo::-*
0
6 24
4.96 X IO-$ 5.40 x 10-Z
2.18 x 10-Z 2.15 X 1O-2
1.26 X 10-l 1.18 x 10-l
4.15 x 10-l 4.20 X 10-l
15
6 24
4.70 x 10-S 4.76 X 1O-3
2.23 X 1OF 2.26 X 1OP
1.19 x 10-l 1.24 X 10-l
4.53 x 10-l 4.38 x 10-l
25
6 24
5.24 X lo+ 4.76 X 1O-3
2.00 x 10-a 1.95 x 10-Z
1.31 x 10-l 1.15 x 10-l
3.98 x 10-l 4.43 x 10-l
35
6 24
5.35 x 10-s 4.95 x 10-3
2.00 x 10-2 2.12 x 10-z
1.26 X 10-l 1.16 X 10-l
4.25 X 10-l 4.33 x 10-l
50
6 24
5.25 X lo+ 4.93 x 10-X
2.26 X 1OF 2.00 x 10-Z
1.12 x 10-l 1.23 X 10-l
4.00 x IO-’ 3.78 x 10-l
4.80 X lO-a 4.85 X 1OV 4.99 x 10-s
2.00 x 10-z 2.25 X lo+ 2.12 x 10-Z
1.10 x 10-l 1.29 x 10-l 1.21 x 10-l
3.94 x 10-l 3.82 X 10-l 4.15 x 10-l
4.98%
5.67%
5.58%
5.71%
65
Sample weight increase (mg)
6 24 Mean Coefficient of variation
Previous studies of the water sorption isotherms of bacteria and bacterial spores (4, 8) and, in particular, determinations of the heats of adsorption of water molecules onto spores (1, 3) have encouraged suggestions of localisation of the absorbed water molecules by strong interaction with specific groups in the cell constituents. These data would lend support to our suggestion for reversible and irreversible dehydration sites within the spore. TABLE
1 T0rr
10 T0l.r
Biological Measurements tion and Reequilibration
After Dehydra-
For the drying processes to be of use in subsequent studies with bacterial spores, it was essential that samples of spore suspension, after dehydration under different conditions and subsequent reequilibration, contain numbers of spores similar to those in samples of undried suspension, within the limits of normal sampling error. Fur5
TOTAL COUNT ML+ X lO-‘O FOR QUINTUPLICATK SBMPLES OF DRIED AND UNDRIED SPORES OF Bacillus megaterium Sample number
Sealed in enoxia
1 2 3 4 5 Mean
Dried for 24 hr at 65°C Reequilibrated to 10 Torr aqueous “&por pm*F3ule Sealed in Sealed in Sealed in oxygen anoxia oxygen
Unequilibrsted
7.91 7.04 7.86 7.24 7.56 7.52
6.84 7.01 7.31 6.65 7.36 7.03
7.95 7.29 7.61 7.91 7.09 7.57
6.65 6.92 6.96 7.44 7.43 7.08
7.02 7.20 7.75 7.75 7.40 7.42
CHARACTERISTICS
OF THE VACUUM
thermore, if spores were not lost from the sample during the experimental treatments, the changes in spore weight observed during these treatments could be considered to reflect accurately changes in water content of the sample. Ten samples of standard spore suspension were exposed to the most severe of the experimental dehydration treatments used, i.e., 24 hr at 65°C. Five of the samples were sealed in anoxia, and oxygen was admitted to the remaining vessels before sealing. A further ten samples were dried under identical conditions and reequilibrated to 10 Torr aqueous vapor pressur prior to sealing in anoxia or in oxygen. Immediately after sealing, each sample was resuspended in sterile water and a total spore count was performed on each resultant suspension. Total spore counts were also carried out on quintuplicate samples of the undried spore suspension. The data obtained in these experiments are summarized in Table 5. A statistical test for equality performed on the total spore counts recorded in Table 5 indicated that the differences between the mean total number of spores in undried sampIes and in dried reequilibrated or oxygen-treated spores were within the limits of error associated with normal sampling procedures, indicating that there was no significant loss of spores from the samples during any of these treatments. The vacuum dehydration apparatus we have described has been characterised using physical parameters, and the drying and reequilibration conditions produced with the equipment have been shown to be reproducible with respect to sample temperature/drying-time profiles and drying rate/drying time curves. The biological evidence that use of the apparatus results in no significant loss of spores from samples confirms the suitability of the apparatus for studying changes induced in bacteria by dehydration and rehydration.
APPARATUS
69
SUMMARY
A vacuum apparatus has been described that has enabled sampIes of bacterial spore suspensions to be dehydrated at defined temperatures between 0 and 65°C with facilities for reequilibration of the dried samples to aqueous vapor pressures between 5 x 1O-4 and 10 torr and subsequent exposure to dry gases, The apparatus has been characterized using sample temperature/drying-time profiles, and drying rate/ drying-time curves, and the reproducibility of the dehydration and rehydration techniques has been established. Biologica data have confirmed the suitability of the apparatus since no loss of spores from samples has been observed during any of these experimental treatments. On the basis of measurements recorded during rehydration of dried spores, it is suggested that dehydration occurs at specific sites in the spore which are of two types, 1) reversibly dehydrated (rehydratable) and 2) irreversibly dehydrated (nonrehydratable), REFERENCES 1. Koga, S., Echigo, A., and Nunomura, K. Physical properties of cell water in partially dried Saccharomyces cereuisiae. Biophys. J. 6, 665-674 ( 1966). 2. Luyet, B. J. An attempt at a systematic analysis of the notion of freezing rates and at an evaluation of the main contributory factors. Cryobiology 2, 196-205 (1966). 3. Maeda, Y., Fujita, T., Sugiura, Y., and Koga, S. Physical properties of water in sporesof Bacillus megaterium. J. Gen. Appl. Microbial. 14, 217-222 ( 1968). 4. Marshall, B., and Murrell, W. G. Biophysical analysis of the spore. J. Appl. Bacterial. 33, 103-129 ( 1970). 5. Meryman, H. T. Freeze-drying. In “Cryobiology” (Meryman, H. T., Ed.), p. 609. Academic Press, New York, 1966. 6. Nei, T., Araki, T., and Souzu, H. Studies of the effect of drying conditions on residual moisture content and cell viability in the freeze-drying of micro-organisms. c11JObiology 2, 68-73 (1965). 7. Nei, T., Souzu, H., and An&i, T. Effect of residual moisture content on the survival of freeze-dried bacteria during storage
70
SOPER AND DAVIES
under various conditions. Cryobiology 2, 276-279 ( 1966). 8. Neihof, R., Thompson, J. K., and Deitz, V. R. Sorption of water vapor and nitrogen gas by bacterial spores. Nature (London) 216, 1304 (1967). 9. Rowe, T. W. G. Freeze-drying of biological materials: Some physical and engineering aspects. In “Current Trends in Cryobiology” (Smith, A. U., Ed.), p. 61. Plenum Press, London, 1970.
10. Soper, C. J., Whistler, J. M., and Davies, D. J. G. The response of bacterial spores to vacuum treatment. II. Germination and viability studies. Cryobiology. 13, 71-79 (1975). 11. Suzuki, M., Sawada, T., and Obayashi, Y. Reduction of the survival rate of BCG in the course of drying, relative to its dehydration curve. In “Freezing and Drying of Micro-organisms” (T. Nei, Ed.), p. 81. University Park Press, Manchester, England, 1969.
13, 61-70 (1976)
The Response of Bacterial Spores to Vacuum Treatments. I. Design and Characterization of the Vacuum Apparatus G. J. SOPER School of Pharmacy
AND
and Pharmacology,
Freeze-drying including secondary drying has been used for many years as a routine method for preserving microorganisms. The techniques have also been extensively used as pretreatments in studies on the response of bacteria to adverse physical and chemical agents. During these studies it has often been suggested that damage is induced in microorganisms by the dehydration and rehydration treatments per se. Unfortunately the treatments employed have been many and varied, and in the majority of instances the design and physical characteristics of the apparatus used have only been briefly documented. As a consequence, comparison of published data is rarely possible, and attempts to reproduce reported results have invariably proved difficult. We have commenced a detailed investigation of the changes induced in bacteria and bacterial spores by vacuum dehydration and subsequent rehydration. For these studies it has been necessary to develop an apparatus that would enable microorganisms to be (a) dehydrated at defined pressures between atmospheric and lOmE Torr, under controlled thermal conditions; (b) reequihbrated to defined aqueous vapor pressures in the absence of permanent gases; and (c) exposed to controlled gaseous environments. The equipment needed to be such that a number of samples could be treated Received January 15, 1975.
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
D. J. G. DAVIES University
of Bath,
Bath, United
Kingdom
together under identical conditions and individual samples removed, if required, at intervals during the treatments. Furthermore, for the apparatus to be of use for biological studies it is essential that there should be no physical loss of organisms from samples during the treatments and that each treatment should be completely characterized in terms of physical parameters, such as pressure, operating temperatures and sample weight-loss, to ensure reproducibility. The theoretical and practical considerations involved in the design of an “ideal” freeze-drying and secondary drying apparatus have been extensively detailed by Rowe (9) and Meryman (5). To attempt to build laboratory-scale equipment complying with these optimal requirements would not have been practicable. The alternative was to construct an ,apparatus that would fulfil the requirements listed above, and then to characterise it fully, using both physical and biological parameters. In this communication we give details of the design and characterisation of the vacuum apparatus that is being used in our studies. Bacterial spores have been subjected to different treatments with this apparatus with careful monitoring of pressure, temperature and weight changes throughout the whole procedure and we include data on these physical effects induced by the treatments. Some biological effects concerned with changes in the viability and germination characteristics of
SOPER AND DAVIJZS
FIG. 1. The vacuum apparatus.
the spores are given in the second paper of this series. MATERIALS
AND METHODS
Test Organism A standard suspension of Bacillus megaterium ATCC 8245 spores in sterile water, containing approximately 8 x lOlo viable spores ml-1 was used as the biological test system to assess the suitability of the vacuum apparatus. The preparation of the spore suspension and the techniques used to determine the total and viable counts of spore samples are described in the accompanying paper (10). Vacuum Apparatus This was constructed in 1.5mm wall thickness Pyrex glass and is illustrated in Fig. 1. The main section of the apparatus was 150 cm long and 3.5cm id. and was evacuated by a Speedivac G.M.2 glass mercury diffusion pump (Edwards High Vacuum Ltd.) backed by a GDRl twostage rotary vacuum pump ( A.E.I. Ltd.). The vacuum line was closed at each end by lo-mm stopcocks (A and J).
Twelve sample-vessels were attached to the conical manifold ( M ), which was of lo-cm diameter, 5-cm height, by means of BlO cones mounted radially. The manifold could be isolated from the vacuum line by the lo-mm stopcock (H) . The sample vessels were constructed from 7.5-mm id. Pyrex glass tubing. One end was sealed by fusion of the glass to give a closed flat-bottomed tube 12 cm long. The sample under test was measured into it, and the open end was fused onto a B.10 socket. The vessel was then constricted to 2-mm i.d., for a length of 5 mm, at a distance of 8 cm from the base. The environment surrounding the sample vessels in position on the manifold was controlled by a constant-temperature bath. The temperature of the bath was designated the “drying temperature.” A solid carbon dioxide/acetone cold-trap at -78°C (G) and a liquid nitrogen coldtrap at -196°C (T) were incorporated into the vacuum line at distances of 100 and 150 cm, respectively, from the manifold. Pressure measurement. Pressure was monitored continuously by means of a Pirani gauge (Edwards High Vacuum Ltd.) and a Bayard Alpert ionisation gauge
CHARACTERISTICS
OF THE VACUUM
( U.H.V. Ltd.). Pirani gauge-heads were mounted on either side of cold trap G, and a third Pirani gauge-head together with the ionisation gauge-head was mounted directly above the manifold outlet. A McLeod gauge and a simple mercury manometer were incorporated via lo-mm ports fitted with 2-mm stopcocks (C, D.). Temperature measurement. This was by means of copper-constantan thermocouples manufactured from 0.4-mm plasticcoated wire sheathed in fiber glass. One thermocouple was located in the centre of the samples with the leads from it being led out through the waII of the sample vessel below the constriction. A second thermocouple recorded the temperature of the bath surrounding the sample vessels. Measurement of sample weight changes. An electronic Micro-Force Balance Mark IIB (C. I. Electronics) was remotely mounted in a Pyrex glass vacuum-bottle attached to the vacuum line at joint K. This enabled sample weight-changes in the range of 1 pg-100 mg to be recorded automatically and continuously during any treatment. The balance pans were constructed of aluminium foil and were manufactured to the same internal dimensions as the sample vessels. Reequilibration apparatus. This enabled samples, after dehydration, to be exposed to water vapor over ice or water at known temperatures in the absence of T.I\BL,E 1 COMPOSITION OF CONST.%NT TEMPERATURE AND CORRESPONDING I~EEQUILIBRATION AQUEOUS VAPOR PRESSURES Constant
temperature
composition
BATHS
bath
Solid carbon dioxide/acetone Melting Z-chloroethanol Melting chlorobenzene Ammonium nitrate/ice Water
-78 -G9 -45 -16 10
5 x lo-’ 2 x 10-s 5 x IO--* 1 10
APPARATUS
63
permanent gases. The apparatus consisted of a double vapor trap of IO-mm i.d. Pyrex glass fitted with a 2-mm L-type three-way stopcock (E). Sterile glass-distilled water was placed in Q and degassed under vacuum over into arm R. Maintaining the degassed water at a defined temperature under vacuum produced the required reequilibration aqueous vapor pressure in the system. The composition of the constant-temperature baths and the corresponding reequilibration aqueous vapor pressures are recorded in Table 1. Exposure to controlled gaseous environments. Gases could be introduced into the apparatus via a O-250 ml min-l Rotameter flow tube and a fine needle-valve (Edwards High Vacuum Ltd. ). Gases were admitted to the sample vessels at a flow rate of 200 ml min-l and were dried by passage through cold-trap G. Operation
of the Apparatus
Dehydration. With cold-trap G in position and all stopcocks cIosed except that to the pumping system (A), the pressure in the apparatus was reduced to 1O-2 Torr, by use of the rotary vacuum pump. The sample vessel containing the thermocouple and 11 other sample vessels, each containing 0.06-ml volumes of standard spore suspension were attached to the manifold. An additional 0.06-ml sample was placed on the Micro-force balance. Constant-temperature baths were placed around the sample vessels and the vacuum bottle. After 5 min, thermocouple readings showed that the samples had equilibrated to the drying temperature, and the stopcocks to the manifold and the balance were opened. Drying was timed from this point. Reduction of pressure in the sample vessels caused degassing and snap freezing of the samples within 1 min of opening the stopcocks. When the pressure in the system had again reached 10s2 Torr, the liquid nitrogen cold-trap (T) and the mercury diffusion pump were brought into
SOPER
AND
DAVIES
decrease in pressure resulted in a rapid decrease in sample temperature (A-B). Subsequent degassing of the sample is shown by a slower, and irregular cooling rate (B-C). After degassing the sample underwent classical “pseudo-freezing,” as described by Luyet (2). The unfrozen material cooled rapidly until the initial invasion of ice occurred (C-D). At this FIG. 2. Schematic diagram of the sample temstage, the latent heat of fusion that was perature/drying-time profile obtained during generated was not dissipated as rapidly as vacuum drying of Bacillus megaterium spores it was formed and a sharp rise in temperaat 65°C. ture to the freezing point resulted (D-E). operation. Dehydration was continued at The temperature of the frozen sample then reduced pressures below 10e5 Torr for de- followed a typical cooling curve to its minimum value (E-F). fined periods up to 24 hr. Reequilibration and exposure to conPrimary drying (the removal of water by trolled gaseous environments. Reequilibrasublimation) commenced when ice was tion when required was carried out as folfirst formed and continued until the temlows. At the end of the drying period coldperature of the sample was at equilibrium trap G was removed and, when it had with that of the surrounding bath. The reached room temperature, the pumping sample temperature therefore increased system was isolated. The stopcock to the during the primary stage and the sample requilibration apparatus was opened, and temperature/drying-time profile took the the samples were left to reequilibrate with form of a classical warming curve (F-G). the water vapor at the defined aqueous During the final stage of the profile, the vapor pressure for 1 hr. sample temperature remained constant at To obtain samples under anoxic con- the chosen drying temperature. This repreditions, the sample vessels were sealed sented isothermal desorption or secondary after the dehydration treatment or after drying. reequilibration, by fusion of the glass at Five sample temperature/drying-time the constriction. On occasions, dry gases profiles were recorded at each drying temwere admitted to the samples, immediately perature, and the mean values for measureafter drying or reequilibration, prior to ments from these profiles are given in sealing. Table 2. For each measurement the five individual values obtained at a particular RESULTS AND DISCUSSION drying temperature were statistically inPhysical Measurements During Dehydradistinguishable at the 5% probability level. tion The time from opening the samples to The drying temperatures investigated the vacuum line until they reequilibrated were 0, 15, 25, 35, 50 and 65°C. Samples to the temperature of the bath, tA - tG, i.e., were dried for 24 hr at each temperature. primary drying, was dependent upon the Sample temperature measurements. The drying temperature, a longer time being sample temperature/drying-time profiles required at low temperatures than at high recorded at all drying temperatures can be temperatures. A similar dependence on represented by the schematic diagram drying temperature was seen in the time shown in Fig. 2. required to reach the minimum temperaWhen the sample vessels were first opened to the vacuum line (A), the initial ture ( tA - tF) and in the value of the
CHARACTERISTICS
OF THE TABLE
ME,ISUREMENTS
FROM SAMPLE megaterium
Bacillus
VACUUM 2
TEMPERATURE/DRYINGTIME SPORE SUSPENSION DRIED
PROFILES OBTAINED WITH AT DIFFERENT TEMPERATURES Drying 15
0
Minimum temperature of sample prior to freezing (D) Time for sample to freeze (tn - tD) Minimum tempersture of frozen sample (F) Time for frozen Time for drying
sample to reach minimum temperature (tA - TV) sample to m-equilibrate to the temperature (tr - to)
(“Cl (min) (“C)
-14
1.0
-27
10
5.5
(min)
35
18.5
-25.5 3.5 12
OF
(“C)
-13
1.03
-30
SAMPLES
35
-11.8
1.0
(min)
minimum sample temperatures ( F). In contrast, the sample temperature prior to freezing (D) was -13 * 1.2”C regardless of drying temperatures, showing that in all cases the spores underwent 1214°C of supercooling. At all temperatures except 65°C the time taken to achieve this minimum ( tA - tn) was 0.98 * 0.05 min. At 65°C the time was shorter, 0.78 min, due to evaporative drying during cooling. Sample weight changes. Sample weight/ drying-time profiles obtained at all drying temperatures were similar in shape and show the expected decrease in sample weight with increase in drying time. A condensed version of a typical profile obtained at a drying temperature of 35°C is shown in Fig. 3. For ease of comparison the sample weight/drying-time profiles were treated according to the method of Suzuki et al. (11) and expressed in the form of drying rate/drying-time curves (Fig. 4). Replicate curves obtained at each drying temperature were superimposable. Each curve exhibited an initial steady drying rate during the 5-min period in which the sample equilibrated to the drying temperature. Since the samples were in air at this stage it was expected that water loss would be by simple evaporation and would result in an activation energy for the water removal process comparable to the heat of vaporisation of pure water (1.05 x lo4 cal mole-l). The experimental value for activation energy
temperature 25
-13
0.93 -33.5
65
APPARATUS
50
65
-11.8 0.92
- 12.5
-23
-21.5
0.78
2.25
1.5
1.08
9.5
6.5
5.25
calculated from the initial drying rates (Table 3). 1.107 x lo4 cal moIe-I, confirmed this. When samples were opened to the vacuum line, the pressure changes that resulted in degassing also produced faster,
I
-2
cn
I
I
I
0
2
4
, IO
I 8
I 12
(fni,*)
I 14
(mind
I 04mg I> 23.5
23 DRYING
24
(hrr)
TlME
FIG. 3. Condensed portions of a sample weight/ drying-time profile obtained with a sample of Bacillus megaterium spores during vacuum drying at 35°C.
66
SOPER
AND
DRYING
FIG. 4. Drying dried at different 65°C (A).
rate/drying-time temperatures.
TIME
(hours)
curves for samples of Bacillus 0°C (+), 15°C (A), 25°C (
drying rates until a maximum rate was reached. These fast drying rates (Table 3) were maintained relatively unchanged for a defined period, after which they decreased rapidly until they again became constant. The duration of the fast drying rate gives an indication of the extent of primary drying. A clear or defined division between the primary drying stage and the secondary drying stage cannot be obtained, since a certain amount of secondary drying must inevitably occur, once primary drying has commenced. This is particularly illustrated at a drying tem-
irregular
TBBLE
DAVIES
3
FROM DRYING RATE/DRYING TIME PROFILES FOR Bacillus megaterium SNORES
l
megaterium spores vacuum ), 35°C (X), 50°C (O),
perature of 0°C where the end of the fast drying-rate period cannot be defined. The final drying rate at high drying temperatures was so slow that its precise measurement was not possible. However, the observation that there was always a positive drying rate and that the sample was still losing weight after 24-hr drying at 65°C casts doubt on the validity of the techniques used by previous workers whereby the residual moisture content of a sample was calculated with reference to the weight after a 3-hr drying at 60°C under a vacuum of either 10-l or 1O-5 Torr, this being considered as the weight of a completely dry sample (6, 7, 11).
MEASUREMENTS DRIED Drying
temperature (“C) 0 15 25 35 50 65
AT DIFFERENT
TEMPERATURES
Drying
rate (mg min-1)
During initial equilibration
During fast dyqzte
Not measurable 9.59 x 10-z 1.91 x 10-l 4.0 x 10-l 9.52 x 10-1 1.88
1.41 2.05 2.35 2.59 3.43 4.16
Physical tion
Measurements
During
Rehydra-
After dehydration at different temperatures for defined periods, samples were sequentially exposed to reequilibration aqueous vapor pressure of 5 X W4, 2 X 1O-s, 5 x 10-2, 1 and 10 torr for 1-hr periods prior to sealing in anoxia or in oxygen. Sample temperature measurements. Reequilibration of spore samples and sub-
CHARACTERISTICS
OF THE VACUUM
6o:y9 m
(0) (b)
(cl
r--i
I
I
I
0
20
40
RE-EQUILIBRATION
TIME
I
(mins)
FIG. 5. Sample weight/reequilibration-time profiles obtained with samples of Bacillus megutetium spores vacuum dried at 25°C and reequilibrated to different aqueous vapor pressures. (a) 2 X 10-’ Torr; (b) 5 X lo-’ Torr; (c) 1 Torr; (d) 10 Torr.
sequent admission of dry oxygen to the sample vessels prior to sealing had no measurable effect on sample temperature. Sample weight changes. Typical sample profiles for weight/reequilibration-time those samples obtained after 6-hr drying at 25°C are illustrated in Fig. 5. Under all conditions, no change in sample weight was observed after reequilibration to 5 X lo-* Torr. With other high reequilibration aqueous vapor pressures a rapid initial increase in sample weight occurred, followed by a period of reduced weight increase up to a maximum sample weight. The maximum value (Table 4) was obtained after the I-hr equilibration period, indicating that reequilibration was complete. These weight increases were unaffected by the addition of dry gases after reequilibration. Provided that removal of water has taken place to a point below the aqueous vapor
67
pressure of the reequilibration bath it would be expected that, after sufficient time, samples would reequilibrate to the same level and would attain the same weight. The weight of water taken up by the spores should therefore be a function of spore “dryness” and consequently of the degree of drying. In fact, the experimental results show that for each reequilibration level the increase in sample weight at the end of rehydration is constant and independent of the drying conditions. The rehydration process therefore results in a specific weight of water being taken up by the spore sample rather than the sample attaining a defined weight level. To explain these findings, we would suggest that water is being removed from specific sites within the spore and that the sites are of two types: Those at which dehydration is reversible sand those at which dehydration is irreversible. If the number of specific sites at which dehydration is reversible is a function of the aqueous vapor pressure of the surroundings, then, during reequilibration, water would only be taken up at these “rehydratable” sites, and thus the increase in weight of the spores would be a function only of the reequilibration aqueous vapor pressure and would be independent of the drying conditions. Furthermore, the number of sites at which dehydration is reversible and thus the weight of water taken up by the spores would be expected to increase with increase in reequilibration aqueous vapor pressure. The observation that the time taken for the sample to attain constant weight is independent of the reequilibration aqueous vapor pressure, and of the drying conditions, wouId further suggest that the available sites in the spore are readily accessible to the rehydrating water vapor and that ease of rehydration is more likely to be a function of the molecular configuration of the sites rather than of their position within the spore.
(d) 60
APPARATUS
SOPER AND DAVIES
68
TABLE
4
WEIQHT INCREBSES AFTER REEQUILIBRATION TO DEFINED AQUEOUS V.~POR PRESSURES FOR SAMPLES OF Bacillus megaterium SPORE SUSPENSION DRIED UNDER DIFFERENT CONDITIONS Drying temperature (“C)
Drying time (hr)
2 XTor
5 xTo::-*
0
6 24
4.96 X IO-$ 5.40 x 10-Z
2.18 x 10-Z 2.15 X 1O-2
1.26 X 10-l 1.18 x 10-l
4.15 x 10-l 4.20 X 10-l
15
6 24
4.70 x 10-S 4.76 X 1O-3
2.23 X 1OF 2.26 X 1OP
1.19 x 10-l 1.24 X 10-l
4.53 x 10-l 4.38 x 10-l
25
6 24
5.24 X lo+ 4.76 X 1O-3
2.00 x 10-a 1.95 x 10-Z
1.31 x 10-l 1.15 x 10-l
3.98 x 10-l 4.43 x 10-l
35
6 24
5.35 x 10-s 4.95 x 10-3
2.00 x 10-2 2.12 x 10-z
1.26 X 10-l 1.16 X 10-l
4.25 X 10-l 4.33 x 10-l
50
6 24
5.25 X lo+ 4.93 x 10-X
2.26 X 1OF 2.00 x 10-Z
1.12 x 10-l 1.23 X 10-l
4.00 x IO-’ 3.78 x 10-l
4.80 X lO-a 4.85 X 1OV 4.99 x 10-s
2.00 x 10-z 2.25 X lo+ 2.12 x 10-Z
1.10 x 10-l 1.29 x 10-l 1.21 x 10-l
3.94 x 10-l 3.82 X 10-l 4.15 x 10-l
4.98%
5.67%
5.58%
5.71%
65
Sample weight increase (mg)
6 24 Mean Coefficient of variation
Previous studies of the water sorption isotherms of bacteria and bacterial spores (4, 8) and, in particular, determinations of the heats of adsorption of water molecules onto spores (1, 3) have encouraged suggestions of localisation of the absorbed water molecules by strong interaction with specific groups in the cell constituents. These data would lend support to our suggestion for reversible and irreversible dehydration sites within the spore. TABLE
1 T0rr
10 T0l.r
Biological Measurements tion and Reequilibration
After Dehydra-
For the drying processes to be of use in subsequent studies with bacterial spores, it was essential that samples of spore suspension, after dehydration under different conditions and subsequent reequilibration, contain numbers of spores similar to those in samples of undried suspension, within the limits of normal sampling error. Fur5
TOTAL COUNT ML+ X lO-‘O FOR QUINTUPLICATK SBMPLES OF DRIED AND UNDRIED SPORES OF Bacillus megaterium Sample number
Sealed in enoxia
1 2 3 4 5 Mean
Dried for 24 hr at 65°C Reequilibrated to 10 Torr aqueous “&por pm*F3ule Sealed in Sealed in Sealed in oxygen anoxia oxygen
Unequilibrsted
7.91 7.04 7.86 7.24 7.56 7.52
6.84 7.01 7.31 6.65 7.36 7.03
7.95 7.29 7.61 7.91 7.09 7.57
6.65 6.92 6.96 7.44 7.43 7.08
7.02 7.20 7.75 7.75 7.40 7.42
CHARACTERISTICS
OF THE VACUUM
thermore, if spores were not lost from the sample during the experimental treatments, the changes in spore weight observed during these treatments could be considered to reflect accurately changes in water content of the sample. Ten samples of standard spore suspension were exposed to the most severe of the experimental dehydration treatments used, i.e., 24 hr at 65°C. Five of the samples were sealed in anoxia, and oxygen was admitted to the remaining vessels before sealing. A further ten samples were dried under identical conditions and reequilibrated to 10 Torr aqueous vapor pressur prior to sealing in anoxia or in oxygen. Immediately after sealing, each sample was resuspended in sterile water and a total spore count was performed on each resultant suspension. Total spore counts were also carried out on quintuplicate samples of the undried spore suspension. The data obtained in these experiments are summarized in Table 5. A statistical test for equality performed on the total spore counts recorded in Table 5 indicated that the differences between the mean total number of spores in undried sampIes and in dried reequilibrated or oxygen-treated spores were within the limits of error associated with normal sampling procedures, indicating that there was no significant loss of spores from the samples during any of these treatments. The vacuum dehydration apparatus we have described has been characterised using physical parameters, and the drying and reequilibration conditions produced with the equipment have been shown to be reproducible with respect to sample temperature/drying-time profiles and drying rate/drying time curves. The biological evidence that use of the apparatus results in no significant loss of spores from samples confirms the suitability of the apparatus for studying changes induced in bacteria by dehydration and rehydration.
APPARATUS
69
SUMMARY
A vacuum apparatus has been described that has enabled sampIes of bacterial spore suspensions to be dehydrated at defined temperatures between 0 and 65°C with facilities for reequilibration of the dried samples to aqueous vapor pressures between 5 x 1O-4 and 10 torr and subsequent exposure to dry gases, The apparatus has been characterized using sample temperature/drying-time profiles, and drying rate/ drying-time curves, and the reproducibility of the dehydration and rehydration techniques has been established. Biologica data have confirmed the suitability of the apparatus since no loss of spores from samples has been observed during any of these experimental treatments. On the basis of measurements recorded during rehydration of dried spores, it is suggested that dehydration occurs at specific sites in the spore which are of two types, 1) reversibly dehydrated (rehydratable) and 2) irreversibly dehydrated (nonrehydratable), REFERENCES 1. Koga, S., Echigo, A., and Nunomura, K. Physical properties of cell water in partially dried Saccharomyces cereuisiae. Biophys. J. 6, 665-674 ( 1966). 2. Luyet, B. J. An attempt at a systematic analysis of the notion of freezing rates and at an evaluation of the main contributory factors. Cryobiology 2, 196-205 (1966). 3. Maeda, Y., Fujita, T., Sugiura, Y., and Koga, S. Physical properties of water in sporesof Bacillus megaterium. J. Gen. Appl. Microbial. 14, 217-222 ( 1968). 4. Marshall, B., and Murrell, W. G. Biophysical analysis of the spore. J. Appl. Bacterial. 33, 103-129 ( 1970). 5. Meryman, H. T. Freeze-drying. In “Cryobiology” (Meryman, H. T., Ed.), p. 609. Academic Press, New York, 1966. 6. Nei, T., Araki, T., and Souzu, H. Studies of the effect of drying conditions on residual moisture content and cell viability in the freeze-drying of micro-organisms. c11JObiology 2, 68-73 (1965). 7. Nei, T., Souzu, H., and An&i, T. Effect of residual moisture content on the survival of freeze-dried bacteria during storage
70
SOPER AND DAVIES
under various conditions. Cryobiology 2, 276-279 ( 1966). 8. Neihof, R., Thompson, J. K., and Deitz, V. R. Sorption of water vapor and nitrogen gas by bacterial spores. Nature (London) 216, 1304 (1967). 9. Rowe, T. W. G. Freeze-drying of biological materials: Some physical and engineering aspects. In “Current Trends in Cryobiology” (Smith, A. U., Ed.), p. 61. Plenum Press, London, 1970.
10. Soper, C. J., Whistler, J. M., and Davies, D. J. G. The response of bacterial spores to vacuum treatment. II. Germination and viability studies. Cryobiology. 13, 71-79 (1975). 11. Suzuki, M., Sawada, T., and Obayashi, Y. Reduction of the survival rate of BCG in the course of drying, relative to its dehydration curve. In “Freezing and Drying of Micro-organisms” (T. Nei, Ed.), p. 81. University Park Press, Manchester, England, 1969.