Preservation of Microorganisms

ROBERT J. HECKLY Naval Biosciences Laboratory

School of Public Health, University of California, Berkeley, Califwnia

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Criteria for Preservation. .....................

A. B. Functional Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Maintenance of Full Genetic Complement . . . . . . . . . . . . .

111. Preservation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Direct Transfer on Culture Media ....................

C. In Distille . . . . . . . . . . . .

E. Dehydrated.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ability to Reproduce.. . . . . . . .: . . . . . . . . . . .

...........................

IV. Culture Collection Practices . . . V. Industrial or Commercial Practices . . . . . . . . . . . . . . . . . . . . . . .

VI. Procedures for Selected Groups . . . . . . . . . . . . . . . . . . . . . . . . . . A. Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Fungi, Yeasts, and Actinomycetes D. Viruses and Bacteriophages

VII. Summary . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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14 15 29 31 34 34 35 41 42 47 47

1. Introduction

Maintenanbe of cultures is a problem common to many areas of microbiol- ogy. The microbiologist needs to have a convenient method for maintaining organisms, for without such tools he or she is out of business. Similarly, a number of industries must maintain the cultures used in the manufacture of their product, whether it be beer, wine, antibiotics, bread, or milk products, such as cheese or buttermilk. It would be desirable to be able to define the conditions for optimal survival of each of the various organisms but this is not yet possible.

As it is not generally feasible to preserve each organism in a culture collection under more than one condition, there is a scarcity of comparative data on long-term storage of a variety of organisms. The preponderance of publications on optimizing culture preservation methods is written by mic- robiologists interested in maintaining relatively few species, often only one strain. Those studying the mechanisms of action of freezing and thawing or lyophilization often use only one or two strains in their studies.

This author will attempt to summarize information on the various methods used to preserve microorganisms. There are many factors to be considered

1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24

Copyright IQ 1978 by Academic Press, Inc. All rights of repdudion m any form reserved.

ISBN 0-12402634-4

2 ROBERT J. HECKLY

and there is no single “best” method applicable to all situations, but by comparing the results of the various procedures it is hoped that the task of selecting procedures for the preservation of specific cultures can be simplified.

Organisms are able to survive under a wide variety of adverse conditions. In fact, they seem to be everywhere and are sometimes difficult to eliminate. However, some organisms are not hardy and preservation of cultures with special characteristics can be a problem. The total property of a culture may depend upon several variants within the culture. Therefore, it is important that all, or at least a representative sample, of the cells be retained in a viable state.

II. Criteria for Preservation

A. ABILITY TO REPRODUCE

Quantitative measurements, such as colony-forming units (cfu) or plaque- forming units (pfu), provide much information about the quality of the pres- ervation technique. If these assays are made before and after storage, they provide an objective means for selecting the optimum method. Unfortu- nately, too many investigators do not have the time to make quantitative measurements of viability. Instead, they depend on a growth or no-growth test; i.e., the entire contents of a vial may be transferred to growth media which are then examined for signs of life. Reconstitution of replicate samples can yield a degree of quantitation. For example, Antheunisse (1972) reported survival after various storage periods based on the percentage of vials that yielded viable cultures. Obviously, such survival rates cannot be compared with survival rates based on the number of cells in a sample that have formed colonies.

Selection of the proper medium for evaluating ability to reproduce should receive serious consideration. Judging from the number of papers published on injury and repair in bacteria, it appears that a significant proportion of stored cells are injured and are unable to initiate growth under the ordinary conditions. Injury is defined as sublethal or repairable damage, whereas mutation is a permanent change. Some injured cells are no longer able to grow on minimal media but are able to form colonies on complete media, such as trypticase soy agar (Sinskey and Silverman, 1970; Gomezet al., 1973; Gibson et al., 1965). In other instances the injured cells required only supplementation with aspartate (Kuo and MacLeod, 1969) or pyruvate (Baird-Parker and Davenport, 1965) to grow. A loss of salt tolerance (Morichi and Irie, 1973) or an increase in the lag phase (Beker, 1972) have also been

PRESERVATION OF MICROORGANISMS 3

attributed to cell injury. In other instances, the proportion of injured cells was determined by the use of special selective media (Ray et al., 1971a, b, 1972; Janssen and Busta, 1973a,b). The percentage of injured cells in pre- served cultures has been as high as 95% (Janssen and Busta 1973a; Ray et al., 1971a; Gibson et al., 1965).

B. FUNCTIONAL PROPERTIES

Viability, even if it is based on number of organisms surviving, is not an entirely satisfactory criterion for evaluating effectiveness of culture preserva- tion. Sirks et al. (1974) demonstrated that the efficacy of two different types of freeze-dried tuberculosis vaccine made from the avirulent bacillus of Calmette and Guerin (BCG) in guinea pigs was not correlated with the percentage of cells surviving the lyophilization and storage. Immunogenic properties of the live vaccine were altered by the drying. Similarly, Heckly et al. (1958) found that although 40% of the lyophilized Yersinia pestis cells survived after 9 months, only 0.3% of the infectivity was retained. There- fore, such preparations would not provide a reliable challenge for testing efficacy of plague vaccines, as apparently there was considerable sublethal damage to the cells. Mitchell and Enright (1957) also demonstrated that the number of viable organisms provided a poor index to preservation. They observed that although the leavening ability of dried yeast was lost, there was no loss of viability. It is particularly important that criteria other than number of viable cells be used in the development of preservation methods if the culture is to be used directly without subculturing. Effectiveness of the preservation method used in the cheese industry has been evaluated by simulating procedures used in making cottage and cheddar cheese (Speckman et al., 1974; Keogh, 1970) or cultured buttermilk (Lamprech and Foster, 1963). Direct measurement of acid development and proteinase ac- tivity has also been used (Cowman and Speck, 1965; Speck and Cowman, 1970 Gibson et al., 1966). Gibson et al. (1966) found that although glycerol and dimethyl sulfoxide (DMSO) were effective cryoprotective agents for maintaining high viability of freeze-thawed Streptococcus cremoris, acid production during subsequent incubation was retarded. In contrast, malic acid protected viability and stimulated acid production after thawing. Johannsen (1972) found a good correlation between survival after freezing Lactobacillus leichmannii in malt extract and ability to produce lactic acid but she did not study the effects of other suspending media. Tanguay (1959) used biochemical activity to evaluate the quality of preservation. Even after 12 months storage at -40°C the response of the thawed organisms was identi- cal with that of the nonfrozen control in the assay procedures for lysine, inositol, tetracycline, and various vitamins. Stapert and Sokolski (1968)

4 ROBERT J. HECKLY

found that although only 33% of L. leichmannii survived freezing and thaw- ing, they obtained a normal dose-response curve for vitamin B,, analysis if they used three times the number cells in the test. Davis (1963) also demonstrated that if cells remained viable they were functional. He found that a high percentage of the lyophilized rhizobial cultures were viable after 21 years and capable of effective nodulation of the hosts after having been stored for 21 years. These few examples show that biochemical or biological activity, immediately after recovery from storage, may be an appropriate criterion for evaluating preservation. Viability assays should not be aban- doned because they can provide sensitive and quantitative measures of quality control which can be used to predict when the culture should be reprocessed. If the original number of viable cells is in the order of 10s/ml a decrease of even 1 log/year should not be a cause for concern, but if loss in titer approaches 2 or 3 logyear, the culture may easily be lost in 5 years.

c. MAINTENANCE OF FULL GENETIC COMPLEMENT

Implicit in the concept of stock culture preservation is the fact that the genetic composition of the progeny is the same as that of the original culture. Ordinarily, cultures are not studied in detail because it is nearly impossible to determine for each culture that there has been no genetic alteration. Under most conditions only gross changes would be noted, such as pigmen- tation (Servin Massieu and Cruz-Camarillo, 1969). Kubica et al. (1977) con- cluded that although mycobacteria stored at -70°C for 2-5 years appeared to be sluggish in diagnostic tests, culturing restored their vigor and key dif- ferential features were retained. However, Harrison and Pelczar (1963) re- ported some genetic changes in two lyophilized strains of Bacterwides that had been stored at room temperature for 5 years. They observed changes in morphology, temperature requirements, and fermentation reactions. It was noted that the number of viable cells was probably very low at that time, because after three additional years of storage no viable cells could be recov- ered.

In view of the fact that in many collections only a few organisms might survive storage to provide the inoculum for subculturing, it would seem that there might be a tendency to select mutants. However, properties of most cultures tend to be retained because it has been stated that auxotrophic mutants are not as hardy as the parent prototrophs (Webb, 1969). If it is true that auxotrophic mutants are less stable than the prototrophs, it is extremely important that methods used to preserve mutants, such as the Ames tester strains of Salmonella (Ames et al., 1975), yield maximum survival and do not alter DNA.

In studying the preservation of a special mutant of Penicillium chrysogenum, MacDonald (1972) observed that storage of this culture at 4°C

PRESERVATION OF MICROORGANISMS 5

led to the development of a subpopulation with substantially lower penicillin production. Such spontaneous mutation and selection might be expected in liquid storage. Fortunately, these changes were not observed in cultures stored at -196°C. As a test for mutagenic effects of lyophilization on fungi, Mehrotra et al. (1970) compared the productivity of 100 single-spore cultures before and after the lyophilization of nine different industrial strains. These strains were used to produce organic acids, antibiotics, and enzymes. Since the productivity of lyophilized spores was not significantly different from that of spores that had not been dried, it was concluded that none of the 900 spores suffered a significant genetic change.

111. Preservation Methods

A. DIRECT TRANSFERS ON CULTURE MEDIA

Agar slants are the most common method for maintaining the working inoculum, but stab or broth cultures are preferred by some workers, particu- larly for anaerobic organisms. Since there is an increased possibility of muta- tion with each subculture, frequent transfers are undesirable for long-term preservation of organisms. Mutation frequency in most cultures is low but Watko and Heddleston (1966) reported that in as few as 2 months, agar slants of Pasteurella multocida dissociated to provide a mixture of fluorescent, blue, and sectored colonies.

B. UNDEROIL

Many organisms survive reasonably well when agar slant cultures are covered with mineral oil and this procedure is used frequently by teachers to carry cultures for class use. Some cultures that did not survive lyophilization well were maintained under oil in the Agricultural Research Service (ARS) collection (Hesseltine et al . , 1960; Hesseltine and Haynes, 1974). Nadirova and Zemlyakov (1971) reported %year survival under oil by Pseudomonas, Bacillus, and Escherichia. Mutation during the prolonged storage is possible because it is believed that bacteria can continue to reproduce under these conditions, but Nadirova and Zemlyakov (1971) have concluded that morphological and cultural characteristics, as well as physiological prop- erties, remain unchanged in 3 years’ storage. However, all organisms cannot be stored successfully under oil. Yamasato et al. (1973) reported that the percentage of cells surviving 3.5 years’ storage varied from 77 to 90% for most of the organisms studied but only 25% of the Gluconobacter were recovered. Furthermore, if the slant is not completely covered, it dries up. Since survival for more than 1 year cannot be assured, considerable labor is

6 ROBERT J. HECKLY

involved in making regular transfers. The greatest disadvantage of using oil- covered slants is that it is a messy method. The method described by An- theunisse (1972) may be considered to be comparable to the oil overlay method but it is not as messy. After the cultures had grown out on the agar slant, he simply removed the cotton plug and replaced it with a rubber stopper. He reported 60-100% survival of 36 species for 3 years, with many surviving 10 years. Only Acetobacter, Aerobacter, and Streptomyces failed to survive well.

A patent for drying organisms in corn oil was issued to Johnston (1962). In his method, water was removed by bubbling dry air through the oil at 35°C and cells were finally collected by filtration. Since this author has not seen any application of this method, he presumes that it is not widely used.

C. IN DISTILLED WATER

Many organisms die rapidly when suspended in distilled water. However, cultures of Pseudomonas solanacearum in distilled water have been reported to survive for more than 10 years at room temperature. Surprisingly, these same organisms died rapidly when stored under refi-igeration (Berger, 1970). McGinnis et al. (1974) recommended distilled water for preservation of stock cultures of fungi and presented data showing that 93% of the cultures sur- vived storage for 4 years at room temperature. The fungi that failed to survive were poor sporulators (about 6 4 % of the collection). They also reported that storage in water suppressed pleomorphic changes and that no genetic changes were detected. Tanguay and Bogert (1974) found that both Saccharomyces cerevisiae and Sarcina lutea survived well when suspended in dilute phosphate buffer at 4°C for 4 months, and even after 1 year 2-19% survived.

D. FROZEN

With the improvements in refrigeration systems and the greater availability of liquid nitrogen, freezing is becoming the method of choice for both short- and long-term storage of viable microorganisms. Disadvantages of liquid nitrogen are that it is relatively expensive and requires constant surveillance if automatic filling and alarm systems are not used. Mechanical refrigeration can also be expensive and it is subject to both electrical and mechanical failure. However, with proper safeguards this is not a serious problem.

1 . Techniques

The general practice is to seal ampules by fusing the neck with a flame, but this may leave a small channel (Greiff et al . , 1975). When the ampule is

PRESERVATION OF MICROORGANISMS 7

immersed in liquid nitrogen enough of the liquid may enter during pro- longed storage to explode the vial when it is warmed suddenly. Therefore, Simione et al. (1977) used screw-capped plastic ampules to minimize the hazard. These appear to be satisfactory for the preservation of a variety of organisms.

In some studies it has been desirable to be able to use frozen material as the inoculum for each experiment and, in these instances, large numbers of vials are required. Instead, bits of culture can be scraped off the frozen surface with a sterile applicator stick without thawing the entire culture (Ames et al., 1975). A more sophisticated tool was described by Bullen (1975) for scraping the surfaces of frozen culture in a closed vial. The purpose of this was to minimize the biological hazard when working with pathogenic mic- roorganisms.

Two other techniques should be mentioned because they are convenient and provide relatively uniform inocula. Cox (1968) obtained satisfactory re- sults by freezing drops of culture in liquid nitrogen. Nagel and Kunz (1972) coated small glass beads with cells suspended in the bacterial culture to which an equal volume of horse blood had been added. Sterile forceps were, used to remove a single pellet or bead without disturbing the remaining culture. Both have the advantage of thawing rapidly when dropped into a tube of warm broth.

A convenient technique for preserving fungi involves freezing organisms on agar plugs. As described by Dietz (1975), cultures are grown on agar plates and plugs are cut by pressing short sections of sterile paper drinking straws into the culture. By repeating the operation, several plugs of agar, with the overlaying culture, can be collected in each section. These pieces of drinking straw are then placed in a vial and frozen in the gas phase of a liquid nitrogen freezer. For retrieval, a straw is placed in a petri plate and when thawed sufficiently, one plug is removed and the remaining plugs are re- turned to the freezer.

2. Considerations

An attempt will be made to consider the various factors identified as having affected the survival of frozen organisms, even though these factors are interrelated and cannot be studied as independent variables.

a. Age of culture. The physiological condition of microorganisms has been considered by many investigators to be a factor in determining their ability to survive stress. It is generally accepted that cells from the maximum stationary phase cultures are more resistant to damage by freezing and thaw- ing than cells from the early or midlog phase of growth. The percentage of cells surviving is also increased by an increase in cell density, possibly be- cause lyzed cells can yield cryoprotective substance (Bretz and Ambrosini,

8 ROBERT J. HECKLY

1966). This generalization is probably not valid for all organisms, particularly viruses, in view of the observations by Nyiendo et al. (1974), who have found that the percentage survival of lactic Streptococcus bacteriophages is not correlated with original titer before freezing. Heckly (1961) pointed out that nutrition during growth, culture density, and age were equally important factors in the survival of lyophilized bacteria. However, the effects of these functions are not easily separated. By using spent growth media (the filtrate from a stationary phase culture) as a diluent, Packer et al. (1965) demon- strated that for Escherichia coli, the phase of growth, the state of aerobiosis, and the density of the culture had no effect on the degree of susceptibility to death by freezing and thawing. Instead, it appeared that the media were changed by the organisms and that sensitive cells were protected by suspen- sion in the spent growth media. Since the protective effect was demonstrable at relatively high dilutions, some of the observed variations in the killing effect of freezing and thawing may be due to incomplete washing of cells. For practical purposes it makes little difference whether the cells or the media is changed; the fact remains that cells from young cultures do not survive freezing and thawing as well as those in mature cultures at a high cell density.

b. Rate offieexing and thawing. For a number of years it was consid- ered essential to freeze organisms rapidly to obtain high survival. Therefore, methods were developed to achieve ultrarapid freezing 'and it was shown that a variety of organisms survived ultrarapid freezing and thawing (Doeb- bler and Rinfret, 1963). They calculated that cooling rates of several hundred degrees per second were obtained by spraying the culture into liquid nitro- gen from a 26 gage hypodermic needle. Similar warming rates were obtained by slowly sifting the fine pellets into saline at 37°C. However, many inves- tigators who have studied the problem of freezing rate have now found that slow freezing and rapid thawing generally yield the highest number of viable cells (Stalheim, 1971; Johannsen, 1972; Bank, 1973; Mazur, 1966, 1970; Raccach et al . , 1975; Torney and Bordt, 1969). Exceptions to this generalized observation have been reported. Levy (1969) found that the highest survival of Mycobacterium leprae was obtained by rapid freezing and slow thawing, but this might not be significant because M . leprae did not seem to be particularly sensitive to the rate of thawing. Furthermore, his data were not entirely consistent.

Mazur and Schmidt (1968) found that for yeast, there was an interaction between cooling velocity and warming velocity; i. e., reasonable survival was obtained at high freezing rates if the thawing was rapid enough. Rapidly frozen Pseudomonas fluorescens also survived well when thawed rapidly (Ashwood-Smith and Warby, 1971a).

PRESERVATION OF MICROORGANISMS 9

It has been found that there is an optimum cooling rate for maximum survival that varies with the nature of the cell (Mazur, 1970, 1977; Raccach et al., 1975). The optimum cooling rate for S. cerevisiae was about 7"C/min (Mazur and Schmidt, 1968), but Wellman and Stewart (1973) reported a higher mutation rate of brewing yeasts when the cultures were frozen at 9-17"C/min than when frozen at 1"C/min. Meyer et al. (1975) found that for other yeasts, the optimum rate was between 4.5 and 6.5"C/min. Raccach et al. (1975) have demonstrated that the optimum cooling rate may be depen- dent upon the final storage temperature. When Acholeplasma laidlawii was cooled to -20°C the optimum rate was between 8 and 1O0C/min, whereas when the culture was cooled to -70°C the optimum rate was about 16"C/min.

Despite evidence just cited, cooling at 1"C/min seems to be widely used largely because it is impractical to determine the cooling rate for every organism. Since freezing rate is not a critical factor for survival, a controlled rate freezer, which is relatively expensive, is probably unnecessary. Satisfac- tory results have been obtained by placing the samples in a slightly insulated container in a -70°C freezer, as described by Swoager (1972).

c. Storage temperature. Liquid nitrogen provides the lowest practical temperature (-196°C) for storing microorganisms and, because viability is preserved so well, it is used extensively for all sorts of organisms (Swoager, 1972; Clark and Klein, 1966; Norman et al., 1970; Hwang, 1970; Butterfield et al., 1974). Most quantitative studies on survival of organisms in liquid nitrogen have involved only short-term storage but prospects for prolonged survival are good. Keogh (1970) stored Streptococcus lactis and S. cremds for 13 months without a significant change in number of viable cells. Norman et al. (1970) preserved some strains of Mycoplasma for as long as 9 years, but they gave no data on viability. Butterfield et al. (1974) reported that practi- cally all of the fungi stored for up to 8 years in liquid nitrogen were viable. They mentioned problems with loss of viability of only two organisms, Paracoccidioides brasilensis and Basidwbolus. These may require special freezing conditions. Clark and Klein (1966) found that although as much as 99% of the original titer of bacteriophages was lost on freezing, there was no further reduction in titer after 3 5 years of storage in liquid nitrogen. It appears that most of the damage leading to the loss of infectivity of phage or viability of other organisms occurs during the freezing and thawing steps and not during the storage period. Therefore, if survival is good after a few months of storage, the samples can be expected to survive for many years.

Because liquid nitrogen storage is relatively expensive it would be advan- tageous for viability to be preserved at higher temperatures. There have been many studies on the effect of temperature on survival of organisms. It is evident that there are marked differences in sensitivity of the various groups

10 ROBERT J. HECKLY

or kinds of organisms. About 80% of the cells in frozen cultures ofhctobacil- lus acidophilus were viable after 6 months at either -10, -20, or -60°C (Dug- gan et al., 1959) and some mycobacteria also survived quite well at -20°C (Gruft et al., 1968; Kim and Kubica, 1972). However, the mammalian tuber- cle bacilli and a few other species of mycobacteria survived much better at -70°C than at -20°C. Practically 100% of the Mycobacterium tuberculosis culture was viable after 25 months at -70°C (Kim and Kubica, 1972, 1973).

About 90% of the L forms of either Neisseria meningitidis or Streptococcus pyogenes survived storage at -196°C for 1 year, but neither survived 6 months at -20 (Stewart and Wright, 1970). Similarly, Cowman and Speck (1965) found that although 7040% of the lactic streptococci survived 2 months at -196"C, fewer than 2% survived at -20°C.

Tanguay (1959) reported that a number of different organisms used in microbiological assays appeared to survive satisfactorily for 1 year at -40°C. However, it was subsequently shown that in the absence of cryoprotective agents less than 1% of S. cereuisiae survived 1 year at -40°C (Tanguay and Bogert, 1974). Cox (1968), also without adding cryoprotectants, obtained 80% survival ofE. coli K-12 HfrC after 1 month at -7O"C, whereas at -15°C the preparations were sterile. After an extensive study of 259 strains belong- ing to 32 genera, Yamasato et al. (1973) concluded that, although many organisms survived well after 4.5 years at -28"C, much higher survival was generally obtained at -53°C.

Influenza and syncytial viruses also have been found to survive better at temperatures below -65°C than at -20°C (Rightsel and Greiff, 1967; Law and Hull, 1968). Measles virus appeared to be unusual because there was greater loss of infectivity at -40°C than at -20°C (Greiff et al . , 1964); at -65"C, how- ever, the virus was adequately preserved.

Using electron microscopy with freeze-fracturing and freeze-etching pro- cedures, Bank (1973) demonstrated that large intracellular ice crystals formed within 30 min when frozen yeast was warmed rapidly from -196 to -40°C. Some recrystalization occurred at temperatures as low as -45°C. As will be discussed in Section 111, D,3, recrystalization of intracellular ice is considered to be lethal for cells. The critical temperature is dependent on a number of factors, but -70°C appears to be sufficiently low to preserve most organisms.

d . Cryoprotective agents. Glycerol has been the most widely used addi- tive or suspending medium for all types of organisms. For example, at the American Type Culture Collection (ATCC), mycoplasma and fungi that do not survive lyophilization are frozen in 10% glycerol (Norman et al., 1970; Hwang, 1968). Brewing yeasts also were well preserved in 10% glycerol

PRESERVATION OF MICROORGANISMS 11

(Wellman and Stewart, 1973). Glycerol was superior to other substances for preservation of lactic cultures by freezing (Baumann and Reinbold, 1966).

Although glycerol was effective without penetrating cells, the presence of intracellular glycerol increased survival on freezing and thawing of E . coli. However, intracellular glycerol alone failed to protect (Nath and Gonda, 1975). The ATCC have used 10% glycerol to preserve bacteriophage (Clark and Klein, 1966) but they have adopted the practice of using rapid freezing without additives because better recovery is obtained for a number of freeze-sensitive phage (Clark and Geary, 1973). Others also have obtained good survival of bacteria, fungi, and yeast without additives (Cox, 1968; MacDonald, 1972; Wellman, 1970; Wellman and Walden, 1971). Keogh (1970) also have considered additives to be unnecessary for S. Zactis and S . cremoris, but this may have been the result of the high cell concentration (1012/ml).

The presence of glycerol actually may be detrimental to some organisms. Barnhart and Terry (1971) found that as the glycerol concentration was in- creased, the percentage of Neurospora crassa surviving freezing decreased from 2435% to less than 1%. However, suspension of the conidia in DMSO increased survival to about 50%. Hwang and Howells (1968), using a less quantitative measure of viability, also found that DMSO was more effective than glycerol for most of the eight fungal cultures tested. Two of the species survived equally well in glycerol and DMSO.

A mixture of 10% glycerol with 5% of either lactose, maltose, or raffinose provided the best overall protection for S. cerevisiae, Pseudomonas aureofa- ciens, Streptomyces tenebrarius, and four species of algae (Daily and Higgens, 1973). Their conclusion was based on the results of testing over 50 compounds and combinations.

In addition to the above citation, a few other examples showing that the effectiveness of the suspending media varies with the organisms may be of interest. Yamasato et al. (1973) found that for most species of Acetobacter and Gluconobacter, a 10% solution of honey was superior to 10% glycerol. Syncytial virus was found to be maintained best by freezing in a 44% sucrose solution (Law and Hull, 1968), and calcium lactobionate has been recom- mended for preserving measles virus (Greiff et al., 1964). Bretz and Abrosini (1966) found that of the various substances from lyzed E. coli cells, only the carbohydrate fraction preserved viability of E . coli. A comparison of its protective effect with sucrose, or other carbohydrates, would have been of interest. Nonionic detergents, as well as glycerol, protected Enterobacter aerogenes from freezing damage (Calcott and Postgate, 1971). The degree of protection offered by the detergents Tween 80, Triton WR 1339, or Mac- rocyclon decreased as cell concentration was increased. Apparently, the

12 ROBERT J. HECKLY

protection was dependent upon the ratio of cells to detergent. In contrast, protection by glycerol was dependent upon its absolute concentration and was independent of cell density.

It has been shown that using polyvinylpyrrolidone and dextran, cryo- protection increased with molecular weight to a maximum protection at about 90,OOO Daltons (Ashwood-Smith and Warby, 1971b; Vitanov and Petukhov, 1973), but these high molecular weight materials were not compared with other substances, such as glycerol or DMSO.

Viability of leptospires was well preserved by freezing in a mixture of 10% rabbit serum with 5% or 10% glycerol (Torney and Bordt, 1969). They did not consider the additives independently. The work of Janssen and Busta (1973a) would indicate that proteins could be protective because most frac- tions of milk offered some protection for Salmonella anatum. Whey was deleterious even though it contained protein.

3. Nature of Cryoinjury

It seems that the problem of identlfying the nature of damage caused by freezing is similar to that of blind men trying to characterize an elephant. The conclusions depend to a large extent on how the subject is approached. Early workers, such as Proom and Hemmons (1949) or Luyet (1951), be- lieved that cells were killed by freezing because ice crystals penetrated the cell wall, but probably this is not true. Mazur (1961) demonstrated clearly that in spite of high mortality (more than 99.99% killed), rapidly cooled yeast cells remained as intact morphological entities when thawed. The cells did lose their vacuole, which was correlated with viability.

It now appears that there are at least two types of injury that can result from freezing of cells. Litvan (1972) believes that the injury produced by slow cooling rates is a result of dehydration and that rapid cooling causes membrane rupture. Mazur (1966, 1970, 1977) has identified these as solution effects, caused by slow freezing, and intracellular freezing, which occurs when a cell is frozen rapidly. The intracellular ice per se is not lethal since rapidly frozen cells survive thawing ifwarmed rapidly enough (Mazur, 1966). Furthermore, Farrant et al. (1977), using Chinese hamster fibroblasts, con- cluded that the damage from intracellular ice occurred during rewarming and was osmotic in nature. On the basis of structural changes in yeast, Bank (1973) also concluded that recrystalization was responsible for death in rapidly cooled cells. For baker’s yeast, he identified the lethal temperatures (recrystalization stage) as being between -40 and -5°C.

Slow cooling can prevent formation of intracellular ice but such cells suffer from solute concentration effects. Cryoprotective agents, such as glycerol, act to minimize solution effects. However, as stated by Maxur (1977), “Under-

PRESERVATION OF MICROORGANISMS 13

standing the nature of these solution effects and their role in freezing injury now represents the major challenge in modern cryobiology.”

There is considerable evidence that damage to the cell permeability bar- rier is associated with death in frozen and thawed cells. Calcott and Mac- Leod (1975a) demonstrated that release of cellular constituents, ultraviolet (UV) absorbing material, potassium, and P-galactosidase were correlated with loss of viability. Also, without being particularly concerned about the mechanics of freeze-thaw damage, Bretz and Kocka (1967) and Ray et al. (1972) found that injury involved the lipopolysaccharide of the cell wall. Since the repair process was not inhibited by either actinomycin, chloram- phenicol, or cycloserene, Ray et al. (1972) concluded that the process did not involve RNA, protein, or mucopeptide synthesis; only adenosine triphos- phate (ATP) was required. Other evidence that the cell membrane might be involved was provided by Gilliland and Speck (1974) and by Smittle et al. (1972), who found that incorporation of Tween 80 into the growth medium increased the resistance of Lactobacillus bulgaricus to freezing damage. Pro- tection was not conferred when Tween 80 was added to the suspending medium as Calcott and Postgate (1971) had reported earlier for E . aerogenes. However, both groups postulated that the detergent affected the cell mem- brane to increase its resistance to freezing damage. Along the same lines, Raccach et al. (1975) found that oleic acid enrichment of A. Zaidlawii in- creased the percentage of cells surviving freeze-thawing which was attrib- uted to a change in the composition of the membrane.

The two types of freezing damage demonstrated by Swartz (1970, 1971a,b) were not correlated with the two types of injury mentioned previously. One he identified as being oxygen dependent, which was mediated by free radi- cals. The radiation-resistant strain, E . coli B/r, could repair this damage in the presence of oxygen but the radiation-sensitive strain, E . coli B,l, could not. The other type of damage was oxygen independent, which he identified as a single-strand deoxyribonucleic acid (DNA) break. However, Ashwood- Smith et al. (1972), using slightly different procedures with radiolabeled thymidine, concluded that freezing and thawing did not break DNA of E . coli. Earlier, Ashwood-Smith (1965) concluded that freezing and thawing were not, in themselves, mutagenic to E . coli since he could not demon- strate any reversion from auxotrophy to prototrophy. More recently, Crom- bach (1973) showed that freezing and storing extracted DNA at -21°C for up to 1 year did not affect the thermal denaturation (melting point) or hybridiza- tion capabilities, which would indicate that freezing did not break DNA. Small differences in procedure, such as freezing rate, may be the cause of the conflicting findings regarding the effect of freezing on DNA. Wellman and Stewart (1973) observed that the biochemical properties of Saccharomyces

14 ROBERT J. HECKLY

uvamm were well preserved when frozen at 1"C/min but when the culture was frozen at 9-17"C/min the percentage of respiratory-deficient mutants increased markedly, despite maintenance of high viability.

E. DEHYDRATED

Since water is required for metabolic activity, it is logical that dehydration should prevent changes in microbial cultures. Indeed, this is an effective method for culture preservation, and over the past 60 or 70 years many papers have been published describing techniques to simplify drying methods or to improve survival of organisms. This author will not attempt to review all of these, but the following discussions should provide a basis for selecting applicable methods.

1 . Soil

Soil is a natural reservoir for many microorganisms, and they can be recovered after prolonged storage. Sterile soil has been used to induce sporulation of both aerobic and anaerobic bacilli. Azotobacters were recov- ered from soil stored for 13 years at room temperature in the laboratory (Vela, 1974); Coccidioides immitis was recovered &om soil stored in the laboratory for 8 years (H. B. Levine, personal communication, 1977). Disad- vantages are that quantitation is difficult and soil is a variable commodity not easily defined. However, the method continues to be used largely for fungi and anaerobic spore-forming organisms. The fungi and spore-forming bac- teria apparently survive in the spore state, but the vegetative cells probably survive because of their low metabolic rate and large amount of stored energy. Despite the applicability to certain requirements, this technique does not appear to be suitable for general use in all cultures.

2. Silica Gel

Perkins (1962) first used silica gel for preserving fungi, and the method was since applied successfully to many other organisms. The procedure is simple and requires no special equipment. Silica gel, in small cotton- plugged tubes, is dried and sterilized by heating in an oven to 175°C for 1.5-2 hours. Details of the procedure were given by Grivell and Jackson (1969). Most of their cultures survived over 2 years, but three failed: Thiobacillus thipams, Chlamydomonas eugametos, and Euglena gracilis. The procedure used by Parina et al. (1972) differed in that they placed the granules of silica gel into the growth flasks for the last 24 hours of incubation. The silica gel and adherent yeast were then dried in a vacuum desiccator. Nearly 100% survival was obtained after 12 months with all three of the

PRESERVATION OF MICROORGANISMS 15

yeasts tested. More recently, Trollope (1975) reported, on the basis of 33 strains of bacteria and 22 fungi, that the survival period was increased two to three fold by storage of the silica gel at 4°C. However, even at 4"C, fewer than 60% of the bacteria and 36% of the fungi were viable after storage for 4 years. Since he did not count number of viable cells but only growth or no growth, this meant that many strains would have been lost if this were the only collection. Although silica gel and glass are similar, Miller and Simons (1962) reported that after drying on perforated glass beads, only 13 of 202 bac- terial cultures failed to grow after 21 years at about 10°C.

3. Cellulose

A convenient method for drying cultures on filter paper strips was de- scribed in detail by Hopwood and Ferguson (1969). They placed thin strips of filter paper, saturated with a suspension of organisms in skim milk, into small tubes (6 x 100 mm). After the neck was constricted to facilitate sub- sequent sealing under vacuum, the tubes were attached to a manifold and dried at 0.01 torr. After 1-year storage at 37°C there was no apparent loss of viability of Streptomyces. Indications are that the method is applicable to other organisms as well. Annear (1964) followed essentially the same proce- dure using small tufts of cellulose or quartz wool, except that he suspended the bacteria in 10% sodium glutamate. Up to 59% survival of Salmonella ndolo was reported after 2 years' storage at room temperature. One-inch pieces of cotton string also have been used as a carrier for preserving a variety of organisms (G. D. Searle and Co., 1976). It is difficult to make a quantitative determination of survival on any of these cellulose carriers but it appears that viability is as least as good as in the conventional lyophilization procedure and considerably more convenient.

F. LYOPHILIZED

Lyophilization, or freeze drying as some prefer to call the method, is the total process of freezing and sublimation of the water from the frozen prepa- ration. This author will not review the historical developments or the proce- dure since this has been covered in considerable detail previously (Heckly, 1961).

Lyophilization is considered by many microbiologists to be the method for preserving cultures. Indeed, many thousands of cultures are successfully maintained by lyophilization for long periods of time under vacuum. The advantages of lyophilization are that most organisms survive drying and cultures are easily stored. Also, most cultures can easily be shipped at room temperature without significant loss of viability, even though long-term sur- vival may require a lower storage temperature.

16 ROBERT J. HECKLY

1 . Equipment

Except for greater use of direct drive, gas ballasted vacuum pumps there have been few basic improvements in lyophilization equipment in the last 10 years. The equipment and supplies being manufactured by the various com- panies differ in convenience features but all are roughly comparable. When a system is selected the volume of material to be processed, as well as the nature of the organisms, should be considered. Since many organisms are sensitive to oxygen, it is recommended that a manifold-type unit be selected for drying the cultures. However, a centrifugal system, such as manufac- tured by the W. Edwards Company, London, is widely used. With secon- dary drying on a manifold for sealing under vacuum, this system seems to be effective for most organisms. Unfortunately, the commercial equipment is relatively expensive, but elaborate units in attractive cabinets are not essen- tial for successful lyophilization. It is convenient to have condensers cooled by mechanical refrigeration but the condenser temperature may limit the ultimate vacuum attainable. If the pressure is to be reduced below 0.01 torr, the condenser surface must be below -50°C. For this reason, and because the initial costs are lower, dry ice cooled condensers are widely used, especially in “homemade” units. Since only small volumes are needed for stock culture preservation, a small condenser cooled with dry ice is usually adequate. Except for the vacuum pump, a practical lyophilization unit can be assem- bled in the laboratory (Heckly, 1961). A 3550 liter/min pump is usually adequate, but it should be capable of an ultimate pressure of less than 0.01 torr (10 pm Hg), even though the vapor pressure of water at -40°C is about 0.1 torr.

It is desirable, but not essential, to have a vacuum gage in the system. McLeod-type gages are widely used, although a thermocouple vacuum gage is more useful since it can provide a continuous indication of vacuum, which facilitates finding leaks in the system.

Heavy wall natural gum rubber tubing (?h inch i.d.) is much more conven- tient to use than heavy wall pressure tubing. Ampules made by sealing off 10-cm sections of 9-mm standard wall Pyrex tubing are quite satisfactory for culture preservation. A disadvantage is that the 9-mm tubing is a bit difficult to seal under vacuum. This can be overcome by heating and constricting the neck slightly (to about 4 5 mm i.d.) after the ampule is filled. These ampules accommodate 0.1 ml of culture.

A novel method for freeze drying without a vacuum system was described by Wagman and Weneck (1963). In their system, cold dry air was forced through a bed of pelletized culture in a closed recirculating system. They suggested that increased viability was obtained because this method permit- ted more uniform drying by avoiding regional overdrying. The method,

PRESERVATION OF MICROORGANISMS 17

obviously, is not suitable for the usual stock culture preservation, but it may be used in industry for yeast or lactic starter cultures.

2. Factors Affecting Survival

The effects of various factors influencing the survival of bacteria during lyophilization and subsequent storage have been described in considerable detail (Heckly, 1961). The conclusions presented at that time are still basi- cally valid and applicable. Since then, there have been many more reports of the application of lyophilization to the preservation of all types of organisms. Unfortunately, too many of these reports fail to mention details of the methods used. Despite the diversity of methods or approaches, an attempt will be made to summarize the findings. It is hoped that this review will facilitate selection of techniques to meet specific requirements.

a. Type of organism. The size and complexity of the organisms are sig- nificant factors in determining the ability of the organisms to survive lyophilization, since damage of any vital structure or function is lethal. Al- though animal tissue culture cells are routinely preserved by freezing in liquid nitrogen, the large cells are extremely sensitive to drying. In only one instance have viable cells been demonstrated to survive lyophilization (Dam- janovib et al. , 1975). Except for some unicellular forms, algae cannot gener- ally be preserved well by lyophilization, and in the mycelial phase fungi also do not survive lyophilization (Hesseltine et al . , 1960).

Because bacteria are larger and have a more complex structure than viruses, it seems that viruses should be the more resistant. However, it appears that viruses are generally more sensitive to lyophilization than are bacteria. This difference may be attributed to the ability of cellular forms to repair damage caused by freezing or drying, whereas viruses and phage cannot. Since viruses depend on the host cells for energy, they must be infective to survive.

Among the viruses there are correlations between morphological type and sensitivity to lyophilization. The larger viruses, which were identified as belonging to type A group by Clark and Geary (1973), were demonstrated to be more sensitive to lyophilization than the smaller viruses (group B). Right- sel and GreifT(1967) arranged viruses into eight different groups on the basis of physical and chemical properties. Infectivity of most viruses within a group was similarly affected by freezing and drying.

Bacteria can be divided into three broad catagories: spores, gram-positive vegetative cells, and gram-negative bacteria. All types of spores are inher- ently extremely resistant to dehydration and survive lyophilization well. Steel and Ross (1963), and many others, have observed that gram-positive

18 ROBERT J. HECKLY

bacteria survive better than gram-negative organisms when lyophilized and stored under comparable conditions.

The ability to withstand drying probably is a genetically stable trait, since there is no indication that resistant strains can be selected for easily. The progeny obtained fiom lyophilized cultures, even after many cycles of lyophilization and regrowth, did not survive drying any better than the original cultures (R. J. Heckly, unpublished). Skaliy and Eagon (1972) found that fieshly isolated cultures of Pseudomonas aeruginosa were no better adapted to withstand the stress of desiccation than were the laboratory strains.

Haynes et al. (1955) reported that they were unable to preserve anaerobic organisms by lyophilization, but the problem might have been one of tech- nique. Lupton et al. (1961) reported that with suitable precautions to ex- clude air, all of the anaerobes processed in a 2-year period grew on reconsti- tution. White et al. (1974) also reported high survival (up to 10%) of lyo- philized anaerobic organisms isolated from bovine rumen. Furthermore, in a more extensive study of 19 strains of strict anaerobes, Phillips et al. (1975) ob- tained high viability (10-100%).

Marine bacteria, in spite of their normal habitat, are probably as resistant to lyophilization as other bacteria. Floodgate and Hayes (1961) reported that all of 45 lyophilized strains tested survived 2 years, and Greig et al. (1970) reported that all but 9% of their dried cultures survived 10 years. Highest survival was among corynebacteria and micrococci, and the lowest was with the vibrios and photobacteria.

b . Physiological age. The age of the culture can have a profound effect on survival of bacteria. A well-nourished cell in a culture at the maximum stationary phase is usually the most resistant cell. It is possible that some properties ascribed to mature cultures may have been due to changes in the growth media, as described by Packer et al. (I%), but age of the cell per se must be a significant factor. There are a few instances in which young or log-phase cultures survived lyophilization better than old cultures (Proom and Hemmons, 1949; Amarger et al . , 1972; Lingg et al., 1967). Although young cultures of Rhizobium meliloti survived drying better than older cul- tures, the stationary phase survived storage in the dried state at 30°C better than young cultures (Amarger et al., 1972).

Perhaps the results of an experiment summarized in Fig. 1 may help explain some of the contradictory reports regarding the effect of culture age and survival after lyophilization. It is obvious that when frozen rapidly, the mature (24-hour) culture survives markedly better than any of the younger cultures. Young cultures (3-7 hour) were so sensitive that no viable cells were demonstrable when frozen rapidly. In contrast, at least loo0 times as

PRESERVATION OF MICROORGANISMS 19

A g e o f c u l t u r e (h r )

4

FIG. 1. Effect of culture age and freezing rate on survival of lyophilized Serratiu murcescens. Organisms were grown in chemically defined medium at 30°C with shaking. At intervals, samples were removed and frozen rapidly by immersion in a dry ice-thanol bath or frozen slowly in a -20°C freezer chest. All samples were dried overnight and reconstituted with distilled water for viability assays.

many cells survived when the 12-hour-old culture was frozen slowly prior to lyophilization than when it was frozen rapidly.

c. Cell concentration. Although there are some exceptions, as discussed previously (Heckly, 1961), increasing the bacterial cell concentration usually results in an increased percentage of cells surviving lyophilization. A similar concentration dependence was observed with bacteriophage T4 (Shapira and Kohn, 1974). If the preparation contained 1O1O particles per milliliter, as high as 10% survived lyophilization; whereas at an initial lo9 particles per milliliter, less than 0.1% survived. It has been postulated that a high survival rate is obtained with high initial bacterial cell concentrations because the majority of the cells are protected by substances released by the lysis of a few cells. It has been shown that cell lyzates are cryoprotective (Bretz and Amro-

20 ROBERT J. HECKLY

sini, 1966) but the observations by Speckman et al. (1974), using lactic bacteria, tend to indicate that the observed effects may not be due to an increase in solutes as a consequence of lysis. They found that with an initial 109/ml, only 10% of the bacteria survived, even in a rich medium consisting of 5% gelatin, 5% sodium citrate, 10% sucrose, and 2% sodium glutamate. In contrast, with an initial concentration of 10"/ml, virtually 100% of the cells survived.

Damjanovik and Radulovik (1967) similarly found that the survival of Lac- tobacillus bifidus increased from 26 to 99% as cell concentration was in- creased from lo7 to 10g/ml. Their medium, which consisted of 8% sucrose, 5% skim milk, and 1.5% gelatin, was rich and also should have offered protection regardless of cell concentration. Lion (1963) observed that the protective effects of various substances, including glucose and thiourea, were dependent on cell concentration and went on to suggest that protection might be affected by a sort of physical or mechanical oxygen barrier.

d . Suspending medium. The suspending medium is perhaps the most studied factor. The basic considerations have been discussed at length (Heckly, 1961) but there are some additional observations that may merit mentioning. Skim milk continues to be a popular suspending fluid and gen- erally yields satisfactory results. Although a large number of marine bacteria survived in a mixture of 20% dehydrated skim milk with 5% aged sea water, a small group of purple pigmented bacteria failed to survive when lyophilized in milk (Ohye and Gunderson, 1970). These purple bacteria survived when they were lyophilized in a medium containing 25% sea water, 5% peptone, and 5% yeast extract. Fisher (1963) also found that skim milk was inferior for preserving Chromobacterium lividurn. When lyophilized in skim milk, no viable cells were found after 10% years at room temperature, but when suspended in "Mist. desiccans," a term applied by Fry and Greaves (1951) to a mixture of serum and glucose broth, or in nutrient broth or broth containing yeast extract, about lo5 viable cells were recovered.

A number of investigators found that the protective effect of skim milk was improved by adding solutes. Addition of either ascorbic acid or thiourea markedly improved survival of S. lactis (Sinha et al . , 1974a) and Green et al. (1970) added 5% sucrose and 5% sodium glutamate for Klebsiella. Danilova and Kudryavtsev (1971) improved survival of Serratia marcescens, E. coli, and P. jluwescens by adding 5% sucrose and 5% lactose.

Although skim milk protected some strains of blue-green algae, Corbett and Parker (1976) found that only lamb serum gave consistently good recov- ery. Since there is so little difference between the animal sera, it is difEicult to explain why no viable cultures have been obtained from Synechococcus c e d r m m and other blue-green algae when dried from horse serum, beef

PRESERVATION OF MICROORGANISMS 21

serum, or fetal bovine serum. Corbett and Parker (1976) mentioned that resistance of the blue-green algae to freezing appeared to correlate with resistance to lyophilization. As mentioned in Section II1,D on freezing, DMSO is a superior cryoprotective agent and perhaps should be tried for those organisms that are particularly sensitive to lyophilization. However, since concentrated DMSO is toxic to microorganisms, special precautions are required to prevent concentrating DMSO as water is removed. Greiff et al. (1976) found that by maintaining the sample temperature at -50°C using the proper ratio of DMSO to albumin, a dry cake was obtained. Under these conditions the DMSO is apparently immobilized as the water is removed and may not be toxic to organisms.

Both glucose and sucrose solutions tend to produce a glazed surface, collapse of the ice structure, and foam but this does not seem to affect the survival of bacteria. After 10 years' storage, Annear (1974) recovered leptos- pires that were lyophilized in 10% glucose. He mentioned that, although other suspending media were tried, none gave consistently reliable results. Sucrose has been recommended as a general purpose additive replacing skim milk (Heckly, 1961) and, when compared with either lactose or glucose as suspending media, ten times as many mycoplasma have been recovered using sucrose (Yugi et al . , 1973). Although sucrose alone conferred signs- cant protection, even higher survival of P . fluorescens and Salmonella new- port was obtained with 0.1 M sucrose plus 0.2 M sodium glutamate and 0.02 M semicarbazide (Marshall and Scott, 1970; Marshall et al., 1974). The rationale for adding semicarbazide was that it could react with carbonyl compounds. Berman et al. (1968) obtained their best results by adding 0.07% glutamate and 2.5% human serum albumin to 8.2% sucrose in 0.01 M phosphate solution. Only a few investigators advocate using sodium gluta- mate alone for lyophilization of bacteria, but Obayashi (1961) and Annear (1964, 1970b) report greatly enhanced heat stability of bacteria dried in glutamate. Annear (1964) obtained high survival in cultures heated to G"C, but not when heated to 100°C. It was believed that this was due to in- adequate drying because stability was increased if drying was completed by immersing the sample in boiling water for 30 min before it was removed from the vacuum system (Annear, 1970b). This additional heating produces a white foam which he thinks is an important indicator. In the foam, both S. ndolo and S . marcescens survived well for 3 days of heating at I0O"C.

Sodium glutamate is also an effective suspending media medium for the lyophilization of viruses. Scott and Woodside (1976) found that glutamate alone, or with sucrose, most effectively stabilized pseudorabies virus, and Suzuki (1973b) found glutamate best for vaccinia virus. The media which Calnek et al. (1970) selected contained sucrose, phosphate, and serum albu- min in addition to sodium glutamate.

22 ROBERT J. HECKLY

In a comprehensive study using 62 strains of organisms, including Strep- tococcus, Lactobacillus, Escherichia, and Serratia, Morichi (1970) tested the activity of 112 compounds. Good survival after lyophilization was obtained using glutamic, aspartic, and malic acid and several other additives, but poor survival was obtained with glutaric acid, glutamine, proline, and L-threonine. Surprisingly, DL-threonine gave good survival, which was at- tributed to its greater solubility. Sucrose, lactose, and glucose were among the sugars that protected organisms on lyophilization, but contrary to the findings of Redway and Lapage (1974), inositol was not as effective. On comparing the effects of adding 15 different carbohydrates or mixtures to horse serum, Redway and Lapage (1974) found that addition of arabinose, glucose, and xylose generally offered the least protection. The highest sur- vival was consistently obtained with inositol for all of the organisms tested, which included Bacillus cerus, Haemophilus suis, Neisseria gonorrhoea, and Vibrio met schnikovii. Trehelose and sucrose gave reasonably good survival.

This review of the substances that have been studied as additives for increasing the survival of microorganisms is by no means complete, yet it is obvious that this subject presents a complex problem. It appears that most sugars act to protect organisms during the freezing and drying state and that glutamic acid and perhaps other amino acids or proteins serve to protect organisms at elevated temperatures.

Another variable decting survival of lyophilized bacteria that has not been confirmed by other investigators concerns temporal relationships. It has been demonstrated that the number of cells that survive lyophilization and storage can vary as a function of the time between mixing of the culture with “additives,” such as ascorbic acid or propyl gallate, and freezing of the sam- ple (Heckly et al . , 1967; Heckly and Dimatteo, 1975). This factor may be responsible for some inconsistencies in data since, as is shown in Fig. 2, a delay of as little as 30 sec between freezing of samples can result in more than ten fold difference in the number of cells surviving after lyophilization.

e. Rate of fi-eezing and drying temperature. The rate of freezing can be critical and deserves consideration. As discussed previously in Section 111, D, sensitivity of frozen cells to slow warming is dependent upon the freezing rate. Because the ice temperature in most frozen specimens during drying is usually above -40°C, recrystalization of intracellular ice is probable. Rapid freezing, which may produce intracellular ice, can be recommended only if the vacuum system is adequate to maintain the sample below -50°C.

The critical temperature above which recrystallization can take place de- pends on several factors, as discussed in Section II1,D. If the sample is to look good, i.e., retain the bulk and general shape of the ice cake, the tem- perature must be maintained below the eutectic temperature of the sample.

PRESERVATION OF MICROORGANISMS 23

0 30 60 120 3M) 600 S e c o n d s b e t w e e n m i x i n g a n d f r e e z i n g

FIG. 2. Effects of propyl gallate on viability of lyophilized Serratia murcescens. Centrifuged cells were resuspended in distilled water and mixed with an equal volume of 0.4% propyl gallate. At the indicated times 0.5-ml samples were rapidly frozen by transferring bottles cooled in dry ice. After all samples were collected they were lyophilized. Half of the samples were rehydrated after 1-day storage and the other half after 7 days at room temperature. (From Heckly et al., 1967, with permission.)

As demonstrated by Fateeva et al. (1970), the effect may not be entirely cosmetic. They determined that the eutectic temperature for yeast was -23°C and that high survival was obtained if it was kept below this tempera- ture during drying. However, their conclusion is not entirely valid, because high survival has been obtained with a number of organisms by drying from the liquid state (Annear, 1970a).

Unless contraindicated by experience with a particular system, the rec- ommended procedure would be to freeze slowly and to dry at the lowest practical temperature.

f Extent of drying. Most workers agree that residual moisture content is a factor influencing the survival of organisms, but there still is no agree- ment as to what constitutes optimum moisture content. This author believes that the highest survival is usually associated with the lowest moisture con-

24 ROBERT J. HECKLY

tent, as has been demonstrated for a number of microorganisms. These included S. murcescens and E . coli (Dewald et al., 1967), phytopathogenic bacteria (Samosudova, 1965), Bacillus popilliae (Lingg and McMahon, 1969), Shigella (Damjanovik, 1974), and vaccinia virus (Suzuki, 1973a; Sparkes and Fenje, 1972). In contrast, influenza virus is apparently most stable at 1-2% residual moisture (GreiE and Rightsel, 1967; Greiff, 1971). Some of the disagreements might be attributable to differences in suspending medium, since Robinson (1972) found that smallpox vaccine was unstable at low re- sidual moisture if the nitrogen content of the preparation was low.

Another possible cause for discrepancies in studies on the effect of moisture may be related to the presence of variable amounts of oxygen. The deleterious effect of oxygen on influenza virus was considerably less at 2.5% moisture than when dried to 0.06% (Greiff, 1971). Chen et al. (1966) also found that even though reducing the moisture content of baker’s yeast (S. ceriuiseue) from 8% to 4 4 % greatly improved thermostability in a nitrogen atmosphere, the drier yeast was more sensitive to oxygen. At least in these instances, water tends to increase resistance of cells to oxygen.

Most researchers use weight loss on heating, usually to 100°C, to measure moisture but this is not specific since some preparations contain significant amounts of other volatile substances besides water. Problems associated with the various other methods for determining moisture were discussed pre- viously (Heckly, 1961), but a new method which was specific for water was described by Robinson (1972). In his method, water was extracted with benzene and the water content of the benzene was determined by gas-liquid chromatography. Brevelt and van Kerchove (1975) claim superior results using ethanol instead of benzene for the extraction. Since gas chromatog- raphs are now common laboratory tools, this may become a standard method.

g . Storage atmosphere. The effects of oxygen on dried organisms was discussed in considerable detail at an AIBS Symposium in Michigan (Heckly, 1978), but certain aspects will be reviewed. Lion and Bergmann (1961) and Heckly and Dimmick (1968) demonstrated that in the absence of protective additives very small amounts of oxygen were toxic to dry bacteria, and Dewald (1966) found that the rate of inactivation was proportional to the oxygen tension. Although influenza virus was more stable in an inert atmo- sphere, oxygen was not particularly deleterious (Greiff and Rightsel, 1969). Either the suspending fluid protected the influenza virus or it might have been less sensitive to the effects of oxygen.

The addition of almost any substance, such as inositol, sucrose, sodium glutamate, ascorbic acid, skim milk, or serum, reduces the sensitivity of

PRESERVATION OF MICROORGANISMS 25

dried bacteria and viruses to oxygen. As discussed in Section V, antioxidants, such as butylated hydroxyanisole (BHA), can protect dried yeast against the effects of oxygen. Butylated hydroxyanisole has not been used extensively in preserving other organisms. The rate of inactivation of dry bacteria by oxy- gen immediately after drying is sufficiently slow to permit short exposure to air. However, it has been shown that cells stored under vacuum for as little as 2 weeks, are inactivated extremely rapidly (Heckly and Dimmick, 1968). Fewer than lo6 cells were viable after less than 1 min exposure to air, whereas replicate samples rehydrated under vacuum (no exposure to air) had 108 viable cells. Admittedly, these cells were dried without protective additives, but this pointed up the desirability of rehydrating lyophilized cultures as rapidly as possible after the ampules were opened.

Since oxygen is deleterious, it is important that ampules be properly sealed. Grieff et al. (1975) showed that the sealing of gas-filled glass ampules was frequently defective and recommended neoprene dissolved in toluene for sealing the leaks. They do not mention sealing of evacuated ampules, but this author believes that this is not a problem if the thin tip, obtained as the evacuated ampule is separated from the manifold, is heated to fuse the tip into a small ball. However, after a few weeks storage it is well to check the effectiveness of the seal with a high-voltage spark tester. Since this produces ultraviolet light, it is advisable to keep the radiation to a minimum.

h. Temperature of storage. As will be discussed in Section 111,F,4, “Ac- celerated Storage Tests,” the temperature coefficient for the inactivation of organisms is rather high and depends upon a number of fictors. These include moisture content, presence of oxygen, nature of the protective addi- tive, and nature of the organism. Spores generally survive as well at room temperature as at 4”C, but it is advisable to store most organisms at 4°C or lower. Although survival is increased at subzero temperatures, most collec- tions of lyophilized organisms are stored at 4°C because it is adequate and considerably less expensive than -60°C or even -20°C storage.

i. Method of reconstitution. The effects of temperature and composition of the reconstitution have been reviewed previously (Heckly, 1961) and there has been little additional work since then. Choate and Alexander (1967) found that the number of viable cells of Spirillum atlunticum surviving lyophilization was increased almost 10,000-fold by reconstituting the lyophilized cells with 24% sucrose instead of distilled water. They also re- ported that regardless of the rehydration media, low-temperature rehydra- tion provided the highest survival rate. It has been noted that high osmotic pressure was generally conducive to obtaining maximal recovery, but some

26 ROBERT J. HECKLY

organisms were adversely affected by high osmotic pressures (Heckly, 1961). Unfortunately, it appears that no single method provides for maximal sur- vival of all organisms.

Viability assays should be made on all reconstituted cultures for several reasons. (1) Such assays provide a basis for anticipating when to reprocess the culture. (2) Examination of discrete colonies or plaques provides a measure of quality control. Morphological variants or gross contamination can be detected. (3) Initiating a culture from a number of identical colonies insures a pure culture. Since it is possible for the lyophilized sample to become con- taminated during any one of the lyophilization steps, cultures should never be initiated by transferring the entire sample to broth. Under some condi- tions a single contaminant may become the predominant organism in the culture. This consideration is particularly important when only a few of the original organisms persist.

3. Lyophilizatwn Damage

As with higher life forms, any one of many events can kill or inactivate a microorganism. In lyophilization, organisms are subjected to possible injury by freezing, slow warming to sublimation temperature, dehydration, and possibly exposure to oxygen. Even in the absence of oxygen, cells stored at room temperature appear to suffer from some sort of solid-state rearrange- ments (Heckly and Dimmick, 1968). Most of the studies on lyophilization damage have been done with bacteria, probably because more is known about them. Bacteria are certainly easier to work with than are viruses and fungi.

There is considerable evidence that lyophilization can damage bacterial membranes (Ray et al. 1971b; Webb, 1960; Calcott and MacLeod, 1975a,b), but proteins and RNA have also been identified as being damaged by the process (Mitie, 1976; Morichi and Irie, 1973). Under some conditions, bac- teria damaged by lyophilization can recover, and the variability in rates may reflect differences in the type of injury. The time required for cells to re- cover from injury varied from about 30 min (Morichi and Irie, 1973; Beker, 1972) to nearly 8 hours (Gomez et al., 1973; Sinskey and Silverman, 1970). Ray and Speck (1972) and Ray et a2. (1971b) demonstrated that repair re- quired ATP synthesis and that temperature was a factor in recovery. At 15"C, it required at least 120 min, whereas repair was virtually complete in about 30 min at 25°C. Israeli and Shapira (1973) concluded that the death of E. coli was not caused by damage to DNA, RNA, or protein synthesis per se; instead, interference with control mechanisms was responsible for cell death. Israeli et al. (1974) demonstrated that lyophilization ofE. coli injured the membrane transport system for 0-nitrophenol P-thiogalactopyranoside.

PRESERVATION OF MICROORGANISMS 27

If oxygen were excluded, the damage could be partially repaired after rehyd- ration. Oxygen also was shown to inactivate nicotinamide adenine dinucleotide oxidase in lyophilized E. coli (Lion and Avi-Dor, 1963). They further showed that the enzyme activity was lost when a cell-free extract was dried and exposed to air.

Several workers have demonstrated that cells may suffer genetic damage (Ashwood-Smith and Grant, 1976; Qu6villon et al., 1964; Servin-Massieu and Cruz-Camarillo, 1969; Webb, 1967). At least some of the loss of viability on lyophilization was attributed to DNA strand breakage by Ohnishi et al. (1977), because the strain that could repair radiation-damaged DNA (E. coli B/r) had higher survival than the radiation-sensitive strain (E. coli BPI). However, they went on to point out that cell damage was not restricted to strand breaks of DNA.

Unless high viability is retained after lyophilization, it is difficult to dif- ferentiate between the mutagenic effect and the selection of spontaneous mutants by lyophilization. Damjanovii. (1972) found no evidence that lyophilization had any influence on the reversion rate of mutant Shigellu vaccines. However, he demonstrated that in a mixed culture, lyophilization effected a great deal of selection (Damjanovii., 1973). Gupta (1975) found that lyophilization of BCG also seemed to be selective. After lyophilization, the percentage of the nonspreading colony types was higher than before drying. Webb (1969) also observed that lyophilization was selective. Since he has found that auxotrophs ofE. coli are more sensitive to damage than the parent prototrophs, extra care should be taken in preserving mutant strains by lyophilization to minimize concentrating spontaneous “back mutations.”

Free radicals are probably not a factor in dehydration damage because it has been shown that lyophilization does not produce a significant number of free radicals (Heckly and Dimmick, 1967). However, free radicals are pro- duced by cells exposed to oxygen (Dimmick et d., 1961; Lion et d., 1961; Heckly et al. , 1963; Heckly and Dimmick, 1968; Kuznetsov et aZ., 1975). A strong correlation between loss of viability and free radical production has been demonstrated (Heckly and Dimmick, 1968). No free radicals were demonstrable as long as the cells remained viable, even in the presence of oxygen. It is tempting to consider the action of free radicals as the cause of death in dry bacteria exposed to oxygen, but it has been demonstrated that loss of viability precedes free radical formation (Cox and Heckly, 1973). Perhaps bacteria can exclude oxygen as long as they are “alive,” and only after the cells die can oxygen diffuse into the cell and produce free radicals by reacting with cellular components. This is compatible with the concept that at least some of the damage caused by drying is reversible and that oxygen reacts to make the damage permanent (Novick et al., 1972; Israeli

28 ROBERT J. HECIUY

and Shapira, 1973; Israeli et al . , 1975). Oxygen can react with many dry biological systems (Heckly, 1972, 1976). Unfortunately, the chemistry in- volved has not been identified.

4 . Accelerated Storage Tests

It usually takes years to observe significant loss of viability of lyophilized preparations, particularly under favorable storage conditions. Therefore, storage at elevated temperatures has been used both for developing or im- proving methods and for predicting survival.

Moisture content is extremely important and can be critical at the higher storage temperatures. At 4"C, Sparks and Fenje (1972) found that at 6.7% moisture smallpox virus was rapidly inactivated, but there was little dif- ference in stability of the vaccine at moisture contents ranging from 0.36 to 4.8%. However at 37"C, there was a graded response, and the highest survival of virus was obtained at the lowest moisture content. Qualitatively the same results were obtained by Suzuki (1973a), who found high survival of vaccinia virus at the lowest moisture content tested (0.97%) at either 37 or 45°C. Suzuki (1973b) also found that at 45"C, glutamate was the most effec- tive additive for vaccinia virus. Recently, Scott and Woodside (1976) simi- larly observed that glutamate, alone or with other additives most effectively stabilized pseudorabies virus at 20 and 37°C.

Unless it is shown that the loss of viability or infectivity fits an Arrhenius equation (a plot of the log of inactivation rate versus ID'), extrapolation or prediction of survival at lower temperatures is not valid. Greiff and Rightsel (1965) described such an accelerated storage test for predicting stability of measles virus. They showed that the activity of measles virus decreased in a reasonably linear manner when stored at 28, 36, and 45"C, and that the data fit a straight line on an Arrhenius plot. Beardmore et al. (1968), using the accelerated storage test of G r e 8 and Rightsel (1965), predicted influenza virus, lyophilized in allantoic fluid, to be stable indefinitely at room tempera- ture.

Application of accelerated storage tests to bacterial preparations are more extensive. Although Redway and Lapage (1974) used only 30 and 45°C for B . cerus, H . suis, N . gonorrhoea, and V. metschnikovii, others used higher temperatures. On the basis of short-term incubation of dried L. bififus at 36, 45, and 65"C, Damjanovid and Radulovid (1968) accurately predicted sur- vival after 203 days' storage at 4°C. Survival of L. acidophilus at 4 and 20°C also was predicted accurately on the basis of accelerated storage tests at 50, 60, and 70°C by Mitid et al. (1974). Similarly, Damjanovid (1974) predicted stability of a live Shigella vaccine. Annear (1964) used 37, 45, and 100°C to compare the protective effect of various substances on lyophilized S. ndolo.

In a description of the procedures used by the Czechoslovak National

PRESERVATION OF MICROORGANISMS 29

Collection of Cultures, Sourek (1974) mentioned that samples were heated to 75 and 100°C for 30 min and checked for viability. No data on the results of these tests were given, only the long-term survival of a large number of different organisms at 4°C was listed. Presumably, if adequate numbers survived the heating, the lyophilization was considered satisfactory for stock culture preservation. This may not be valid because Maister et al. (1958) found that survival of S. murcescens at 80°C was not correlated with survival at lower temperatures. However, Obayashi et al. (1961) reported a good correlation between results of heating dried samples to 100°C and viability after storage at lower temperature. Even higher temperatures are needed for spores. Molin (1977) heated Bacillus subtilis var. niger, and B. stearothermophilus to 190°C. Although the data fit an Arrhenius plot well only at low moisture content, he predicted a D value (time for 90% reduction) of about 4 years at 0°C.

Only one report on the use of elevated temperature for stability testing of dried fungi was found. Rogan and Terry (1973) incubated lyophilized cul- tures of P. chrysogenum at room temperature (RT), 37, 45, and 60°C. With these data they constructed Arrhenius plots from which they predicted the rates of viability loss at RT. A comparison of predicted values with experimen- tal data at room temperature storage with 12 additives demonstrated that the test permitted reliable estimation of shelflife.

The accelerated storage test is a useful tool, but considerably more check- ing of predicted values for a wider range of organisms needs to be done. Fortunately, as far as stock culture preservation is concerned, inaccuracies have been on the short side; i.e., survival has been better than predicted.

IV. Culture Collection Practices

The procedures used to preserve large culture collections are not neces- sarily optimal, but generally they are adequate. Obviously, it is not possible to determine optimum conditions for each group of organisms. The Ameri- can Type Culture Collection (ATCC) does have an active research program to improve preservation methods for the more sensitive organisms. Lyophilization is used widely, although some organisms do not survive well. These are frozen. Although the ATCC maintains all of their stock viruses and most phages in liquid nitrogen, specimens are lyophilized for distribution (Clark and Geary, 1969, 1973). The ATCC uses two methods for lyophiliza- tion. For hardy organisms a number of tubes is lyophilized in a batch system, only 1 strain per container. The small tubes are then placed in larger test tubes containing a desiccant and label. These are then constricted, evacu- ated, and sealed. For those organisms that appear to be damaged by even short exposure to air, the ampules are attached individually to a manifold and

30 ROBERT J. HECKLY

sealed under original vacuum by fusing the glass. All are stored at about 4°C. Survival data for 26 strains of Mycoplasma at the ATCC, after up to 10

years’ storage, were reported by Norman et al. (1970) and Norman (1973). In general, better recovery was obtained from the liquid nitrogen than &om the dried preparations, yet 105-106 organisms remained viable when lyophilized in 12% sucrose. Clark and Klein (1966) tabulated infectivity data on 26 strains of bacteriophge after 3-5 years’ storage in liquid nitrogen at the ATCC. More recently, Cjark and Geary (1973) reported on preservation of a large number of bacteriophages by lyophilization. Berge et al. (1971) success- fully Iyophilized several enteroviruses but they did not present any long- term storage data.

Although most of the 4500 strains in the fungal collection of the ATCC are maintained by lyophilization, some do not survive well. Hwang (1968, 1970) reported that 74 of the 104 sensitive strains were successfully frozen in 10% glycerol and survival to 18 months was tabulated These strains were sub- sequently reported to have survived after 42 months at -196°C (Hwang, 1970). Although DMSO has been shown to be more effective than glycerol for some species (Hwang and Howells, 1968), glycerol may continue to be the standard protective agent. Butterfield et al. (1974) recently summarized results in an extensive table showing survival of lyophilized as well as frozen cultures. Some lyophilized strains survived 32 years of storage but the longest test period reported for storage in liquid nitrogen was 8 years.

The Northern Regional Research Laboratory, Peoria, Illinois, has not used liquid nitrogen for preserving their culture collection. Instead, it has relied largely on lyophilization for the preservation of fungi. The procedure, as described by Haynes et al. (1955), was to suspend the organisms in bovine serum and seal the ampules under vacuum. An extensive list showing rela- tive vigor of the lyophilized cultures after 8-17 years’ storage at 4-10°C was published by Hesseltine et al. (1960). Of 363 strains tested, 331 were viable after 17 years. Subsequently, Ellis and Roberson (1968) summarized the results of viability tests on 447 strains. Many of those stored for 23 years were viable.

The Indian Type Culture Collection used either calf serum or skim milk as a protective agent. Results after 2 years’ storage of 38 strains were published by Sarbhoy et al. (1974). Only 26 of the 38 strains survived. They did not indicate that there was any difference in survival between those in serum or those in skim milk.

The lyophilization method used by the Institute of Tropical Medicine in Antwerp, as described by Bosmans (1974), employs a suspending medium consisting of 10% sucrose, 5% peptone, and 30% ox serum. Details on sur- vival were not given but he did list the fungi and yeasts that had survived 10 years’ storage. He mentioned that other methods, such as covering agar

PRESERVATION OF MICROORGANISMS 31

cultures with oil or freezing cell suspensions were used, but no data were given.

The National Collection of Type Cultures, in London, initially used horse serum but it has been superceded by “Mist. desiccans” of Fry and Greaves (1951). Steel and Ross (1963) reported on survival of some 100 strains of bacteria surviving lyophilization and storage for 10 years but only in general terms.

Miller and Simons (1962) reported on the standard method used by the Department of Microbiology, Woman’s College of Pennsylvania. Bacteria suspended in defibrinated rabbit or horse blood were dried on perforated glass beads over calcium chloride at room temperature. After 21 years, only 13 of 202 cultures had failed to grow. Considering the presence of oxygen and high temperature, this was a high survival rate. The Department of Microbiology, the Ohio State University, preserves its culture collection in liquid nitrogen using either DMSO or glycerol (Swoager, 1972). No long- term survival data were given.

Since 1939, about 3000 cultures have been preserved at the University of Buenos Aires, Argentina, using a simple desiccation system (Soriano, 1970). A small amount of the bacterial culture was introduced into a small tube and closed with a cotton plug, which in turn was placed in a larger tube contain- ing a dehydrating substance, such as potassium hydroxide. The larger tube was fitted with a rubber stopper and capillary tube so that after evacuation to 0.01-0.05 torr, it could be sealed by fusion. Soriano (1970) indicated that despite the fact that room temperature rose to 30°C, some 75% of the tubes retained viability for up to 30 years.

The Czechoslovak National Collection of Type Cultures uses a system comparable to that described for the American Type Culture Collection. Sourek (1974) tabulated survival of 122 organisms after storage for as much as 23 years.

Skim milk was used in the lyophilization of all microorganisms at the Institute of Microbiology, Academia Sinica, Peking (Research Group of Cul- ture Collection, 1975). Extensive tables showed that reasonably good sur- vival was obtained after 4-8 years, even though the ampules were stored at room temperature. Lyophilization is also used extensively in Russia (Konev and Kuzmina, 1975; Kuznetsov and Rodionova, 1971) and in Turkey (Cetin, 1970).

V. Industrial or Commercial Practices

Applied aspects of culture preservation can be found in the various indus- tries using microorganisms. Some do not have a culture preservation pro- gram. Instead, they use a perpetual culture system. The sourdough French

32 ROBERT J. HECKLY

bread bakeries in San Francisco, California, have been identified as the only bakeries in the United States to carry portions of the sponge as inoculum for the next dough (Reed and Peppler, 1973). However, there are probably many others who use this method because such information is not generally published. The unique flavor of San Francisco sourdough bread has been attributed to a mixed culture of Lactobacillus sanfiancisco and an acid- tolerant yeast, Saccharomyces exiguus (Sugihara et al., 1971; Klein and Sugihara, 1971). Efforts are being made to develop a method for preparing these organisms in a dry form that preserves activity and facilitates distribu- tion of the culture. The dried preparation would be used as the primary culture.

Although a San Francisco brewery also routinely uses the perpetual cul- ture system, most of the brewing industry uses pure cultures prepared from preserved cultures. For example, Anheuser-Busch prepares the cultures in a central laboratory. Cultures are shipped to plants at approximately weekly intervals. Such practices obviously insure minimizing contaminants in the final fermentation. These breweries, as well as other companies producing yeast, maintain stock cultures in the lyophilized state and on malt agar, either with or without mineral oil overlay.

Fresh yeast is supplied to the larger bakeries in metropolitan areas as fresh pressed yeast cake without any requirements for storage. Active dry yeast (ADY), which can be easily stored, is now widely used for home baking and by the more isolated bakeries. However, it is necessary that yeast not only remain viable and able to reproduce without a significant lag period but also remain biochemically active. As with other microorganisms mentioned pre- viously, many factors affect the survival of yeast. These are discussed, with details of the commercial yeast production methods, by Reed and Peppler (1973). Briefly, yeast is grown aerobically to maximize cell yield and nutri- ents are restricted slightly to increase stability of the cells. After the yeast is washed and concentrated by centrifugation, it is pressed into cakes. Com- pressed yeast cake loses only about 5% of its initial activity in a week but it cannot be stored for much more than a month. After that time, mold con- tamination is likely to become excessive.

For production of active dry yeast, the filter cake is extruded in the form of thin ribbons and dried in air at 4040°C. Less than 5% of the activity is lost in the drying process but in air, the loss is nearly 7% per month. However, if it is packed in vacuum, or in a nitrogen atmosphere, losses are reduced to about 10% per year. The deleterious effects of oxygen can be minimized by adding an antioxidant, such as butylated hydroxyanisole (BHA), to the yeast before drying (Chen et al., 1966). The optimum concentration, on a dry yeast basis, was 0.1% BHA and 1% sorbitan monostearate. It was also noted that yeast was more active when it was rehydrated with water at 4045°C.

PRESERVATION OF MICROORGANISMS 33

The dairy industry is dependent on preserving microorganisms because the likely contamination with bacteriophage makes perpetual culture uneco- nomical. Not only is a variety of organisms needed, but bacteria with dif- ferent phage types must be available to maintain production. Since few production plants can afford a microbiology staff, several companies have been established to provide starter cultures. Chr. Hansen’s Laboratory, Inc., has been providing starter cultures to the industry since 1893 (Sellars, 1975). Initially, liquid cultures were delivered to plants in their immediate area, but expansion of business required some form of preservation to insure that the starters were active. They now use liquid nitrogen. Cultures frozen in liquid nitrogen survive well, and in some instances stored cultures were slightly more active than before or immediately after freezing (Sellars, 1975).

The Marshall Division of Miles Laboratories, Inc., Madison, Wisconsin, ships dairy cultures frozen in dry ice with a recommendation that they be stored between -48°C and -80°C. Microlife Tecnics, Sarasota, Florida, pre- serves their stock cultures in liquid nitrogen, but activity of the commercial starter cultures is retained satisfactorily at -29°C.

Christensen (1977) commented that the Marshall Dairy Laboratory’s old freeze-dried culture program was inadequate, but the DPL Culture Service, San Francisco, California, has a thriving business lyophilizing lactic cultures for the manufacture of buttermilk, cheese, and yogurt. Use of special pro- prietary additives is claimed to yield dried cultures that can be stored with little loss of activity. Advantages of lyophilization are that cultures can be stored and shipped economically and activity is not lost if a shipment is delayed.

Industrial strains of bacteria, molds, and yeasts are maintained by the Marshall Division of Miles Laboratories, Inc., at Elkhart, Indiana, by lyophilization in double-strength skim milk (C. E. Brownewell, personal communication, 1977). These are lyophilized on a manifold system after rapid freezing and are stored at 44°C. Pfizer, Inc., Groton, Connecticut, also preserves the majority of its stock cultures by lyophilization using skim milk and the double-tube system similar to that used by the ATCC. After 20 years of storage a lyophilized culture of Streptomyces rimosus still produced oxytetracycline in the same quantity as before (L. H. Huang, personal com- munication, 1977).

Prior to 1963, stock cultures at Squibb Institute for Medical Research, New Brunswick, New Jersey, were lyophilized (Fortney and Thoma, 1977). Because survival of frozen S. griseus stored at -40°C was higher than in lyophilized cultures, storage over liquid nitrogen was instituted. Under these conditions there was not loss of viability. Ross Laboratories, in Colum- bus, Ohio, also maintain their cultures in liquid nitrogen using the method described by Swoager (1972).

34 ROBERT J. HECKLY

The Upjohn Company, Kalamazoo, Michigan, maintains stock cultures of actinomycetes and fungi on soil and others over liquid nitrogen. The freezing procedures, as described by Dietz (1975), are of interest. For bacteria, broth cultures or distilled water suspensions of agar growth are dispensed into small ampules and frozen without additives. Fungi and related organisms are grown on agar plates and plugs are cut and frozen, as described in Section III,D, with no added fluids. There have been no viability problems to date and cultures hnction normally in fermentations and bioassays.

Unfortunately this section is not complete because a number of companies have declined to provide any information on culture preservation methods.

VI. Procedures for Selected Groups

A. ALGAE

The preservation of algae has received little attention. Tsuru (1973) re- ported a high percentage (6045%) survival of a variety of algae at -196"C, with glycerol and DMSO being equally effective as protective agents. He concluded that the addition of suspending agents to algal cultures resulted in greater viability for most of the green algae but showed little effect on the blue-green algae. Algae varied markedly in their resistance to freezing; in fact, nearly 1,OOO times as many ChZoreZZa protothecoides cells survived freezing and thawing as C. fusca (Morris, 1976a). However, in the exponen- tial growth phase, the resistant species was damaged by cooling from 25 to 0°C as well as by freezing and thawing. A study of the effect of cooling rates, from 25 to O"C, showed that maximum survival was obtained at a cooling velocity of about 4"C/min. Morris (1976b) went on to show that the growth temperature affected freezing tolerance. Cells grown at 20°C were the most sensitive to freezing and the highest survival was obtained at 4°C with 24-day incubation. Morris (1976~) found that resistance of Prototheca spp. to freez- ing also was affected by the growth temperature. Incubation at 4°C yielded the most resistant cells but even the most resistant cells failed to survive rapid freezing, whereas 90% survived slow freezing (O.S"C/min). Some lyophilized Nostoc muscmm algae can be stored for 5 years at 25°C with no loss of viability (Holm-Hansen, 1967) but other algae were not that resistant. Corbett and Parker (1976) reported consistently good recovery of various b;ue-green algae when suspended in lamb serum. Since they reported only growth or no growth, it is difficult to evaluate their data. They did test a number of other additives, including skim milk, which yielded no viable cells when rehydrated. Tsuru (1973), using 10% skim milk with 1% monosodium glutamate, obtained 0.03-0.08% survival of six different algae

PRESERVATION OF MICROORGANISMS 35

TABLE I STORAGE DATA ON SELECTED ALGAE

Storage

Organism and Temp. preservation method (“(2) Years Viable“ References

Anabaena sp. Lyophilized in lamb serum Dried on silica gel in milkb

Lyophilized in milk Lyophilized in milk + 1% glutamate Frozen in 10% DMSO or 10%

Chlorella sp.

glycerol Nostoc muscmm

Frozen in 10% glycerol or DMSO Lyophilized in milk + 1% glutamate Lyophilized in milk

Stichococcus bacillaris Lyophilized in milk

Synechoccus cedrorum Lyophilized in lamb serum

26 2 4

25 5

- 196

- 196 +5 25

25

26

0.25 2

5 0.25

0.25

0.25 0.2 5

5

0.25

100% +

0.007% 0.08%

85%

60-70% 0.05%

+ 10-5% to 10-3s

100%

Corbett and Parker (1976) Grivell and Jackson (1969)

Holm-Hansen (1967) Tsuru (1973)

Tsuru (1973)

Tsuru (1973) Tsuru (1973) Holm-Hansen (1967)

Holm-Hansen (1967)

Corbett and Parker (1976)

=Percentage of original viable cells or qualitative measure of growth (+). *In all instances “milk’ refers to skim milk.

when rehydrated after 3 months’ storage. Takano et al. (1973) failed to obtain any survival of a rather sensitive blue-green alga, Spirulina platensis, when suspended in any of 18 different materials, including bovine albumin, skim milk, and sucrose. They did obtain viable cells with gum arabic using a modification of Annear’s peptone-plug method (Annear, 1956).

Dried algae, as well as other organisms, are adversely afLected by oxygen (Holm-Hansen, 1967). No significant effect of storage temperature (-26, 4, and 26OC) on survival of two of the algae tested was found.

Some data on the preservation methods used with algae and results ob- tained are summarized in Table I. This is not intended to be a complete list but it should serve as a useful guide.

B. BACTERIA

Since so much of the discussion on preservation methods has involved bacteria as test organisms, it seems redundant to consider these again. Fur- thermore, the lyophilization of bacteria has been reviewed in considerable

36 ROBERT J. HECKLY

detail (Heckly, 1961). Table I1 summarizes some of the data on preservation of bacteria that has been published since 1961. Survival data using different preservation methods are given for a few organisms largely for comparative purposes.

Several investigators have published extensive tables showing survival characteristics of many bacteria under various conditions. Trollope (1975) reported on the use of anhydrous silica gel for preserving 33 bacterial species. Cultures were stored at 4°C and at room temperature for up to 3.7 years. Antheunisse (1972) tabulated survival data of 36 species stored on agar slants in sealed tubes. These were stored at room temperature for up to 10 years. Although the storage period was only 2 months, the report by Sinha et al. (1974b) may be of interest. They compared the survival of 23 strains of lyophilized lactic acid bacteria held in both air and vacuum at 30°C. Iijima and Sakane (1973) reported on survival of 16 genera dried from the liquid state and stored at 5 and 37"C, However, the longest storage period was only 6 months. The Research Group of Culture Collection (1975) reported on the survival of organisms lyophilized in skim milk. Only a few of the cultures stored for 16 years at 5°C failed to grow. Most of the data, however, per- tained to bacteria stored 4-7 years at 536°C. Survival data on 122

TABLE I1 STORAGE DATA ON SELECTED BACTERIA

Storage

Organism and Temp. preservation method" ("C) Years Viableb References

Archromobacter spp Under parafin oil

Frozen 15% glycerol Lyophilized in mist.

Frozen 15% glycerol Lyophilized in mist.

desiccansc

desiccans" Amtobacter

Dried on silica gel in milk On agar in sealed tubes Cysts on agar slow dried Cysts in soil

Bacillus popilhe Lyophilized in 5%

glutamate + 0.5% tragacanth

1

- 29 RT

- 29 RT

2 4 RT RT RT

RT

2

2 2

10 10

2 3-10 10 10

0.5

+

+ 10%

%

0.02%

+ 78% + +

2 -25

Floodgate and Hayes (1961)

(1961) Floodgate and Hayes

Greig et al. (1970) Greig et al. (1970)

Grivell and Jackson (1969) Antheunisse (1972) Vela (1974)

Lingg et al. (1967)

PRESERVATION OF MICROORGANISMS 37

TABLE I1 (continued) STORAGE DATA ON SELECTED BACTERIA

Storage

Organism and Temp. preservation methoda (“C) Years Viableb References

Lyophilized and mixed with soil RH < 22%

Bacteriodes (8 spp.) Lyophilized in horse

serum + 7% glucose

Frozen in 15% glycerol

Under oil

Burdetella pertussis

Curynebacterium spp.

10% Lingg and McMahon (1969) RT 1

4-6 3 10-100% Phillips et al. (1975)

Eckert and Flaherty (1972)

Floodgate and Hayes

Floodgate and Hayes

Floodgate and Hayes

Greig et al. (1970)

(1961)

(1961)

(1961)

- 70

1

- 29

RT

RT

37

4

2

2

2

10

4

+ +

+ 40%

2-100%

+++

Frozen in 15% glycerol

Lyophilized mist.

Lyophilized mist.

Dried from liquid in 5%

desiccansc

desiccans‘

peptone + 5% glutamate

Escherichia coli Frozen-no additive Lyophilized in milk +

5% sucrose + 5% lactose

Agar slant under oil

Annear (1970a)

- 70 RT

2 2

30% 50%

cox (1968) Danilova and Kudryavtsev

(1970)

RT

5

3

0.5

Nadirova and Zemlyakov

Iijima and Sakane (1973) (1971)

+

42% Dried from liquid in 0.1 M PO, + 3% glutamate

Lyophilized in sucrose + glutamate + polyvinyl pyrrolidone (5% each)

Klebsiella

Lactobacillus acidophilus Lyophilized in 3%

glutamate Lyophilized in 3%

glutamate Lyophilized in 8% lactose

+ 1.2% peptone Frozen concentrate in

whey

RT 0.9 6-22% Green et al. (1970)

37

5

20

-20

0.5

1.0

1.6

0.6

0.01%

100%

5%

86%

Obayashi et al. (1961)

Miti6 et al. (1974)

Duggan et al. (1959)

.. I

38 ROBERT J. HECKLY

TABLE I1 (continued) STORAGE DATA ON SELECTED BACTERIA

Storage

Organism and Temp. preservation method" (T) Years Viable* References

Lactobacillus bijidus Lyophilized in 8%

sucrose + 5% milk + 1.5% gelatin

Leptospira canicola Frozen 10% rabbit

serum Leptospira pomona

Frozen 10% bovine Leptospira interrogans

Frozen in 10% glycerol Dried on quartz fibers in

10% glucose Mycobacterium (BCG)

Lyophilized in 1.5%

Mycobncteriuin leprae

Mycobacterium tuberculosis

glutamate

Frozen-no additive

Frozen in milk Frozen in milk

Frozen in milk Frozen in milk Frozen in various media

Mycobacterium tuberculosis Lyophilized in 1%

glutamate Lyophilized in serum

and 10% lactose Myocbacterium (9 strains)

Lyophilized in milk On agar slants sealed in

Mycobacterium tuberculosis

tubes Mycoplasmu (L form)

Frozen-no additive Lyophilized-no

Frozen-no additives Lyophilized in 2%

bovine albumin

additive

4

- 196

- 196

- 196 4

37

- 60

-20 - 70

- 20 - 70 - 70

RT

RT

RT RT

- 70 - 20

- 70 4

0.6 60%

2

2

0.5 10

0.1

0.23

3 3

4 3 5

16

18

5 3-10

1 1

3.5 3.5

22%

0.7%

1% 919

50%

25%

5% 100%

+ 100% 100%

4 4

515

2433 80-100%

10% 0.02%

>W% 100%

Damjanovib and Radulovib (1968)

Torney and Bordt (1969)

Torney and Bordt (1969)

Stalheim (1971) Annear (1974)

Sirks et al. (1974)

Levy (1971)

Kim and Kubica (1972)

Gruft et al. (1968) Kim and Kubica (1973) Kubica et al. (1977)

Slosarek et al. (1976)

Slosarek et al. (1976)

Gruft et al. (1968) Antheunisse (1972)

Stewart and Wright (1970) Stewart and Wright (1970)

Addey et al. (1970) Addey et al. (1970)

PRESERVATION OF MICROORGANISMS 39

TABLE I1 (continued) STORAGE DATA ON SELECTED BACTERIA

Storage

Organism and Temp. preservation method" ("C) Years Viableb References

Lyophilized in 2% Lyophilized in milk

Lyophilized in 12%

Frozen culture medium Frozen culture medium

Dried from liquid in 5%

Mycoplam sp.

sucrose

Neisseria

glutamate + 5% peptone

Pseudomonas Agar slant under oil Agar slant in sealed tubes Dried from liquid in 0.1

M PO4 + 3% glutamate Dried in silica gel in milk Dried from liquid in 5%

peptone + 5% glutamate

Lyophilized in milk + 5% sucrose + 5% lactose

Salmonella Dried from liquid in 5%

peptone and 5% glucose

Lyophilized in 0.1 M sucrose + 0.2 M gluta- mate + 0.02 M semi- carbazide

Dried on cellulose tufts in 10% peptone + 10% glutamate

Sarcina lutea Suspended in 0.02 M

Frozen in 15% glycerol

Dried from liquid in 3%

PO, buffer

SerratM murcescens

37 - 26

4

- 20 - 70

25

RT RT

5

2 4 25

RT

25

25

RT

3 5

- 40

5

3.5 4

8-10

0.2 0.2

4

3 3-10 0.5

2 4

2

2

5

2

1

1

0.5

1% +

(est.) 10%

10-4% 1%

++

+ 95% 52%

+ +++

40%

90%

80%

96

19%

2%

74%

Addey et al. (1970) Kelton (1964)

Norman (1973)

Raccach el al. (1975)

Annear (1970a)

Antheunisse (1972) Iijima and Sakane (1973)

Grivell and Jackson (1969) Annear (1970a)

Danilova and Kudryavtsev (1970)

Annear (1970a)

Marshall and Scott (1970)

Annear (1964)

Tanguay and Bogert (1974)

Iijima and Sakane (1973)

continued

40 ROBERT J. HECKLY

TABLE I1 (continued) STORAGE DATA ON SELECTED BACTERIA

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ______- ~ ~

Storage

Organism and Temp. preservation method' ("C) Years Viableb References

glutamate + 0.1 M PO, Streptococcus cremoris

Lyophilized in fortified milkd

Streptococcus lactis Frozen in skim milk Frozen in skim milk Lyophilized in fortified

Frozen at 10"/ml Streptococcus spp.

Frozen in milk Frozen in milk Frozen in milk at pH 7

milkd

Thiobacillus ferrooxidans Frozen-no additive

Vibrio Under oil

Frozen in 15% glycerol

Lyophilized

Frozen in 15% glycerol Lyophilized in mist.

desiccansc Dried from liquid in 0.1

M PO4 + 3% glutamate Yersinia pestis

Lyophilized in 0.01 M PO4 + 2.5% albumin + 8% sucrose

37

30

-20 - 196

30

- 196

- 23 - 196 - 20

150

1

-29

RT

-29 RT

5

- 20

0.5

0.2

0.2 0.2 0.2

1

0.5 0.5 0.7

3

2

10 10

0.5

1

0.5%

0.3-7%

1.6% 90% 50%

100%

330% 76-91% 1546%

1%

f

+, -

0.1%

- < 10-88

0.6%

100%

Sinha et al. (197413)

Speck and Cowman (1970) Speck and Cowman (1970) Sinha et al. (1974a)

Keogh (1970)

Gibson et al. (1966) Gibson et al. (1966) Lamprech and Foster

(1963)

Manchee (1975)

Floodgate and Hayes

Floodgate and Hayes

Floodgate and Hayes

Greig et al. (1970) Greig et al. (1970)

Iijima and Sakane (1973)

(1961)

(1961)

(1961)

Berman et al. (1970)

"In all instances "milk refers to skim milk. bPercentage of original viable cells; or qualitative results are indicated by (-) for no growth to

(+ + +) for heavy growth; or fractions, such as 3/4, indicate that growth was obtained in three of four samples tested.

e"Mist. desiccans" is a mixture of 3 parts serum and 1 part broth with 7.5% glucose added (Fry and Greaves, 1951).

dMilk fortified with ascorbic acid, thiourea, and ammonium chloride (0.5% each).

PRESERVATION OF MICROORGANISMS 41

strains of bacteria in the Czechoslovak National Collection of Type Cul- tures were tabulated by Sourek (1974). Some of these bacteria were still viable after 23 years at 4°C. Survival data after 2 years’ storage of 45 lyophilized and frozen cultures of marine bacteria were first tabulated by Floodgate and Hayes (1961). Lyophilized cultures were stored at room tem- perature and frozen cultures were held at -29°C. Subsequently, Greig et al. (1970) reported on the survival of these cultures after 10 years of storage.

C. FUNGI, YEASTS, AND ACTINOMYCETES

Some members of this diverse group of microorganisms are difficult to preserve, but most are extremely hardy. H. B. Levine (personal communica- tion, 1977) found viable C . immitis in soil samples from Woodland, Califor- nia. These had been stored in screw-capped bottles at room temperature for more than 8 years. The record for “naturally preserved’ organisms is proba- bly that described by Seaward et al. (1976). They obtained viable actinomy- cetes from samples that were 1,890 years old. Therefore, it is not surprising that lyophilization is widely used for preserving fungi and has been the only method used in some laboratories.

Because some fungi in their collection failed to survive the freeze drying, the ATCC began storing cultures in liquid nitrogen 8 years ago (Butterfield et al., 1974). Wellman and Stewart (1973) also mention that since brewing yeasts tend not to survive lyophilization well, they have used liquid nitrogen storage and reported high viability after 3 years. Similarly, Squibb Institute for Medical Research discontinued the use of lyophilization in favor of freez- ing and storage over liquid nitrogen (vapor phase) for preserving fungi (Fort- ney and Thoma, 1977). Hesseltine et al. (1960) stated that fungal cultures without spores never survived lyophilization, but they reported that 15 cul- tures failed despite the presence of spores. Sarbhoy et al. (1974) found that 12 of their lyophilized cultures failed to grow after 1 year of storage. These results indicate that it is extremely important that viability of lyophilized fungal cultures be checked at rather frequent intervals to preclude loss of the culture, at least until it is found that numbers of viable cells remain high.

Several procedures have been used to freeze fungi. The method used by Wellman (1970) and by Wellman and Walden (1971) was to grow the or- ganisms on agar slants in the small cryogenic ampules which were heat sealed and rapidly frozen without additives by immersion into liquid nitro- gen. They considered this to be necessary for the osmotic mutants of Neurospora that were glycerol sensitive (80-97% survival versus 1839%) when frozen in 10% glycerol solution. The procedure used by the ATCC for preserving cultures not amenable to lyophilization, as described by Hwang (1968, 1970), was to transfer small plugs of fungal growth with agar to am-

42 ROBERT J. HECKLY

pules containing 10% glycerol. These were frozen slowly (about l"C/min), and cultures were thawed rapidly by swirling the ampules in a 3840°C water bath. The use of glycerol and rapid thawing may not be necessary for fungi because Dietz (1975) has obtained satisfactory results using an agar- plug method without glycerol and with a slow thawing procedure. Addition of suspending liquids to any of a number of rust spores before freezing was harmful, and one of these, Pucciniu stri$wmis, was so sensitive to moisture that to retain viability it had to be vacuum dried before freezing (Cunning- ham, 1973).

Barnhart and Terry (1971) found that with N. crassa conidia, the rate of freezing was not critical provided samples were thawed rapidly. They also reported that survival of conidia was improved 50-fold by using DMSO instead of glycerol as the protective agent. Hwang and Howells (1968) also found that DMSO was generally superior to glycerol but observed that the relative efficacy of protective agents depended on the species involved.

Storage of fungi and yeasts in water has been advocated because it is convenient and inexpensive and minimizes or prevents pleomorphism (McGinnis et aZ., 1974; Bosmans, 1974). In this procedure, pieces of culture were placed in distilled water and stored at room temperature in screw- capped vials. McGinnis et aZ. (1974) claimed that 92-94% of cultures were viable after a year. Those that failed to survive belonged to the following genera: Madurella, Paracoccidioides, and Trichophyton. Bosmans (1974) re- ported viability of some dermatophytes after 4 years in distilled water.

Other methods used for preserving fungi, and the results obtained, are summarized in Table 111. This does not include data from extensive tables on lyophilized cultures showing preservation for up to 32 years that have been published by Hesseltine et al. (1960), Ellis and Roberson (1968), Butterfeld et al. (1974), Antheunisse (1973), and the Research Group of Culture Collec- tion (1975). Survival data of about 23 strains of fungi stored on silica gel were tabulated by Trollope (1975).

No genetic changes were detected in P . chrysogenum cultures stored in liquid nitrogen, but 18% of the conidia of P . chrysogenum stored at 4°C formed a subpopulation with substantially lowered ability to produce penicil- lin (MacDonald, 1972). Except for one report of decreased pigmentation in three lyophilized strains (Kuznetsov and Rodionova, 1971), morphological characters were retained after lyophilization and storage for up to 17 years (Semenov, 1975; Sarbhoy et al., 1974; Hesseltine et al . , 1960; Kapetonovii: and Pavletik, 1972; Ellis and Roberson, 1968).

D. VIRUSES AND BACTERIOPHAGES

In a comprehensive report, Rightsel and Greiff (1967) classified a large number of viruses into eight different groups on the basis of their nucleic

PRESERVATION OF MICROORGANISMS 43

TABLE 111 REPRESENTATIVE DATA ON SELECTED FUNGI

Storage

Organism and Temp. preservation method ("C) Years Viable" References

Aspergillus

serum

serum

Lyophilized in goat

Lyophilized in bovine

Agar slant in sealed tube Sealed in H,O

Dried on silica gel in spent media

Agar slant in sealed tubes In distilled water

Agar slants-no addition Agar slants frozen-no

Candida

Neurospora erassa

additive Penicillium

Lyophilized in goat

Lyophilized in bovine

On agar slant in sealed

Agar slants frozen-no

serum

serum

tube

additive Streptomyces griseus

Lyophilized in goat

Lyophilized in bovine serum

Lyophilized in growth medium

Frozen in growth medium

Dried from liquid in 0.1 M PO, + 3% glutamate

Agar slants in sealed tubes Agar slants in sealed tubes

serum

Sheptomyces

RT

7

RT RT

RT

RT RT

25 - 196

RT

7

RT

- 190

RT

7

5

- 100

5

RT RT

2

23

3-10 4

1

3-10 2

0.5 5

2

23

3-10

4

2

10-13

7

5

0.5

1 3 3-10

>90%

+

80% 111

+

97% 212

5% 98%

90%

+

33%

68%

80%

++

2.5%

100%

65%

67% 0%

Mehrotra et al. (1970)

Ellis and Roberson (1968)

Antheunisse (1972) McGinnis et al. (1974)

Parina et al. (1972)

Antheunisse (1972) McGinnis et al. (1974)

Wellman and Walden (1971) Wellman and Walden (1971)

Mehrotra et al. (1970)

Ellis and Roberson (1968)

Antheunisse (1972)

MacDonald (1972)

Mehrotra et al. (1970)

Hesseltine et al. (1960)

Fortney and Thoma (1977)

Fortney and Thoma (1977)

Iijima and Sakane (1973)

Antheunisse (1972) Antheunisse (1972)

"Percentage of original viable cells; or qualitative results are indicated by (-) for no growth to (+ + +) for heavy growth; or fractions, such as 314, indicate that growth was obtained in three offour samples tested.

44 ROBERT J. HECKLY

acid content, solvent sensitivity, and membrane and pH lability. Most of the viruses in each group had similar resistance to freezing or lyophilization and the effect of additives varied from one group to another. On the basis of trends indicated by the sparse data, they anticipated it to be ultimately possible to correlate physicochemical characteristics of viruses and the ef- fects of freezing on lyophilization. Clark and Geary (1973) also demonstrated a correlation between morphology of bacteriophage and sensitivity to both freezing and drying. No additives were used by Rightsel and Greiff (1967), who noted that the titer of syncytial virus, probably the most sensitive of those tested, decreased markedly while frozen for 1 month at -65°C. How- ever, essentially no loss in titer after 28 months at -70°C was observed when the syncytial virus was stabilized with 44.5% sucrose (Law and Hull, 1968). It was unlikely that the difference in temperature was a significant factor, even though Greiff et al. (1964) demonstrated that measles virus was less stable at -40°C than at either -76 or -20°C.

Freezing has been used extensively for the preservation of bacteriophage. Although freezing in 10% glycerol may reduce the titer of some preparations by as much as 99.9%, Clark and Klein (1966) reported that the titer of almost half of the 26 different bacteriophages tested were unaffected by freezing and storage for 4 years at -196°C. None of the preparations suffered more than a one log loss in titer during storage. Nyiendo et al. (1974) also found that in 15% glycerol, all of a number of lactic streptococcus phages were resistant to freezing.

As mentioned before, glycerol or other substances are necessary for pre- serving viability of bacteria and higher forms but this may not be needed for bacteriophages. A few years ago, the ATCC adopted a rapid freezing method with no protective substances for the preservation of bacteriophages (Clark and Geary, 1973). Better recovery of the freeze-sensitive phages was ob- tained than with glycerol and, furthermore, samples could be lyophilized directly for mailing without further manipulations. It might seem that lyophilization are not a suitable method for preserving phage because less than 1% was recovered (Clark and Geary, 1973). However, the work of Imshenetskii et al. (1970) shows clearly that even though the sensitive T2 coliphage lost 96.5% of the original titer on lyophilization, there was little additional loss during storage at 4°C for 4 years.

Grivell et al. (1971) described a silica gel method for preservation and reported active preparations of lettuce necrotic yellow virus after 2% years' storage at 4°C. At 30°C, none of the preparations was viable after 12 weeks. This method probably results in a relatively high moisture content, which may account for the high temperature coefficient for inactivation of the virus. Residual moisture appears to be critical, particularly at the higher storage temperatures (Sparkes and Fenje, 1972; Suzuki, 1973a). See Section II1,F for a discussion of the effects of moisture.

PRESERVATION OF MICROORGANISMS 45

As with other microorganisms, a wide variety of substances has been tried as protective additives for lyophilization of viruses. Rightsel and Greiff (1967) demonstrated a synergism between calcium lactobionate and serum albumin for poliovirus. Even with optimum concentrations, 1% albumin with 3.8% lactobionate, about 97% of the titer was lost on dehydration. Berge et al. (1971) also found that after lyophilization in bovine serum albumin, dextran, polyvinyl pyrollidone, or polyethylene glycol, less than 10% of the poliovirus infectivity was retained. However, when electrolytes were removed by dialysis or ultrafiltration, there was virtually no loss of poliovirus titer on lyophilization. After 1 month of storage, less than 0.5 log loss was observed. Comparable results were observed with other enteroviruses. In contrast, Fellowes (1968) found that purification significantly reduced the stability of foot-and-mouth disease virus.

Scott and Woodside (1976) tried 15 media for stabilizing pseudorabies virus during lyophilization and concluded that peptone was the least effec-

TABLE IV STORAGE DATA ON SELECTED BACTERIOPHAGES

Storage

Organism and Temp. preservation methodn (“C) Years Viable (%) References

Corynebacteriophage (14 strains) Lyophilized in 3% -25 +2.5 10-100 Came and Greaves (1974)

peptone, 1.7% sucrose, 0.3% glutamate

Escherichia coli phage T-2 Lyophilized lyzate 4 Lyophilized in 10% milk - Dried in lyzate from liquid

Lyophilized in lyzate 4

Lyophilized in lyzate 4

- E . coli phage MS-2

E . coli phage 5d

E . coli phage (14 types) Frozen in 10% glycerol - 196

4 0.5 lmshenetskii et a2. (1970) 0 0.1 Clark and Geary (1973) 0 7 Iijima and Sakane (1973)

4 0.1 Imshenetskii et al. (1970)

4 100 Imshenetskii et al. (1970)

3 4 10-100 Clark and Klein (1966)

2 >10 Engel et al. (1974) Mycobacteriophage (1 1 strains)

Lyophilized in 10% RT glutamate + 1% gelatine

Serratia marcescens phage

Streptococcus phage (15 types) Frozen in 10% glycerol - 196 3 40 Clark and Klein (1966)

Frozen in 15% glycerol - 22 2.5 30-100 Nyiendo et al. (1974)

all instances “milk’ refers to skim milk.

46 ROBERT J. HECKLY

TABLE V STORAGE DATA ON SELECTED VIRUSES

Storage

Organism and Temp. preservation method (T) Years Viable' References

Foot and mouth disease Lyophilized in various

media Lettuce necrotic yellow virus

Dried on silica gel in milk Measles virus

Frozen in 2% DMSOb Polio virus

Lyophilized in 1 M tris buffer

Pseudorabies virus Lyophilized in 5% sucrose

+ 1% glutamate + 4% dextran

Respiratory syncytial virus Frozen in 30% sucrose Frozen in 44.5% sucrose Frozen in 44.5% sucrose

Lyophilized in 5% peptone

Lyophilized in 5% peptone

Vaccinia virus

buffer

or 5% glutamate

37

4

- 76

4-10

4

- 70 - 70 -20

4 3 7

45

0.1

2.5

0.5

1

1

2 2 0.3

0.5

1

130%

+

100%

16%

1%

5% 100%

0.1%

100%

1 4 %

Fellowes (1968)

Grivell et al. (1971)

Greiff et al. (1964)

Berge et al. (1971)

Scott and Woodside (1976)

Law and Hull (1968) Law and Hull (1968) Law and Hull (1968)

Sparks and Fenje (1972)

Suzuki (1973a)

a + indicates some infectivity. bDimethyl sulfoxide.

tive medium. They found that the best suspending medium for preserving infectivity was a mixture of sucrose, phosphate, and glutamate, to which albumin had been added. Calneket al. (1970), as well as Scott and Woodside (1973), also found the above mixture to be the best for herpesvirus. Peptone is not necessarily detrimental because infectivity of vaccinia virus was retained at a high level when lyophilized in media containing 5% peptone (Sparks and Fenje, 1972; Suzuki, 1973a). Corynebacteriophage also was stable when lyophilized in a mixture of peptone, sucrose, and sodium glutamate (Carne and Greaves, 1974).

Fairly extensive tables showing the effects of freezing and lyophilization on bacteriophage were presented by Clark and Geary (1973) and the effects of storage at -196°C of a number of ATCC bacteriophages were given by Clark and Klein (1966). Table IV summarizes results that others have ob-

PRESERVATION OF MICROORGANISMS 47

tained with bacteriophages and similar data on preservation of viruses are given in Table V.

VII. Summary

If cost is not considered a factor, storage in liquid nitrogen is probably the best method for preserving all microorganisms. For some viruses it may be better to freeze the sample rapidly, but slow freezing with a cryoprotective agent is desirable for all other organisms to retain maximum viability or infectivity. Storage at -70°C is nearly as effective as -196°C (liquid nitrogen). Only a few organisms survive well at higher temperatures, such as -20 or -40°C. For short-term preservation (3-12 months) agar slants or stab cultures in tightly capped tubes are usually adequate. For long-term storage of those organisms that can withstand dehydration, lyophilization is the most eco- nomical and reliable method. Oxygen is harmful to some organisms; there- fore cultures should be sealed in glass ampules under vacuum. Although many lyophilized organisms survive well at room temperature, all cultures should be refrigerated. Since excellent survival has been demonstrated at 4"C, the additional expense of using lower storage temperatures is probably not warranted. Certain problems associated with lyophilized cultures must be recognized. Dehydration may be mutagenic and, in addition, the relative number of spontaneous mutants can increase in the rehydrated culture. Some revertants have been shown to survive lyophilization better than the mutant strain. Reconstituted cultures should be diluted and plated to ascer- tain colonial morphology, and by initiating the new culture from a few of the colonies the chances of propagating a mutant or a contaminated culture are minimized.

ACKNOWLEDGMENT

This work was supported by the Office of Naval Research. I wish to thank J . H. Quay for her dedicated assistance in the preparation of this manuscript.

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