Chapter 18

Chapter 18

Collection and Preservation of Frozen Microorganisms

Rosamaria Tedeschi and Paolo De Paoli

Abstract

The storage of the different microorganisms over long periods is necessary to ensure reproducible results and continuity in research and in biomedical processes and also for commercial purposes. Effective storage means that a microorganism is maintained in a viable state free of contamination or genetic drift and must be easily restored without genotypic or phenotypic alterations to its original characteristics and properties. To this end, different techniques have been described and advances in cryopreservation technology have led to methods that allow low-temperature maintenance of a variety of cell types, minimizing the risks of genetic change and are now recommended for long-term storage of most microorganisms. This chapter summarizes the most important steps and components in the process of low- and ultra-low temperatures freezing of bacteria, parasites, yeasts and fungi, viruses, and recombinant microorganisms. Key words: Cryogenic preservation, Long-term storage

1. Introduction

Cryogenic preservation is the act of freezing and storing cells at very low temperatures. The effects of the freezing and thawing process on living cells are not fully understood: when water changes from liquid to solid state, cellular metabolism ceases and, when cells are warmed and water returns to liquid, cellular function resumes. Cell cryopreservation process remains the main method of cell preservation to date, and the high survival rates achieved by this method are of interest from both the biophysical and the practical points of view. Storing over long periods of bacterial or fungal strains, parasites, and viruses allows future research study, and it is essential for clinical, epidemiological, educational, microbiological, and commercial reasons. A special approach is required for the proper storage of recombinant microorganisms.

Joakim Dillner (ed.), Methods in Biobanking, Methods in Molecular Biology, vol. 675, DOI 10.1007/978-1-59745-423-0_18, © Springer Science+Business Media, LLC 2011

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Effective storage means that the organism is being maintained in a viable state, free of contamination, and without alterations in genotypic or phenotypic characteristics, easily restorable to the same condition it was before preservation. Several researchers attempted to develop methods for 100% preservation of freezing– thawing of diverse cellular specimens (1, 2). This literature also includes detailed information on those microorganisms, which remain recalcitrant to cryopreservation methods or that cannot be preserved by freezing (3–6). This chapter presents simple and broadly applied methods, from the clinical-microbiology laboratory point of view, which can be used for long period storage of bacteria, protozoa, fungi and viruses, and genetically modified microorganisms. The chapter includes several important steps or components involved in the process of cryopreservation at low- or ultra-low temperatures.

2. Safety Procedures The infectious and often pathogenic nature of most of the microorganisms, viruses in particular, means that they must be carefully handled by experienced personnel in purpose designed and approved laboratories and following the appropriate safety conditions. Special precautions must be taken during collection, processing, and storage of biological samples. The personnel must be trained and the laboratory must adopt biological safety level II or III, according to the handled pathogens. Guidelines related to biosafety issues are available from the Centre of Disease Control and from the World Health Organization ((7, 8), http:// www.cdc.gov; http://www.who.int).

3. Storage Temperature Liquid nitrogen provides the lowest practical temperatures (−196°C) for storing different sorts of microorganisms and, because viability is preserved so well, it is used extensively. However, because of its relatively expensive cost, there have been many studies on the effect of higher temperatures on survival of various groups or kinds of microorganisms. The critical temperature is dependent on a number of factors, but –70°C appears to be sufficiently low to preserve most organisms (5). Freezing at –20°C is used to keep a few organisms for as long as 1–2 years, most have a poorer survival than at higher or lower temperatures because of the damage caused by ice crystal formation (5) and electrolyte fluctuations (4) at this temperature. At this temperature


of storage, the preservation times vary depending on the medium used (5, 9). For long storage periods, microorganisms must be kept at temperatures of –70°C or lower in electric freezers and in liquid nitrogen storage containers. Close observation of the system that must have an adequate alarm mechanism is essential since any increase in temperature will reduce viability and, if thawing occurs, there are no guidelines for rapid restoration of the storage condition.

4. Storage Vials

Storage vials, supplied in a variety of sizes and from different commercial Companies, must withstand very low temperatures and keep their contents sterile. Plastic (polypropylene) vials are easier to use as compared to glass tubes. The most common sizes used are 1.0–1.8 ml tubes; generally, 0.5–1.0 ml of cell suspension is placed into each tube. The most important factors to be considered include cryotolerance (particularly when material has to be stored at liquid nitrogen temperatures), storage conditions, type of cells, and safety conditions. Usually, vials are conveniently packaged in boxes with grid to allow an easy recovery and picking up.

5. Age of Culture

To ensure the genetic stability of a culture, the number of passages from the original must be minimized. It is generally accepted that cells from the maximum stationary phase cultures are more resistant to damage by freezing and thawing than cells from the early or mid-log phase of growth, and the percentage of cells surviving is also increased by an increase in cell density (5).

6. Cryoprotective Agents

Cryoprotective agents, often added to the cellular suspension, serve to protect microorganisms from damage during the freezing process, storage, and thawing. There are two types of cryoprotective agents: ones that enter the cells, delay intracellular freezing, and minimize the solution effects (glycerol, DMSO) and others that protect the external milieu of the organism (sucrose lactose, glucose, mannitol, sorbitol, dextran, polyglycol) (10). Although there are no absolute rules in cryopreservation and many compounds have been tested and reviewed (11), glycerol and DMSO

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have been widely used and seem to be most effective; generally, the choice of a cryoprotective agent is dependent upon the type of micoorganisms to be preserved (Table 1). Glycerol and DMSO are generally used, after sterilization by autoclaving or filtration, respectively, at a concentration of 5–10% (vol/vol) (12, 13) and are not used together; the optimum concentration varies with the cell type and the highest concentration the cell can tolerate should be used. At the ATCC, mycoplasma and fungi that do not survive lyophilization are frozen in 10% glycerol. Rapid freezing without additives is allowed for long-term survival of protozoa. Of the external products, skim milk is most commonly used. It is purchased from medical product suppliers (e.g. Becton Dickinson, Oxoid) and used, after autoclaving, at a final concentration of 20% (wt/vol) in distilled water, the equivalent of regular milk (3, 14).

7. Preparation of Microorganisms for Freezing

Several factors must be considered when preparing microorganisms for cryopreservation: type of cell, viability, growth conditions, physiological state, and amount of cells. Microbial cells, in particular bacteria and yeasts, grown under aerated conditions show a greater effect of cooling and freezing than nonaerated cells (3). It was demonstrated that cell permeability is greater in aerated cultures and aerated cells dehydrate faster during cooling than nonaerated cells (15). Most bacteria and yeasts are inoculated in a medium that adequately supports maximal growth; cultures are allowed to mature to late growth or stationary phase before being harvested, generally considering that, the greater the number of cells initially present the greater the recovery. Concentrations of at least 107 CFU/ml or higher are recommended (3, 5, 14). Microorganisms can be conveniently harvested from agar slants or plates, or when greater quantities are required, grown in broth cultures and thereafter harvested by centrifugation. In both cases, cells are generally suspended in fresh growth medium containing the cryoprotectant agent and aliquots frozen in volume of 0.2–0.5 ml. Equilibration is the period of time between mixing the cryoprotectant with the cell suspension and the beginning of the cooling process. It should take place at room temperature, and it allows time for the cryoprotective agent to penetrate the cells. For most cells, equilibration should occur for at least 15 min but no longer than 45–60 min (as the cryprotective agent may become toxic to the cells). The optimal equilibration time should be determined empirically for the cells being cryopreserved to maximize later recovery. Once the cells and the cryoprotectant have been combined and dispensed into vials, the next step is to cool the

T T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T T

T

T

T

–b 106/ml

105 to 107/ml

Protozoa

Viruses cell free infected cells

–a 106/ml

107/ml

107/ml

Fungi hyphae spores

Spore forming bacteria

Gram-negative bacteria

DMSO + FCS (10%)

Blood, nutrient broth + DMSO + sucrose DMSO or glycerol or blood + nutrient media

Glycerol, DMSO

Glucose Skim milk, glucose

Sucrose, lactose Sucrose, lactose, glycerol

Skim milk Skim milk

b

−70 to −196

−70 to −196

−20 to −40

−70 to −196

−20 −70 to −196

−20 −70 to −196

−20 −70 to −196

−20 −70 to −196

−20 −70 to −196

Sucrose, glycerol Skim milk, sucrose, glycerol Skim milk Skim milk

Storage temperature (°C)

Cryopreservative

Mycelial masses are prepared for freezing of the hyphae of fungi regardless of cell amounts The number of infectious particles has little effect on the recovery of viruses and bacteriophage

a

107/ml

Streptococci

107/ml

107/ml

Gram-positive bacteria

Mycobacteria

Cell amount

Organism group

1–30

2–30

1–2 2–30

1–2 2–30

3–5 3–5

0.2 0.2–1

1–3 1–30

Storage duration (years)

Table 1 Common procedures for ultra-low temperatures preservation of microorganisms (adaptation from ref. 17)


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s uspension. The rate of cooling is another important parameter: it affects the rate of formation and size of ice crystals, as well as the solution effects that occur during freezing. For a wide variety of cells, a uniform cooling rate of 1°C per min from room temperature is effective. An easy-to-use system, designed to achieve a rate of cooling very close to what recommended, is the Nalgene “Mr Frosty” 1°C freezing container. Furthermore, nowadays, different companies provide freezers with computer-driven programmed cooling/thawing rates that allow a strict and effective control within an optimization of the cooling process. Uniform rates of cooling are required by particularly fastidious bacteria and nonsporulating fungi, while most bacteria and spore-forming fungi will tolerate less-than-ideal cooling rates and can be placed and stored at −60°C. Protists often require even more accurate cooling rates to minimize the detrimental effects of under cooling and the heat released during freezing.

8. Freezing Methods The temperatures at which frozen microorganisms are stored affect the length of time after which they can be recovered. In general, a lower storage temperature implies a longer viable storage period (5). As a good method, it is recommended a slow, controlled-rate freezing at a rate of 1°C per min until the vials cool to temperature of at least −30°C, followed by more rapid cooling until the final storage temperature is achieved (3). When organisms must be stored permanently at −60 to −70°C, the vials can be placed directly into this freezer. However, when organisms are to be stored in liquid nitrogen, it is still recommended that vials be placed initially in a −60°C freezer for 1 h and then transferred to the liquid nitrogen.

9. Thawing and Reconstitution The critical temperatures for the frozen microrganisms to get damaged occur between −40°C and −5°C. The rapid warming through these temperatures improves recovery rates. Recommen dations are to rapidly warm stored material frozen in vials by placing them in a 37°C water bath until ice has disappeared (3, 5). As a general rule, rapid thawing is preferable to slow thawing (http:// www.cryobiosystem-imv.com). Once the vial is thawed, the organism should be transferred immediately to an appropriate growth medium, to minimize exposure to the cryoprotective agent.


10. Procedures for Specific Organisms

Procedures for long-term storage of specific microorganims are described below and summarized in Table 1.

11. Storage of Bacteria

11.2. Cryoprotectant

Low temperature storage greatly reduces genotypic and phenotypic drift for living bacteria and enables cultures to be used as standards, helping to ensure reproducible results in a series of tests or experiments. After storage and thawing, some bacterial species, characterized by a potentially reduced viability and/or stability of antigenic, molecular, and biochemical properties are defined “fastidious microorganisms” (as Haemophilus spp., Neiseria gonorrhoeae, Campylobacter spp.). Several factors are critical to the stability and viability of a bacterial culture undergoing cryopreservation, as cell type, age growth conditions, population size, cryoprotectant used, cooling and storage conditions. 11.1. Growth and Preparation for Bacteria Cryopreservation

Bacteria being cryopreserved should be grown under optimal conditions on the recommended media (ATCC website: http:// www.atcc.org). Cells can be harvested from broth culture (as pellet after centrifugation and by removing the supernatant), agar plates or slants, and suspended in fresh growth medium containing a cryoprotectant. For most bacteria, a concentration of 107 cells/ml is required for good recovery (14); the most commonly used vials are plastic cryovials with volume between 1.2 and 2.0 ml. Growing condition and at which point the bacteria are harvested are the major factors to be considered. Bacteria grown under aerate conditions tolerate the stress of freezing better than statically grown or nonaerated cultures, as reported for E. coli (15). Microbial cells harvested in late log or early stationary phase are generally more resistant to the stresses of freezing than younger or older harvested cells (5). Glycerol at 10% in culture medium (e.g. Trypticase soy broth, TSB) is recommended by ATCC for freezing nonfastidious bacterial cultures. It is important to respect the equilibration time, leaving the cells at room temperature for a minimum of 15 min, but no longer than 60 min, to ensure that the cryoprotectant has enough time to penetrate (13); during this time the operator can proceed to aliquot cell suspension into the cryovials.

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11.3. Cooling and Storage Conditions

Once the cryoprotecant is added, the cultures are ready for cooling. The ideal cooling rate for bacteria is approximately −1°C per min (16) and a programmable-rate freezing apparatus can be used. Most bacteria will withstand less than ideal cooling rates and can be frozen by placing the vials on the bottom of the −60°C to −80°C freezer for 1 h, whereas for “fastidious strains” of bacteria more uniform cooling rates are required. In general, bacteria can be stored at −20°C for 1–3 years, at −70°C for 1–10 years, while freezing in liquid nitrogen at −196°C allows bacteria storage for up to 30 years (Table 1) (17). A simple and useful method based on the use of glass or plastic as carriers to support microorganisms (e.g. MicrobankTM, by Pro-Lab Diagnostics) offers a practical solution for long-term storage of frozen bacterial suspensions (18, 19). The protocol requires that a sterile vial with the cryopreservative fluid, containing approximately 25 porous beads, is open under aseptic conditions and inoculated with young colonial growth (18–24 h) picked from a pure culture to approximately a 3–4 Mcfarland standard; once closed, the vial should be inverted four to five times to emulsify suspension, without using vortex and, at this point, the microorganisms will be bound to the porous beads; the excess medium should be aspirated leaving the inoculated beads as free of liquid as possible. After inoculation, the cryovials are kept at −70°C for extended storage and when a fresh culture is required, a single bead is easily removed from the vial and used to directly inoculate a suitable bacteriological medium, while the vial returns as soon as possible to low storage temperature. By this approach, frozen bacteria may also survive a freezer failure for one or several days (12). The glass capillary method has proved to be another suitable method for preserving strict anaerobes bacteria (20). Considering the group of “fastidious microorganisms”, comparative studies of preservation and storage of Haemophilus influenzae evaluated the influence of different suspension media, with or without cryoprotective agents, the supporting material, the initial inoculum concentration, and thawing time at room temperature of stored strains at −20°C and −70°C (21, 22). Recommendations from the results were: (a) starting with a concentrated inoculum; (b) suspending viable cells in MGY, BHI, or TSB supplemented with 25% glycerol; (c) thawing at room temperature for not more than 3 h. Gelatin-based media or TSB added with 10% glycerol was used in preserving at −20°C N. gonorrhoeae for at least 1 year (9) and a successive evaluation (23) of similar medium with Campylobacter jejuni also shown 85% recovery after 12 months of storage at −20°C, suggesting that this medium may thus be useful for the preservation of a variety of fastidious strains for at least 1 year. Mycoplasmas and Chlamydiae, despite their peculiar growth and metabolic requirements, can be successfully stored by cryopreservation for up to 10 years (24). Helicobacter pylori is also among the bacterial species “difficult to growth” and sensitive to storage conditions as


compared to other intestinal bacteria (25), but previous studies reported about comparison of different cryopreservative media and use of particular procedures that allowed recovery of about 60% of Helicobacter strains after more than 3 years at −70°C (26, 27). The survival rate of bacteria after storage varied significantly according to species, being for Gram-positive higher than that of Gram-negative bacteria, probably because of their greater resistance of the structure and surface components (28). A wide range of aerobic and facultative anaerobic bacteria may survive longtime storage at −70°C to –80°C, especially if repeated freezing and thawing is prevented.

12. Storage of Fungi, Yeasts and Actinomycetes

There are many preservation protocols suitable for fungi as no individual preservation technique was ever applied to all fungi. Although lyophilization is widely used for preserving fungi and has been the only method used in some laboratories, several years ago ATCC began storing cultures in liquid nitrogen, as some fungi in their collection failed to survive the freeze-drying, in particular fungal cultures without spores (5). This storage method appears to approach ideal, although changes in physiology and genetic stability may occur in some isolates (29) requiring optimal and targeted protocols (30). In particular, fungi that cannot be preserved using traditional preservation methods, termed “preservation recalcitrant fungi”, include those that do not readily sporulate in culture (e.g. some members of the Oomycota, some Basidiomycota) and others that are difficult to maintain in culture (e.g. Diplocarpon) or are facultative pathogens (6). Spore-forming fungi require harvesting of spores and suspension of the spores in fresh growth medium with the cryoprotectant. It is very important, during the process of freezing fungal spores, to take care in not delaying the freezing process too long, avoiding germination to occur prior to freezing. For fungi that do not form spores, special procedures for harvesting mycelia prior to freezing must be adopted (13). For fungi with tough mycelia, the culture is harvested from agar growth by cutting and removing agar plugs containing the mycelia and preserving it into fresh growth medium added with the cryoprotectant. Tough mycelia that do not adhere well to agar cultures are grown in broth culture, and the mycelial mass is blended prior to freezing (14). The two approaches aimed at optimizing the cryopreservation of fungi are, first, the use of light cryomicroscope to directly observe the effects of cooling and thawing on the microorganism, and, second, the comparison of the effects of defined preservation regimens on the viability, pathogenicity and morphological, physiological, and genomic stability of replicates before and after preservation (6, 31).

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Regarding yeasts in particular, freezing at −80°C with 10% glycerol or 5% DMSO was the recommended method for proper and long storage of Malassezia spp. (32). Particular studies were also performed analyzing influence of cooling rate and freeze thaw cycles on yeasts viability during freezing (33).

13. Storage of Parasites Cryopreservation at ultra-low temperatures has been applied to several species of parasites, such as T. gondii, Entamoeba histolytica, Trypanosoma cruzi, G. lamblia, and T. vaginalis, and represents the method of choice for long-term storage, mostly considering the disadvantages due to propagation and preservation of parasites in vivo or in vitro, the need for labor, initial isolation, and loss of strains, bacterial an fungal contamination, and changes in the original biological and metabolic characteristics (34). The technical procedures for cryopreservation do not differ significantly from those used for the other microorganisms (3, 14). In general, type and concentration of the cryoprotectant and the rates of cooling and thawing are important factors affecting viability after cryopreservation. DMSO showed the highest cryoprotective effects for many species of protozoa at concentrations between 5 and 12.5%; glycerol is also considered an effective cryoprotectant, even if it permeates the cell more slowly than DMSO, requiring a period of equilibration (34); in particular, its use remarkably improves the survival rate of helmints that have never been successfully cryopreserved. Both rapid and slow cooling methods have been used depending on the parasitic species having high or low freezing tolerance, whereas exposure to a rapid thawing method using a water bath at 35–40°C produce better motility or infectivity for all parasites (34). Additional information on cryopreservation and detailed protocols of selected parasitic species may be found (e.g. E. histolytica (35), Blastocystis hominis (36)). Cryopreservation by vitrification represents another method for long-term storage of parasites, in particular for several species of helminths (37). Blood protozoa, such as Plasmodium spp. or Trypanosoma spp., can be stored from infected blood samples collected at the peak of parasitemia in liquid nitrogen, using 10% glycerol or 5% DMSO, respectively (17).

[AU1]

14. Storage of Viruses Most viruses can be frozen as cell-free preparations without difficulties and do not require controlled cooling (14), whereas those


viruses in viable infected cells do require it. Viruses are noncellular forms of life with small size, simple structure and absence of free water, and consequently are more stable than other microorganisms. Virus infectivity is retained well at temperature below −60°C, and it is significantly reduced in presence of a slow rise in temperature (more than 5°C). In general, larger viruses tend to be less stable than smaller ones; DNA viruses are more stable than RNA viruses (38). Viruses with envelopes are often less stable than nonenveloped viruses at room temperature, although this is not always evident at low- or ultra-low temperatures. Storage of virus suspension at −20°C is not recommended (5), but can be chosen if retention of virus infectivity is not essential and when the sample stored will be used for serodiagnostic purposes, e.g. as antigen in an enzyme-linked immunosorbent assay (ELISA) test, considering that the antigenic activity at this temperature is maintained. Storage in liquid nitrogen allows viruses to survive almost indefinitely but is not the most convenient and cost-effective method. Furthermore, viral stocks stored in liquid nitrogen containers may be exposed to cross-contamination if not preserved adequately in heat shrinkable tubes, and vapor phase containers are best indicated. Proteins are effective protectants for virus cryopreservation and the suspending medium used is, in general, tissue culture medium containing 10% or greater amount of serum or other proteins. Even the exact mechanisms are not deeply known, proteins keep virus infectivity during freezing, buffering capacity against pH changes (being the optimal pH for virus storage between 7.0 and 8.0), assist in colloidal dispersion of the virus particle and reduce or inhibit other processes that damage nucleic acids (38). About the use of particular cryoprotectants, sucrose-phosphate-glutamate containing 1% bovine albumin (SPGA) and hypertonic sucrose has been reported for storing labile viruses, as Respiratory Syncytial Virus (39). In general, it is good practice to use a high titer virus suspension and preserve it in small aliquots of 0.1–0.5 ml in cryotubes that should be frozen rapidly. Thawing frozen virus samples should be carried out just before the virus is to be used and rapidly by placing the cryotubes in a water bath at 37°C. Specimens known to contain viral pathogens may be kept at −70°C to −85°C for several years with reasonable recovery and without changes into the morphological characteristics as documented for enteric viruses in stool specimens (40). Viruses can be stored at ultra-low temperatures without particular treatment also in tissues or blood derived-specimens (serum, plasma, lymphocytes). Storage of peripheral blood lymphocytes in RPMI 1640 added with 10% fetal bovine serum and 10% DMSO is a commonly used method for HIV isolation (41). The effects of multiple freezing and thawing of serum specimens on acid nucleic stability, however, must be taken into account. Plasma stored for up to one year at −70°C showed quite stable

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levels of HIV RNA (42, 43) and viral DNA from TT virus, and HBV was still valuable by qualitative PCR after seven cycles of freezing and thawing (44). Viruses can be also preserved for very long period as nucleic acids. RNA must be precipitated with ethanol, which inhibit the enzymes that breakdown RNA, while DNA can be stored either under ethanol or as dried pellet. This method has several advantages since virus as nucleic acid can be frozen in extremely small volumes, in many aliquots, without the need for large volumes of storage capacity and almost indefinitely, but is not widely used.

15. Storage of Genetically Modified Microorganisms

Genetically modified organisms are widely used for both research and industrial production purposes and consequently regular programs of long-term viability, good preservation, and plasmid retention testing are important requirements. In general, such microorganisms can be preserved in a manner similar to the unmodified host cell, in most of cases (45, 46). In a recent study, cultures of recombinant E. coli strains, cryogenically frozen and stored at −80°C, showed stable viability and high plasmid retention over a period of up to 11 years; lower viability and/or plasmid retention may be likely due to the improper selection of initial colonies (47). The absence of gross structural instability of transfected sequences might be verified at least by restriction patterns analysis of the strains. Different parameters for the preparation and cryopreservation of recombinant microorganisms need to be considered, as suggested for recombinant cultures of Saccharomyces cerevisiae (48). Cryoprotectant concentrations of 2–5% glycerol in water resulted in optimal pre- and post-freezing recovery rates; the addition of the amino acids in the cryoprotectant media appeared to have a protective effect during deep freeze storage. Storage temperatures of −70°C or below and rapid thawing allows good recovery rates.

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20. Hippe, H. Maintenance of methanogenic bacteria. In: Kirsop/Doyle, Eds. Maintenance of Microorganisms and Cultured Cells, II Edition. Academic Press, London (1991). 21. Aulet de Saab, O.C., de Castillo, M.C., de Ruiz Holgado, A.P., de Nader, O.M. (2001) A comparative study of preservation and storage of Haemophilus influenzae. Mem. Inst. Oswaldo Cruz. 96, 583–6. 22. Votava, M., Stritecka, M. (2001) Preservation of Haemophilus influenzae and Haemophilus parainfluenzae at −70 degrees C. Cryobiology 43, 85–7. 23. Gorman, R., Adley, C.C. (2004) An evaluation of five preservation techniques and conventional freezing temperatures of −20 degrees C and −85 degrees C for long-term preservation of Campylobacter jejuni. Lett. Appl. Microbiol. 38, 306–10. 24. Furr, P.M., Taylor-Robinson, D. (1990) Long-term viability of stored mycoplasmas and ureaplasmas. J. Med. Microbiol. 31, 203–6. 25. Ohkusa, T., Miwa, H., Endo, S., Okayasu, I., Sato, N. (2004) Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures. J. Gastroenterol. Hepatol. 19, 200–4. 26. Shahamat, M., Paszko-Kolva, C., Mai, U.E., Yamamoto, H., Colwell, R.R. (1992) Selected cryopreservatives for long term storage of Helicobacter pylori at low temperatures. J. Clin. Pathol. 45, 735–6. 27. Spengler, A., Gross, A., Kaltwasser, H. (1992) Successful freeze storage and lyophilisation for preservation of Helicobacter pylori. J. Clin. Pathol. 45, 737. 28. Miyamoto-Shinohara, Y., Imaizumi, T., Sukenobe, J., Murakami, Y., Kawamura, S., Komatsu, Y. (2000) Survival rate of microbes after freeze-drying and long-term storage. Cryobiology 41, 251–5. 29. Ryan, M.J., Jeffries, P., Bridge, P.D., Smith, D. (2001) Developing cryopreservation protocols to secure fungal gene function. Cryo Letters 22, 115–24. 30. Smith, D., Thomas, V.E. (1998) Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi. World J. Microbiol. Biotechnol. 14, 49–57. 31. Smith, D. (2001) Provision and maintenance of micro-organisms for industry and international research networks. Cryo Letters 22, 91–6. 32. Crespo, M.J., Abarca, M.L., Cabanes, F.J. (2000) Evaluation of different preservation

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33.

34.

35.

36.

37.

38.

39.

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Please expand the genus names at first occurrence in the chapter.

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