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Enrichment Isolation Cultivation and Preservation of Bacteria
Enrichment Isolation Cultivation and Preservation of Bacteria
Preservation of Bacteria
JAN C.GOTTSCHAL
WIMHARDER
RUDOLF A.PRINS
) of Bacillus subtilis had a strong selective advantage over the nonrequiring spontaneous revertant (his
+
)
when grown together in a chemostat in the presence of histidine.
The carryover of genetic information in mixed cultures with strains of Pseudomonas species either capable of using 3-chlorobenzoate but not
4-chlorobenzoate, or containing the TOL plasmid coding for a benzoate-1,2-dioxygenase with broad substrate specificity, has been the principle behind the
experiments of Knackmuss and colleagues when selecting for exconjugants capable of growing on 3- and 4-chlorobenzoate.
Instability of chimeric plasmids encoding commercially important foreign proteins may result in the loss of the inserted genes during culture, a problem in
many biotechnological applications. Fleming et al. (1988) were able to improve the plasmid-stability characteristics of a strain of Bacillus subtilis carrying a
chimeric plasmid (derived from pUB110) encoding chloramphenicol and kanamycin resistance and high-temperature -amylase by growing the organism in
the chemostat in the presence of rising levels of chloramphenicol. Interestingly, the improved strains with higher plasmid stability isolated at the end of the
chemostat run had almost three times higher maximum growth rates as compared to the original strain, which was outcompeted both in batch and in
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chemostat culture. Meinecke et al. (1984) used the chemostat for the isolation of asporogenous strains of Clostridium acetobutylicum. A method for detecting
mutants with altered adhesion ability was proposed by Cowan and Fletcher (1987).
Conservation of Bacterial Cultures
The enormous growth of biotechnology and various branches of microbiology has resulted in an increased interest in techniques for the maintenance and
long-term preservation of cultures.
The maintenance of bacterial cultures usually requires propagating working cultures by subculture and keeping strains of bacteria alive and growing in the
course of investigations or in order to provide inocula of standard strains for industrial processes, certain assays, and tests, and routine investigations. For
long-term maintenance, cultures are usually stored in a lyophilized or deep-frozen from to prolong their viability and to reduce changes due to the occurrence
of mutations. A practical book with information on various techniques and procedures from subculture to storage in frozen nitrogen and freeze-drying is
Kirsop and Snell (1984). The book is an extension of the courses given by the UK Federation for Culture Collections and as such reflects the techniques used
in the United Kingdom.
Maintenance of Working Cultures
Usually, maintenance of working cultures requires the periodic transfer of strains to fresh minimal media, streaked on cotton-plugged agar slants (aerobes) or
stabbed in agar deeps in screw-capped tubes (facultative anaerobes, microaerobes, anaerobes). The cultures can then be incubated and then are stored in the
refrigerator in the dark at 58C to reduce metabolic activity, while maintaining viability. Frequency of transfer should be kept to a minimum, but it has to be
separately determined for each organism: some require transfer after just a few days, while some sporeformers can survive for years. Drying of the agar must
be prevented. To avoid selection, it is advisable to subculture from the whole plate or slant and not just from one colony. For different physiological groups
of bacteria, specific maintenance requirements exist. Working cultures of rumen bacteria and other fermentative anaerobes can be maintained in slants or
sloppy agars (0.71.2%, w/v agar) prepared from nonselective media. The cultures are incubated at 39C until growth is just apparent and then stored at 4C.
Such cultures remain viable for a few weeks at least, although the viable count is decreased.
Lists of maintenance media can be found in culture collection manuals, such as of the Deutsche Sammlung fr Mikroorganismen. Some serious problems
that may be encountered in maintenance of strains are infection, mislabelling of tubes, and plates, mutation and genetic variation, and selection.
Preservation of Stock Cultures
The above-mentioned problems made it necessary to find ways for the long-term storage of bacterial strains in national and international culture collections.
Furthermore, long-term preservation of cultures is of importance to taxonomy, because it allows confirmation and extension of research findings. It is also
essential to many kinds of industrial applications, and it is mandated by patent laws and regulations.
A method of long-term preservation that can be followed with sporeforming bacteria is based on the use of sterile soil or sand. A sample of soil or sand is
sterilized in a screw-capped bottle by autoclaving for several hours on at least two successive days. Then, 1 ml of a suspension of the organism is added, and
the contents are dried in a vacuum desiccator with the cap of the tube loosely closed. When the contents are dry, the cap is closed tightly and the bottle is
stored in the refrigerator. Even easier is the use of sterile filter paper discs or strips soaked with the bacterial suspension. The discs are stored as described for
sand above. One may keep several discs in one screw-cap container and remove one disk at a time, taking care not to contaminate the remaining discs.
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Lyophilization.
Cultures maintained on agar slants at 4C or kept on oil still show slow growth and cell turnover with the potential of genetic and metabolic alterations.
Starter cultures for microbiological research or for processes in industry have to be maintained in a state that allows reproducible rapid growth upon
inoculation. Most strains therefore are stored frozen or as freeze-dried (lyophilized) cultures.
The organisms are placed in an ampoule with suspending medium and frozen at 60 to 80C, most often in a metal chamber with solid carbon dioxide and
ethanol. The water is then removed from the frozen state directly by sublimation in a vacuum. Freeze-dryers consist of a manifold connected to a vacuum
pump capable of reducing the pressure to less than 0.01 mm Hg. The ampoule is sealed while under vacuum and stored in a cool place. The preserved culture
weighs little, takes little space, and can easily be stored, handled, and mailed. The suspending medium is critical for the rate of survival during the
freeze-drying process and for the rate of death of dried bacteria during storage. Among the most commonly used suspending media are serum plus 30%
glucose nutrient broths and sterile skimmed milk fortified with 5% sucrose, sterilized in 3 ml amounts in bijou bottles.
A procedure for the freeze-drying of anaerobes is given by Phillips et al. (1975). A freeze-drying method suitable for the preservation of nitrogen-fixing and
other fragile bacteria was described by Malik (1988a). No loss of diazotrophy, plasmids, or other desirable qualities was observed. References for the
freeze-drying of several bacterial groups can be found in Malik (1988a, 1988b). A simple method for long-term preservation of luminous bacteria (often used
in microbial toxicity tests) has been described by Janda and Opekarov (1989).
Survival of the population during freezing and thawing depends on a number of factors (Calcott, 1986), but growth characteristics and pretreatment and
posttreatment manipulations are as important as the way cells are frozen and thawed. Each type of cell appears to exhibit its own set of optimal conditions for
maximum survival. Irreversible injury will lead to death or loss of viability. The last point is a subjective determination that depends as much on the choice
of the medium composition and incubation conditions (Postgate and Calcott, 1985). Reversible cell damage due to freezing and thawing includes altered
plating ability reflecting both structural and metabolic injury. Damage may occur to the cell's basic structure including the membrane, cell wall, cytoskeleton,
and DNA secondary structure. Damage to the metabolic machinery may include impairment of active transport, protein synthesis, and the induction of the
synthesis of inducible operons. Mutagenic effects of freeze-thaw stress are shown to be related to single-stranded breaks in DNA, an effect that might be
similar to that of ionizing radiation. The damage can be repaired in nutrient media. In Escherichia coli, both the uvrA-, polA-dependent (excision repair) and
the error-prone rec-, polA-dependent (recombinational repair) DNA repair pathways are required for repair of freeze-thaw-induced DNA damage.
Calcott et al. (1983; cited in Calcott, 1986) examined the incidence of plasmid loss from the survivors of freeze-thaw, namely the loss of plasmid RP4 from
Escherichia coli and the loss of plasmid pPL1 from Pseudomonas aeruginosa, two natural non-engineered plasmids known to be extremely stable under a
variety of growth conditions. Freezing and thawing did cure the strains at a low rate of loss in a dose-dependent manner. Frozen storage of the population
itself, although detrimental to survival, did not increase the incidence of plasmid loss. When populations were protected by either glycerol or sucrose viability
of the preparation remained high with minimal loss of plasmids from the culture. A percentage of the survivors of freeze-thaw stress may show metabolic
injury, i.e., the loss of the ability to form colonies on a minimal salts-glucose (poor) medium. This was studied in Escherichia coli and was shown not to
be due to a transient requirement for a particular nutrient that was absent, but rather due to a perturbation of the cell control systems. Cyclic GMP and ppGpp
singly or in combination partly restored the efficiency of plating on a minimal medium of frozen-thawed cells.
Effects of specific harmful agents and potential damage sites in the microbial cell are reviewed by Hurst and Nasim (1984), while Andrew and Russell (1984)
report on problems associated with enumerating and recovering microbes exposed to various forms of stress.
Freezing and Ultrafreezing
For the preservation of cultures of rumen bacteria for up to one year at 20C, Teather (1982) recommended freezing after the addition of 20% (v/v) glycerol
as a cryoprotectant to cultures that have been incubated at 39C for 1224 h. Long-term storage under liquid nitrogen (196C) in the presence of
6. Principles of Enrichment, Isolation, Cultivation, and Preservation of Bacteria
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dimethylsulfoxide is described by Hespell and Bryant (1981), who advocated rapid recovery of deep-frozen cultures by thawing in water at 3235C. Winter
(1983) devised a method for the long-term storage of methanogens as cell suspensions in glycerol contained in double glass vials. Joubert and Britz (1987)
described a method to freeze bacteria on assay discs sealed in glass ampoules.
Culture Collections and Shipment of Cultures
Culture collections and their role in biotechnology and microbiology are described by Lapage et al. (1970), Malik (1987), and Malik and Claus (1987). Iizuka
(1987) provides an overview of new microbial resources and world culture collections. Certain collections are very specific and deal only with bacteria from
a certain habitat, e.g., the Australian Collection of Antarctic Microorganisms (ACAM), or a certain taxonomic group of bacteria, e.g., the Oregon Graduate
Center Collection of Methanogenic Archaebacteria (OGC/CMA), Oregon Graduate Center, 19600 N.W. Von Neumann Drive, Beaverton, OR, USA).
Regulations and guidelines for packaging and requirements for labeling and shipping of cultures and biological materials are described in a reference manual
of the American Type Culture Collection (ATCC). These guidelines and regulations also describe restrictions on quantities and a list of countries that
prohibit movement of perishable biological substances through the postal service (Alexander and Daggett, 1988). Many diagnostic specimens (human or
animal excreta, secreta, blood, tissues, and tissue fluids shipped for diagnosis believed not to contain infectious substances) are exempt from the regulations
defined in the International Mail Manual (IMM 137,2). For International Postal Shipments, noninfectious biological substances that are freeze-dried
(nonperishable) have no special packaging and shipment requirements, except that a high-quality box must be used.
Drying or freeze-drying undoubtedly is the safest and most practical method of preparing specimens for shipment. In air transport, a package may be
subjected to a wide range of temperatures from 40 to +55C. Packages sealed at atmospheric pressure (100 kilopascals) may become subject to reduced
pressures as low as two-thirds of atmospheric pressure (60 kilopascals). Samples sent as liquids, e.g., in test tubes, should be heat-sealed in 5 ml of
polyethylene tubing before shipment, a good packaging practice for all infectious substances.
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