LABORATORY PROCEDURES FOR MICROORGANISMS
M/1998/3.00 Appendix 5.05
PRESERVATION OF BACTERIA BY FREEZING AND LOW TEMPERATURE STORAGE BELOW -70°C ON GLASS BEADS A wide range of aerobic and facultative anaerobic bacteria may survive long-time storage at -70°C to -80°C especially if repeated freezing and thawing is prevented. Feltham et al. (1978) and Jones et al. (1991) described a method based on the use of frozen bacterial suspensions on glass beads. Individual beads may be removed without thawing the whole sample. Alternatively bacterial suspensions frozen in glass capillaries (
PRESERVATION OF BACTERIA BY FREEZING AND LOW TEMPERATURE STORAGE BELOW -70°C ON GLASS BEADS
A wide range of aerobic and facultative anaerobic bacteria may survive long-time storage at -70°C to -80°C especially if repeated freezing and thawing is prevented. Feltham et al. (1978) and Jones et al. (1991) described a method based on the use of frozen bacterial suspensions on glass beads. Individual beads may be removed without thawing the whole sample. Alternatively bacterial suspensions frozen in glass capillaries (see M/1998/3.00 Appendix 5.01) may be stored below -70°C.
The method may be especially useful for preserving pathogenic bacteria since the risk of explosion of vials or capillaries is excluded while removing cultures from a -70°C/-80°C freezing unit.
Pell and Sneath (1984) reported that bacteria frozen on beads may survive a breakdown of the freezer for one or several days.
For documentation of preservation details and viability checks protocol formM/1998/3.00 Appendix 5.01.1 is used.
1 Preservation of beads
1.1 Preparation of beads and vials
2 mm glass embroidery beads are washed in tap water with a detergent, followed by dilute HCl to neutralize alkalinity. The beads are then washed several times in tap water until the pH of the wash water is that of tap water. The beads are finally washed in distilled water. They are then air dried. Glass beads of different colours may be used to differentiate various groups of bacteria.
About 30 beads are placed in 2 ml screw-cap glass vials or screw-cap polypropylene ampoules. The vials are loosely capped and sterilized by autoclaving at 121°C for 20 min. in a beaker, covered by a layer of felt.
1.2 Preparation of suspension medium
10 mI quantities of 15% (v/v) glycerol in nutrient broth (Difco Laboratories) are prepared in tubes and sterilized by autoclaving at 121°C for 20 min. For extremophilic bacteria appropriate growth medium plus 15% (v/v) glycerol has proved satisfactory.
1.3 Labelling of vials
The appropriate culture collection number, the date of freezing and any other pertinent information is written on a self-adhesive label and stuck on the side of each glass vial. The labels may be further secured by wrapping a layer of clear sticky tape around each vial. Polypropylene ampoules are may be directly marked with a cryomarker. To facilitate the retrieval of an required vial or ampule the top of the cap may be colour coded.
1.4 Cultivation of organisms
Bacteria are grown on agar plates with the medium recommended for the strain and at their optimum temperature. If liquid cultures are used for growth, cells have to be centrifuged before suspending in protective medium.
1.5 Preparation of suspension for freezing
Fast growing strains are harvested after about 24 hours of growth. 1 ml (0.5 ml if growth is less vigorous) of the appropriate sterile suspending medium is aseptically pipetted onto the plate. Using a wire loop, the growth is emulsified with the broth to make a suspension as thick as possible.
1.6 Distribution of suspension
With a sterile Pasteur pipette the bacterial suspension is aseptically dispensed into one of the prepared vials. The suspension should be aspirated several times to ensure the air bubbles inside the beads are displaced by the bacterial suspension.
After the beads are thoroughly wetted, the excess suspension should be removed from the bottom of the vial. Excess suspension left in the vial makes it more difficult to remove individual beads when required after storage.
1.7 Freezing and storage of cultures
The vials or ampules are placed in trays of suitable size (for examples Nalgene CryoBoxes 5025-0505). The trays are placed in a commercial freezer capable of maintaining temperatures of -70°C.
Removal of single beads after storage is facilitated if the vials are tapped onto the side of the vial before freezing in the upright position.
Nalgene CryoBoxes have a numerical grid system molded into the box and printed on lid to keep track of cryovial inventory. The box is keyed in one position so grids always match. For documentation of the number of cultures stored in each Nalgene CryoBox protocol form
1.8 Recovery of frozen bacteria
The vial is removed from the cryobox and one bead removed using a sterilized mini-spatula. The vial should be replaced immediately to prevent the remaining contents from thawing. The bead is transferred to a suitable solid medium and moved on the surface of the plate back and forth with a wire loop so that the bacterial inoculum is released. The plate is then incubated under appropriate conditions.
2 Preservation in glass capillaries or polypropylene straws
2.1 The procedure of preparing cell suspensions, filling of capillaries/straws is the same as described inM/1998/3.00 Appendix 5.01 for glass capillaries and M/1998/3.00 Appendix 5.02 for straws.
2.2 Freezing and storage of capillaries or straws is described under 1.7 above.
2.3 Recovery of frozen bacteria is described inM/1998/3.00 Appendix 5.01 for glass capillaries and M/1998/3.00 Appendix 5.02 for straws.
Feltham et al. 1978. J. Appl. Bacteriol. 44, 313-316.
D. Jones et al. 1991. Maintenance of bacteria on glass beads at -60°C to -76°C. In: Kirsop and Doyle (eds.) Maintenance of microorganisms and cultures cells, 2nd edition, p. 45-50. Academic Press.
P.A. Pell and P.H.A. Sneath. 1984. J. Appl. Bacteriol. 57, 165-167.
Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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