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LABORATORY PROCEDURES FOR MICROORGANISMS

Appendix

M/1998/3.00 Appendix 3


PROTECTIVE SUSPENSION MEDIA FOR FREEZING OR (FREEZE)-DRYING

For the protection of cells against damage during freezing and (freeze)-drying as well as during storage microorganisms have to be suspended in a protective suspension medium The composition of these media may differ with the type of organism to be preserved. Some examples of protective suspension media are given below.

For sterility control and use of protective media for preservation see examples given in M/1998/3.00 Appendix 4 and M/1998/3.00 Appendix 5.08.2.

Composition of suspension media for freezing

1 Bacto Nutrient Broth (Difco 0003) 0.8 g
Glycerol 20.0 g
Distilled water ad 100.0 ml
5 ml amounts are distributed in tubes, plugged with cotton stoppers and sterilized at 121°C for 20 min.

2 Bacto Nutrient Broth (Difco 0003) 0.8 g
Distilled water 95.0 ml
Dimethylsulphoxide 5.0 ml
5 ml amounts are distributed in tubes, plugged with cotton stoppers and sterilized at 121°C for 20 min.

3 Any medium, with 5% DMSO
Medium to grow a specific strain, sterile 5.0 ml
Dimethylsulphoxide, sterile 0.27 ml
DMSO (undiluted) is sterilized as appropriate aerobically or anaerobically at 115°C for 10 min

4 Any medium, with 10% DMSO
Medium to grow a specific strain, sterile 5.0 ml
Dimethylsulphoxide, sterile 0.55 ml

DMSO (undiluted) is sterilized as appropriate aerobically or anaerobically at 115°C for 10 min

5 Any medium, with 10% resp. 15% or 20% glycerol
Medium to grow a specific strain, sterile 5.0 ml
Glycerol, sterile 0.55 ml resp. 0.83 or 1,10 ml
Glycerol (undiluted) is sterilized as appropriate aerobically or anaerobically at 121°C for 15 min

For anaerobic bacteria protective media 3 to 5 should be freshly prepared. Just before use glycerol is added. The cryoprotectant-containing medium is then reduced by adding appropriate amounts of cysteine hydrochloride and sodium sulphide from anaerobic stock solutions. Sterile dimethylsulphoxide (DMSO) is added only after the medium has been reduced.

6 10% glycerol
Glycerol (Merck, 87%, p.a.) 287.3 ml
Distilled water 2212.7 ml
Sterilize 15 min at 120°C

7 10% DMSO with 8% glucose
DMSO, sterile 10.0 ml
Glucose 7.92 g
Distilled water 89.0 ml

DMSO (undiluted) is sterilized as appropriate aerobically or anaerobically at 115°C for 10 min

Composition of suspension media for (freeze)-drying

8 Nutrient Broth (Difco 0003) 0,8 g
myo-Inositol1 5.0 g
Distilled water 100 ml
1 myo-inositol may be replaced by other protective compounds

9 Skim Milk, spray dried (Difco 0032) 20.0 g
Distilled water 100 ml
Ampoules are filled with 0.5 ml skim milk and sterilized at 115°C for 13 min. The milk is then freeze-dried to obtain "predried milk plugs". For preparation and sterility control see M/1998/3.00 Appendix 5.11.2

10 Skim Milk, spray dried (Difco 0032) 20.0 g
myo-Inositol1 5.0 g
Distilled water 100 ml
1 myo-inositol may be replaced by other protective compounds myo-inositol may be replaced by other protective compounds

Ampoules are filled with 0.5 ml skim milk and sterilized at 115°C for 13 min. The milk is then freeze-dried to obtain "predried milk plugs". For preparation and sterility control see M/1998/3.00 Appendix 5.11.2

11 Skim Milk, spray dried (Difco 0032) 20.0 g
Distilled water 100 ml
5 ml amounts of milk are distributed in tubes, plugged with cotton stoppers and sterilized at 115°C for 13 min. The milk is then used for the preparation of bacterial suspension.

1212% Skim Milk (Elk, DMV Campina B.V.)
Skim Milk 27.0 g
Distilled water 324 ml
Tyndallize

13 12% Skim Milk (Elk) with 7% trehalose
Skim Milk 27.0 g
Trehalose 22,7 g
Distilled water 324 ml
Tyndallize

14 12% Skim Milk (Elk) with 20% trehalose
Skim Milk 27.0 g
Trehalose 64.8 g
Distilled water 324 ml
Tyndallize

15 12% Skim Milk (Elk) with 7% trehalose and 5% Na-glutamat
Skim Milk 27.0 g
Trehalose 22,7 g
Na-glutamat 16.2 g
Distilled water 324 ml
Tyndallize

16 Chopped meat medium (without meat) 100 ml
Sucrose 20 g
Distilled water 100 ml
The medium is prepared according to the description given in the VPI Manual (1977). 5 ml amounts are distributed under N2 to Hungate tubes and sterilized at 121 °C for 20 min.

For the preservation of anaerobic bacteria, tubes with 5 ml medium (in anaerobic tubes) are supplemented by 0.5 ml of a suspension of FeS.

17 ‘Mist. desiccans’
Horse serum 100 ml
Nutrient Broth (Oxoid CM1) 33 ml
Glucose 10 g
Sterilize by pressure filtration (membrane or Seitz filter). The mixture is dispensed in 5 ml amounts in sterile screw-capped tubes and incubated at 30 °C for 3 days as a sterility check. ‘mist. desiccans’ is stored at 20°C until used.


Guidelines prepared for CABRI by DSMZ, CBS and BCCM, 17 May 1998
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