We are working with Bordetella pertussis and we have some problems to preserve them at -80 ºC. When we try to recover it, all bacteria seems to be dead. We are using skimmed milk, as with the other bacteria we commonly work with. Could you give me any tips?
Try FBS supplemented with about 5% inositol. You can substitute sucrose for inositol, but you will need to use 10% sucrose. You might also want to try freezing them down slowly. Use a freezing chamber to control the rate of temperature drop.
I hope this helps.
CM
you should try in skim milk. We use it instead common glycerol technique. Once you have a clean pellet, just wash it in sterile distilled water, and then put 10% skim milk solution and freeze it at -80°C. We had very good result with it. Have a nice day !
You should use in 15% skim milk because we used this method for pertussis and our culture is good last 5 years.
Bordetella pertussis is a bit tricky and heat labile organism. You can use 15-20% of sterile skim milk and subject it to freeze drying for long term storage. Apart from skim milk you can also use a receipe with a combination of stabilizers and preservatives such as Dextran solution (ready-made), Glutamic acid and Sucrose and then freeze drying. Revive the freeze-dried culture on Bordet-Gengou agar medium supplemented with 15% of sheep blood.
I did freeze drying with the second receipe for long term storage of four serotypes of B.pertussis for vaccine production.
1. Swab several colonies of bacteria from the agar plate using a Dacron swab.
2. Transfer bacteria to a 2-ml cryotube containing 1 ml of Stainer-Scholte broth + 15%
(v/v) glycerol. Immediately put tube in -80°C freezer.
3. Once stock is frozen, do not thaw. To avoid thawing, keep stock tube on ice when
streaking new plates, and return immediately to -80°C freezer.
It is advisable to make a backup stock of each strain in case contamination
occurs, and to have a stock that has not been partially thawed and refrozen many
times.
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