I want to make -80C stocks of Legionella species. Is there some special protocol or can I simply use 25% glycerol which we often use for other Bacterial species?
Why 25 % ? You can use the protocol below and still get good recovery after prolonged time !
Robert
Best
Prepare a solution of 30% glycerol (v/v) by mixing 30 ml of glycerol with 70 ml of water. Transfer the solution to a screw cap glass bottle and sterilize by autoclaving at 121°C for 15 min. Loosen the cap during autoclaving.
Aliquot 500 µl of sterile 30% glycerol into sterile 2 ml sterile microfuge tubes containing 4 mm glass beads*.
Add 500 μl of bacterial culture to the tube and mix with the glycerol using a vortex mixer.
Label the tube with the organism name, strain, date, etc.
Place the tube in the freezer and record its location.
I agree with Robert - 15% glycerol final concentration is fairly standard for bacteria stocks
The ISO 11731 method for legionella has specific instructions about it
6.3.3.4 Glycerol broth
Dissolve 5 g of a commercially available dehydrated nutrient broth in 170 ml of water (6.1) and add 30 ml of glycerol. Mix well and dispense in clean, dry silica-glass bottles in volumes of 2 ml. Sterilize by autoclaving at (121 ± 3) °C for (20 ± 1) min. Store at room temperature until required.
In the past I had good results with skim milk
best regards
@ Francesc Codony!
Is it possible to get a copy of the document which you have mentioned in your post?
Because there are some points which are still not clear.
@Robert Armon!
Is there some specific reason behind the use of lower concentration of glycerol?
Thanks
Hi there,
a cheap and easy alternative that I've used is to suspend in sterile skimmed milk!
hope this helps,
cheers
Richard
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