Hello!
I do not know the best way to lyophilize Pasturella multocida for shipping. It has been grown in TSB (15% glycerol) and is sitting in a 1ml cryovial frozen at -80. The procedure is below. Can someone help me get the easiest procedure for growing the bacteria in a solution that can be freeze dried while keeping the cells alive for shipment to another country?
Thank you!
- 5-20 live individual Pasturella multocida colonies were collected from a Blood Agar plate
- The loop was swirled around in a culture tube with 4mls of TSB (Trpticase Soy Broth plus 15% glycerol)
- The culture tube was incubated for 18-24hours then gently vortexed
- 1ml was aliquoted into four 2ml cryovial tubes and labeled
- These isolates were placed in our -80 freezer
Jamille
Hi Jamille,
The procedure you mentioned is perfectly fine for maintaining a glycerol stock. I would suggest you to adopt the following procedure for lyophilization of P. mutocida culture :
1. Just harvest the culture tube (18 hours growth) by means of centrifugation.
2. Resuspend the pellet in 10-15% skim milk solution.
3. Transfer the skim milk containing culture into sterile ampoules/ vials. If ampoules are being used one small sterile cotton ball should be inserted into the ampoule. The amount should be very less. Say for ampoules it may be 100-250 µl and for vial of 3 ml capacity you may keep a volume of 0.5 ml to 1 ml.
4. Lyophilize the culture. Freezing should be done for at least 8 hours and primary drying for at least 10 hours and secondary drying for 6-8 hours.
5. If using ampoules seal them using flame where as for vials seal with bungs and flip-off seal.
6. test the vacuum in case ampoules are being used using a vacuum sealing tester. It should be done it the upper most part only above cotton ball.
7. Now you can send your ampoules/ vials to any country where ever you wish.
Good luck!
Thank you so much Gaurav!
I do not have any ampoules so I will centrifuge 2ml's of the TSB culture, decant the TSB broth and bring up in 1ml of 10%milk. After freeze drying what should the samples be re-suspended in? Back into the milk? or TSB broth again?
Another researcher said I should grow them in Casein Sucrose Yeast broth with 10% skim milk. If you think the milk alone will work fine I will go with that and not complicate it anymore.
Let me know your thoughts
Thank you!
Jamille
Hi Jamille,
Once you have lyophilized the bacterial suspension you can reconstitute using the same media you were using earlier for its propagation i.e. TSB.
10% skim milk will suffice your requirement for sure. but if you want to include Casein sucrose Yeast broth you can.
Basically this is ATCC formulation 0.75% Tryptic Soy broth, 10% Sucrose, 5% BSA. But since BSA can not be autoclaved so sterilize it by terminal filtration. But my suggestion will be same, do not complicate things too much. you can proceed with 10% skim milk only.
Ampoules would have been a better choice but since if you have not a facility you can proceed with vials.
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