Can Agar cause problem in glycerol stock? I have this specific doubt as I am not getting good bacterial growth upon revival of Clostridium perfringens glycerol stocks.
How are you preserving your stocks? -20 or -80 ºC?
What is your glycerol conservation percentage? 20% or 40%?
Personally I don't have any problems whenever I need to revive my Clostridium stocks. I always preserve them at -20ºC in buffered peptone water + 40 % glycerol. May I also ask you with what specie of Clostridium are you working with? I am asking you this because some Clostridum species are very sensitive to oxygen and this might also influence the percentage of CFU recovered.
#carla carneiro
I am working on clostridium perfringens.For glycerol stocks I am growing on BHI broth and I am taking cultures directly from there to make a 20% glycerol stock.I am preserving the cultures in -20C . I used very small amount of agar agar in the broth so that less oxygen gets dissolved.This is what I have added extra.
You shouldn't have any problem recovering your strains because everything sounds OK to me. I don't add any agar agar to my medium but I don't think that is the problem at all. How many times have you defrosted your stock samples? If your using it more than once it is possible that consecutive defrost may lead to lower recovery percentages.
#Carla
Another possible reason may be the time I am keeping for broth culture. Generally I grow them in small 1.8ml cryovials and I keep them for overnight. I doubt by that by that time very less bacteria remains in viable state.Growing in 1.8ml cryovials with little bit of agar agar does not require Anaerobic jars.
That might be a problem. Clostridium do grow very fast indeed and 1,8 ml sounds a very little amount of medium (well it depends how much you inoculate and the replication rate of the isolate off course...). If you're trying to avoid anaerobic jars , maybe instead of BHiB you should try thioglycollate medium instead which is a semi solid medium and does not need to be incubated in anaerobiosis.
If I were you I would simply increase the volume of broth.
Never.. as C.perfringens is a strict anaerobic pathogenic bacteria it should be required a anaerobic condition to maintain its culture preservation and it was done by using glycerol stock over semisolid agar containing medium.But the concentration of stock is always maintain at about 20 %......
Keeping plates at -70ºC in 40 % glycerol is best solution. Glycerol stock over semisolid agar containing medium if keep at 4 0C release water plate become more humidified.When a lyophilized bacterial strain is purchased, a small portion of the powder is transferred with a sterile pipette, tip or inoculating loop to liquid LB medium and incubated for 2-8 hours in a shaker. A drop of liquid culture is spread on a selective plate and propagated overnight at 37°C to check for presence of selective markers. Select a single colony and grow overnight, Add 180 µl of 87% sterile glycerol to a 2 ml screw-cap culture vial, 5. Add 820 µl of liquid E.coli culture to vial, mix well, freeze in liquid nitrogen and store at -70°C.
I freeze C perfringens in Skin milk, after growing 24-48h in blood agar, but if you want to keep it for very long time meat cooked medium is the best. I recovered C perfringens several years after in this medium.
Luis Llanco at Which temp. did you keep isolates in meat cooked medium?
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