how to preserve staphylococcus in BHI broth- glycerol? - (Jul/27/2007 )
Hello,
I'm supposed to store some staph isolates for a long time in a brain heart infusion broth supplemented with glycerol at -70C. The bacteria is isolated on mannitol salt agar(MSA) . I'm not sure what to do next. Should i take an innoculum from the MSA culture directly and suspended it into the BHIB-glycerol mixture or should i incubate it first in the BHIB for 24 hours then freeze it? When do i add glycerol? Do i add it during the preparation of BHI broth from the dehydrated powder or do i add it at the end after inoculating the BHIB alone? Do i autoclave glycerol alone or after mixing with the BHIB? And, how can effective thawing be achieved, room temperature, water bath,...?
In general, glycerol freezer stocks are made by growing a liquid culture to OD 1 or so and then adding 10-20% (final) glycerol. The glycerol is autoclaved prior to addtion. It is convenient to dilute the glycerol to an 80% solution, which is much easier to pipet (less viscous) prior to autoclaving it.
In your case, you may just be able to add glycerol to the MSA culture and freeze it. If not, then you should inoculate a BHIB broth culture, grow it to late exponential phase, add glycerol and freeze it.
I try to never thaw freezer stocks. Instead, use a sterile loop to scrape a bit of ice from the freezer stock and spread it on a plate or inoculate a culture. Keep the stock on dry ice during this process if possible.
In your case, you may just be able to add glycerol to the MSA culture and freeze it. If not, then you should inoculate a BHIB broth culture, grow it to late exponential phase, add glycerol and freeze it.
I try to never thaw freezer stocks. Instead, use a sterile loop to scrape a bit of ice from the freezer stock and spread it on a plate or inoculate a culture. Keep the stock on dry ice during this process if possible.
Thank you. was very helpful. A colleague of mine does it like this: she isolates the bacteria on MSA. Then, in an Eppendorf tube she puts the desired amount of BHI and glycerol together (each previously sterilized). Then, she takes a few well separated colonies from the MSA and suspends them in the BHI-glycerol mixture till a heavy turbid solution is made and freezes them. Anybody tried it that way? Another thing, currently the deep freezer i'm using is set to - 50C, would that be a problem?
Thank u
Does it work if freeze it immediately with liquid nitrogen?
Unfortunately liquid nitrogen freezing isn't available
Hmm... you can try to just put them in -70 but it is not advisable as slow freezing can cause most of the bacteria to die during the freezing.
I've never had any problem with putting bacteria directly into the -80 freezer. I do typically let them adjust to the presence of glycerol for 1/2 hour before freezing them, but other in our lab omit that step and also have no problems.