Microbial culture preservation

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elizafeb11
elizafeb11's picture
Microbial culture preservation

For bacterial preservation, which percentage of glycerol stock is required at which temperature,say for example at 30% glycerol stock , it is stored at -20, so is there any condition that a particular percentage of glycerol can be store in a particular temperature ?? 

Arvind Singh Pundir
Arvind Singh Pundir's picture
Hi eliza

Hi eliza
there are different conditions varying slightly on species and certain condition for storage of stock in glycerol (cryoprotectant) here are are some refernce for  hope they will guide you

When making a healthy frozen glycerol stock, three factors that hold significant
weight are the phase of growth in which the bacteria are frozen, the percentage of
glycerol concentrated in the frozen solution, and the rate at which the stocks are frozen.
Ideally the bacteria should be in late exponential phase growth, and suspended in 30%
glycerol, 70% photobacterium broth solution, at which point they should be dipped
directly into liquid nitrogen (-196oC) so that they freeze solid in under 15 seconds.
Including glycerol in the solution prevents the formation of ice crystals that could
damage the bacterial cells during the freezing process www.phys.ufl.edu/REU/2007/reports/johnson.pdf
THE PRESERVATION OF BACTERIA BY FREEZING IN GLYCEROL BROTH www.pubmedcentral.nih.gov/picrender.fcgi
Improved Preservation of Treponemna pallidum and Other Bacteria by Freezing with Glycerol www.pubmedcentral.nih.gov/picrender.fcgi
PRESERVATION OF BACTERIA BY FREEZING AND LOW TEMPERATURE STORAGE BELOW -70°C ON GLASS BEADS www.cabri.org/guidelines/micro-organisms/M300Ap505.html
 
 

heehawmcduff
heehawmcduff's picture
I've stored bacteria at -40oC

I've stored bacteria at -40oC in a 10% milk solution and they have recovered well.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi elizafeb11,
The standard concentration of glycerol for bacterial cultures is as follows: final concentration of 15% of glycerol for storage at -80oC. If you add less the cells will likely lyze during the freezing process. If you add more this may lead to some growth inhibition later on.

elizafeb11
elizafeb11's picture
suppose, i have prepare 30%

suppose, i have prepare 30% glycerol stock, then i have taken 750microlitre of culture broth and 750 microlitre of the 30% glycerol stock, so that is to make 1:1 combination to add in 1.5 cryo vial,so is there a chance of inhibition or no growth and again at what temperature i shud store

Ivan Delgado
Ivan Delgado's picture
 

 
Mixing 1:1 would give you a final concentration of 15% glycerol, so you are perfect. No chance for inhibition. You should store at -80oC (minus 80 degrees Centigrade)

JimAch
JimAch's picture
Hi Eliza,

Hi Eliza,

Not sure about other temperatures but in liquid nitrogen 12-15% is the usual concentration, for yeasts & fungal spores/hyphal fragments this works well.

Jim