If I want to make glycerol broth for my isolates, what is the best method to do that and what are the best proportions of the glycerol and the broth?
For bacteria, I have used 25-30% of glycerol and as broth Brain Heart Infusion (BHI). The protocol is very simple: prepare and sterilize tubs with 5 ml BHI, from the plates with your microorganisms take one single colony and resuspend it in BHI. After overnight incubation add 500 μL of the overnight culture to 500μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently vortex for homogenization. The 50% glycerol should be prepared with distillate water and autoclave too. What type of isolates do you have? Because you need to chose the right broth.
For bacteria, I have used 25-30% of glycerol and as broth Brain Heart Infusion (BHI). The protocol is very simple: prepare and sterilize tubs with 5 ml BHI, from the plates with your microorganisms take one single colony and resuspend it in BHI. After overnight incubation add 500 μL of the overnight culture to 500μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently vortex for homogenization. The 50% glycerol should be prepared with distillate water and autoclave too. What type of isolates do you have? Because you need to chose the right broth.
Dear Andrei Bolocan,
I use the same technique as described by Mohamed Sebak. However, by mixing 50% glycerol (prepared in destilled water) 1:1 with your culture, the medium is diluted as well. Thus, I either prepare 50% glycerol with 2 mM MgCl2 (as it is common for E. coli) or artificial seawater/freshwater (salts only, pH adjusted).
I additionally recommend to prepare triplicates and store them in different -80°C freezers.
All the Best
Dear Richard Hahnke,
Thank you for the information about the preparation of 50% glycerol. I used the presented tehnique just for patogenic bacteria as L. monocytogenes and Staphylococcus aures. For lactic bacteria I used MRS broth to replace BHI. All the time I try to use a good enrichment broth.
I used for some strains the tehnique with beads in cryo fluid, but I dont know if it is a semnificative difference between the quality of these methods. Even if, the tehnique with beads is more simple, the costs are the problem, because it is a little bit too expensive.
All the best
Are you interested in examining the bacterial isolates for their ability to utilize glycerol as sole carbon source or for storing them under -70. if it is for storing, I use 15% glycerol for preserving bacteria (E.coli, Lactobacillus, Pseudomonas, Paracoccus, Bacillus). I take 0.75ml of culture at mid log phase and mix with 0.25ml of autoclaved or filtered glycerol (60%). Store them under -70 C.
Dear all,
Can I use BHI + 50% glicerol for preservation of Brucella spp? Do you have better idea?
Hi everyone,
I wanted to preserve Klebsiella pneumoniae, Rhodococcus equi and Mycobacterium tuberculosis. What is the best technique to preserve these bacteria for a longer period of time? Also, I wanted to know how long we can preserve them. Please specify preservation conditions as well if possible. Thanks Thanks
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