I am trying to preservea pure culture of some bacteria in glycerol but after preserving when I am trying to re isolate it, I am not able to find the same bacteria.
Generally 10-20% Glycerol works just fine. If its a pure culture and you are not getting any growth you should make sure you freeze as carefully as possible to protect the bacteria during freezing. Try using something like a Mr Frosty that allows for a more uniform cooling rate.
Yes its completely a pure culture but is there is any possibility of contamination?
If you are freezing a "pure culture" and then you dont get the same pure culture back, something is definitely contaminated. Freezing Media, tubes or plates could be contaminated. Either that or it wasnt pure to begin with.
What is your parameter to say it is the same bacteria or not? Biochemical tests/molecular analysis or based on some metabolite production? Sometimes depending on the conditions and the species, stored bacteria or isolates that have been subcultured "for too long" may loose their ability to produce certain substances.
Also, sometimes we believe a culture is pure, but there may have other slow growing bacteria mixed together and that couldn't be seen while plating because the dominant one overgrew. So, glycerol stock could be contaminated.
Check what Mr. Wood said and try to perform another reactivation of your stocks, this time using also a negative control (put everything used in the process, except your bacteria). And see if the case repeats and in both conditions. If it does, then your media or instruments are contaminated. If it was only shown in the stock culture, then you could perform a molecular identification (16S rDNA) comparing your activated cells and the original culture from which you prepared the glycerol stock and see if they are the same thing.
Best luck!
Freezing of bacteria in glycerol broth helps to store them. For that stab cultures are required to be made by sterilization of strain-compatible agar (like lysogeny broth agar for E. coli). Subsequently the agar can be transferred to screw cap vials using aseptic technique. After solidification of the agar it is necessary to pick a single colony from actively growing culture using a straight wire (sterile). The wire with the bacteria must be deeply plunged into the soft agar several times subsequently followed by incubation at 37°C for 8-12 hours . Tight sealing of the vial is then required which is followed by storing at 4°C.
For long time storage 5-15% (v/v) glycerol is required.
To activate bacteria, only a couple of beads need to be removed from the tube. The tube doesn't need to be thawed. Open the tube and loosen a couple of beads using a sterile pipette tip. Pour the loose beads onto an agar plate and roll them around. Colonies that develop should be re-streaked. Replace the tube into the freezer immediately. If the culture thaws, do not re-freeze it as cells are typically very sensitive to freezing and thawing. Discard the thawed culture appropriately.
Hi,
There are several protocols available on web sites. I am giving here few links.
http://www.thermoscientificbio.com/uploadedFiles/Resources/preparation-ecoli-culture-glycerol-stocks.pdf
http://web.mnstate.edu/provost/Bacteria%20Glycerol%20Stock%20Protocol%202012.pdf
http://www.oardc.ohio-state.edu/stockingerlab/Protocols/GlycerolStocks.pdf
http://www.addgene.org/plasmid_protocols/create_glycerol_stock/
Hope you will find useful.
Thanks,
Ajit
Thank you all
I have isolated the bacteria on selective media and conducted all the biochemical tests and preserve the bacterial culture in 20% glycerol. previously I used to preserve the culture and never found any contamination or anything. But this time may be there is some contamination.
Process for preservation of the bacteria in glycerol and re-isolate the bacteria.
- Follow the steps for Inoculating an Overnight Liquid Culture.
- After you have bacterial growth, add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix.
Note: Make the 50% glycerol solution by diluting 100% glycerol in dH20.
Note: Snap top tubes are not recommended as they can open unexpectedly at -80°C.
- Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life.
- To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria onto an LB agar plate.
- @@Grow your bacteria overnight at the appropriate temperature. Growth conditions, including copy number and growth temperature, can be found on your plasmid's information page. The next day you will be able to start an overnight culture for plasmid DNA prep the following day.
If I take bacterial isolates from a fresh culture plate and put it in to500 ul broth (glycerol broth/tryptica soy broth)media, then add 20% glycerol, would be good for bacterial preservation?
Sangjukta Roy It will work but there is slight growth required in the broth. You need to incubate it for sometime and can preserve it glycerol afterwards.
Thank You!!!
We need to preserve that bacterial isolates, nt need the growth, is the incubation obligatory step..otherwise..can we ignore the step, if I want preserve only?
Another type of bacterial preservation is using skimmed milk. It is good one.
add 10microleter from 24- 48 h bacterial culture to 20-50% sterlized glycerol in ependorf tube ,then keep it at -80 degree centigrate deep freezing
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