Bacterial culture - (Nov/06/2008 )
How to store bacterial culture
Which bacteria, how long and for what reason you want to store it?????
General: for short term storage: use para-film to close the petri dish and put it into a fridge (4°C).
for long term storage: use tubes which can be frozen at -80 °C or in liquid nitrogen, fill them with some liquid culture and add some glycerol; you can alternatively use ready to use systems (I have posted a link to those some time ago, just forgotten where).....
I store bacterial culture as follows: I grow them in a liquid to the density I would want to use them at, then I take 300 ul glycerol stock(50% glycerol, 50% sterile water) and add 600 ul bacterial cell suspension, and keep at -80C.
250ul of glycerol, pipet tips must be cut to make hole bigger for this to work , or u could opt for heating the glycerol to make it less viscous.
then add 750ul of bacterial culture to it . freeze at -80C. done for the day.
some people suggest me to grow the culture and make its pellet and then add 750 ul of LB media but with out antibiotics and into that 750 ul glycerol. Does this LB media after maiking the pellet should be with out antibiotics or with antbiotics.
what you think about this.
regards
then add 750ul of bacterial culture to it . freeze at -80C. done for the day.
then add 750ul of bacterial culture to it . freeze at -80C. done for the day.
we store at teh glycerol stock at -20 C rather than -80C , is that ok? But we have found that after sometimes the culture goes off, the pellet is not formed properly and plasmids cannot be ontained? Has that something to do with the storage temperetaure?
then add 750ul of bacterial culture to it . freeze at -80C. done for the day.
we store at teh glycerol stock at -20 C rather than -80C , is that ok? But we have found that after sometimes the culture goes off, the pellet is not formed properly and plasmids cannot be ontained? Has that something to do with the storage temperetaure?
There is a specific protocol for that. The bacteria reserves are called STAB.
After you made the mini, ore the midi, you take 850ul of the bacteria suspension and you mix it with 150ul of sterile glycerol in a steril 1.5ml eppendorf. Than you have to put the eppendorf in cold ice for at least 30 minutes. After the cold ice, you can keep the bacteria STAB at -80C FOREVER! When you need them, just scrape a small amount in sterile LB medium with the correct antibiotic and they will grow in 16 hours.
Bye bye.
then add 750ul of bacterial culture to it . freeze at -80C. done for the day.
we store at teh glycerol stock at -20 C rather than -80C , is that ok? But we have found that after sometimes the culture goes off, the pellet is not formed properly and plasmids cannot be ontained? Has that something to do with the storage temperetaure?
There is a specific protocol for that. The bacteria reserves are called STAB.
After you made the mini, ore the midi, you take 850ul of the bacteria suspension and you mix it with 150ul of sterile glycerol in a steril 1.5ml eppendorf. Than you have to put the eppendorf in cold ice for at least 30 minutes. After the cold ice, you can keep the bacteria STAB at -80C FOREVER! When you need them, just scrape a small amount in sterile LB medium with the correct antibiotic and they will grow in 16 hours.
Bye bye.
Thank you , i will try this onwards.
Is that ok for all kinds of bacteria ?i am using E.Coli
then add 750ul of bacterial culture to it . freeze at -80C. done for the day.
we store at teh glycerol stock at -20 C rather than -80C , is that ok? But we have found that after sometimes the culture goes off, the pellet is not formed properly and plasmids cannot be ontained? Has that something to do with the storage temperetaure?
There is a specific protocol for that. The bacteria reserves are called STAB.
After you made the mini, ore the midi, you take 850ul of the bacteria suspension and you mix it with 150ul of sterile glycerol in a steril 1.5ml eppendorf. Than you have to put the eppendorf in cold ice for at least 30 minutes. After the cold ice, you can keep the bacteria STAB at -80C FOREVER! When you need them, just scrape a small amount in sterile LB medium with the correct antibiotic and they will grow in 16 hours.
Bye bye.
Thank you , i will try this onwards.
Is that ok for all kinds of bacteria ?i am using E.Coli
E.coli is the easy and hardy one . You should not have any problem with it.