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help me..how to cultivate bacteria from lyophilized cell

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help me..how to cultivate bacteria from lyophilized cell

Postby azarulez » Sun Nov 22, 2009 4:22 am

hi..I'm facing a big problem now. all of my bacteria glycerol stock are died.so, I cannot proceed my research. I only have one solution right now, to recover it from lyophilized cell. i have lots 3-6 months of lyophilized cell. i keep it at room temperature. so, could I cultivate it to get back my bacteria? what is the method and the best medium to cultivate bacteria from lyophilized cell? I hope all of you can suggest me the solution as soon as possible.

thank you..
Last edited by azarulez on Sun Nov 22, 2009 7:52 am, edited 1 time in total.
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Postby JackBean » Sun Nov 22, 2009 6:58 am

If are they really dead, you can't let them grow again.
If they are not all dead (what's probable), you should use appropriate media for your bacteria (E.coli?) with appropriate selection.
If they were all dead and you need some plasmid, they are harboring, I guess, and if you got enough stocks, you could try to purify and transform again...
http://www.biolib.cz/en/main/

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Re: help me..how to cultivate bacteria from lyophilized cell

Postby azarulez » Sun Nov 22, 2009 7:46 am

but i need that bacteria,not only plasmid to proceed my research..I heard some said that we can grow bacteria from lyophilized cell. but I need to know the method to cultivate the bacteria from lyophilized cell.
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Postby JackBean » Sun Nov 22, 2009 10:23 am

As I said, if are the bacteria really dead, you won't make them grow again, no matter, in what culture or whatever will you try...
Can't you transform the same type of bacteria with the plasmid? Why is that so important?
http://www.biolib.cz/en/main/

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Re:

Postby azarulez » Sun Nov 22, 2009 10:47 am

JackBean wrote:As I said, if are the bacteria really dead, you won't make them grow again, no matter, in what culture or whatever will you try...
Can't you transform the same type of bacteria with the plasmid? Why is that so important?


because it's my isolate from environment. I still in progress of identify it and characterize the PHA production of the bacteria. that's why I need the bacteria,not only the plasmid.
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Postby JackBean » Sun Nov 22, 2009 12:44 pm

OK, now I see. Well, why do you think, they are dead? Did you try to grow them on the same media as before?
http://www.biolib.cz/en/main/

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Re:

Postby azarulez » Sun Nov 22, 2009 1:05 pm

JackBean wrote:OK, now I see. Well, why do you think, they are dead? Did you try to grow them on the same media as before?


for the glycerol stocks, yes, they are dead because it's not frozen when I took out from freezer..I tried to culture from glycerol stock so many times,but failed.

But, for lyophilized cell, I haven't try yet..that's why I'm asking here, how to cultivate the bacteria from lyophilized cell. I'm thinking to use nutrient rich medium. is it simply just put some of lyophilized cell into the medium or it has some step before culture it in the medium?
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Postby JackBean » Sun Nov 22, 2009 1:40 pm

But you do not have lyophilized cells!
At what temperature did you have it? That's the effect of glycerol, but that doesn't necessarily mean, they are dead.
http://www.biolib.cz/en/main/

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Re:

Postby azarulez » Sun Nov 22, 2009 2:11 pm

JackBean wrote:But you do not have lyophilized cells!
At what temperature did you have it? That's the effect of glycerol, but that doesn't necessarily mean, they are dead.


As in my first post, I do have the lyophilized cells. I have prepared it about 3-6 months ago and I keep it at room temperature.

I have tried so many times to cultivate from glycerol stocks, but it cannot grow. that's why I want to give a try to culture it using my lyophilized cells.
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Postby canalon » Sun Nov 22, 2009 6:53 pm

Usually freeze dried cells can be resolubilized in any medium or just resupended in water or light buffer and then plated on any agar that is suitable. Although at this step I would avoid any selective medium, just a general non selective agar or medium.
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Re:

Postby azarulez » Mon Nov 23, 2009 2:07 am

canalon wrote:Usually freeze dried cells can be resolubilized in any medium or just resupended in water or light buffer and then plated on any agar that is suitable. Although at this step I would avoid any selective medium, just a general non selective agar or medium.


thank you, canalon. I'll try to use this method. hopefully, I'll get my isolate back!
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Postby canalon » Mon Nov 23, 2009 2:29 am

If you can, try to not use all your lyophilised sample at once. In case it would not work.
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