Viability of frozen bacterial sample - (Mar/22/2008 )

Hello, i have many problems, and hope you could give me some advice.
I'm a college student doing a project in a microbial lab.
the sample i'm getting from is those from hospital clinical origin.
they are storge in -80 degree c refrigerator.
I kind of messed up.
i have about 200 samples that have stood in room temp while i subculture.
they've thawed completely and frozen again.

the problem i have is, how much damage it would do to the clinical samples?
they are about 3ml and 15% is glycerol

the protocol i found on protocol-online is a 1990 one, it wrote that each strain should not be on bench for more than 5 minutes, and it shouldn't even be thawed .

but in my lab, though it was totallly idiotic to let those samples complete thawed and stood in room temp for at least 1hr.
other people in my lab said that they always wait till it complete thawed before they start doing the subculturing.

so, how much damage it would do to the freeze storaged samples?

Is there any way to repair it?
reinoculate and make fresh sample then freeze the fresh ones?

i feel absolutely idiotic and ashamed because according to the seniors in my lab, those clinical samples aren't technically ours, but research assitants'.

another thing is i could possibly contaminated some of them, but i think there's little i could repair there.

hmm

please help
thanks

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to be honest: I dont understand your message completely.

are you saying that you left 200 samples standing in room temperature while you were subcultivating a few of those samples?

ANd you then placed all the samples back in the refrigerator?





Anyway : a general answer



You cant "repair" the samples, but what you can do is the following: subcultivate all the samples and then freeze the new samples.
When subcultivating : check your plates to see if there is any contamination. You should know how the bacteria of fungus looks like you are storaging. So when you see other things, then just scrape off a little bit of your fungus, bacteria you need and put that on a new plate, and keep doing that till you have a clean fungus or bacteria to use and to freeze.

anyway: new samples will be beter then the old ones you thawed and refroze.

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Sorry, i think my english is still quite poor.
i apologize
actually, i left them standing in room temp while i were subcultivating all of them.
what i did is that i only streak the first zone so it'll be faster, after i done them all, i put them back into the -80 degree celcius refrigerator. then streak the second and the third.

from the books and from a protocol online document, i guess if the things wrote in those are true, the damage has probably been done each time any member in my lab subculture the clinical samples.
We all wait it to be thawed.
from the document on protocol online, i guess the samples shouldn't even be thawed at all.
but the seniors in my lab said what i shouldn't do is processing so many samples at a time(which is where i begin to feel completely idiotic),
and should try to keep the samples from standing in room temp for too long.






I have this thought too, and i'm going to have a word with the PI tomorrow, and tell her that i screwed up.

but there is something i also wondered, if i do new samples, wouldn't there be more possible that the descendants have mutations that might made them different from the original sample?

if the PI decide that i should resave all the samples, i guess i would have a lot of tubes to washed. but anyway, it's my fault
thank you, pito

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From where are you?


Ah ok, I see.
Well, to be honest: nobody, but you, would take 200 samples at one to work with haha
Its just a rookie mistake, no sweat.
I find it strange they didnt help you more with it.




true: the more you subcultivate and refreeze, the more changes on mutations...
But its better to risk that then to risk losing all your samples because they are contaminated.
In the end : when the samples are contaminated , they are worthless too...

I would advize you to not start cleaning all 200 samples, just subcultivate 10 orso, see what will grow and if there is no contamination, then leave it like that.
If you have 1-2 plates that are contaminated, then maybe leave it like that too...

It all depends on what your "teacher" or "boss" wants. (whats a PI? for what does it stand?)


Eum, one general idea: how come you know "nothing" or not much about the procedure ? Didnt they help you ?

I mean: I would think that your "teacher" or someone responsible would help you the first few days and explain some things and show you some things so that you know what you are doing?
Dont they have protocols themself that they can give you ? or?

Anyway, when I started working in the lab they showed me what I had to do, then I had to do 1 while they watched me do it and when that was ok, then they left me to work on myself..; But they never just told me to do something I never did before... (at least not when I was new in the lab... after a while you learn enough yourself... but the start is always hard)




I do wonder what your "PI" will say!

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The problem while freezing and thawing is not one of contamination, but that:
1- every time you freeze and thaw new ice cristal are formed and you kill bacteria, so you limit the viablity of the rest of your sample
2- Bacteria can use glycerol as a carbon source, so everytime they thaw they will reduce the amount of glycerol and make 1 even worse

But that does not mean that your glycerol are dead. You can still use them, it's just that the survival rate among frozen bacteria will be worse than it was before.
Ideally the best thing is to restart cultures and freeze them in fresh glycerol. But you are adding 1 or 2 more culture steps (1 agar plate and you freeze a few isolated colonies or 1 liquid culture). But you can just freeze again and pray, even without prayers it works well if you did not leave your plates out for hours.

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If he did indeed work asepticaly then yeah, there would be no contamination.

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Well contamination was not in the question, and I give the OP the benefit of doubt for what I would consider would be the worst sin when working with frozen samples: contaminating your glycerol...

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Sorry for not replying so long.
I'm in Taiwan actually.
well, everyone has their own exp to do, so...

PI, principle investigator?
i just thought people use that word?


Well, i have been in the lab for a semester, though didn't do much culturing procedures.
I was helping a phd student doing her exp, and she had completed the collecting sample part and started to work on the actual exp.
and this time i was collecting the samples for my own project scheduled to be completed next Jan.
the mistake is that i should do small amount of samples each time instead of doing all.
it's really stupid.
well, they taught me how to do it, but just didn't expect me to do this stupid thing, i guess



thanks for your help !!

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ok,

good luck with it.

ah PI means principle investigator, I didnt know that.

I hope the books I send you are usefull.

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yes, they are very useful
thanks alot!!

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