Frozen transformed bacterial cells problems - (Mar/20/2005 )

1. Should I sterilize the cap before opening the cryo vial? Should i also sterilize the opening before recapping it? And what should i sterilize it with?

2. How can i make a new culture from the frozen transformed stock? Should I use a pipette and take some of the frozen transformed cells directly into a new liquid medium and shake it overnight? Or should i add the frozen transformed cells directly on an agar medium plate? Should the cells be thawed first? Can i freeze the rest of the cells back in the freezer or do i have to use them all at the same time?

Thanks for any inputs.

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hi
usually, vials are sterilized. It's better, moreover if you want to do a stock.
In orderto make a new culture, thaw bacterias on ice and stripe it on plate (just pick some liquid by pipette.
and do a second stock following the culture. You will have two stocks and will be more secure.

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The vial which has been frozen in the freezer may get contaminated outside of it, so I still don't need to sterilize the outside of it before using?

When i have opened the cap, the opening may be contaminated too. Should i not wipe it with something before recapping it?

Should the cells be completely thawed before making the streaks on the agar plate?

What should i do with the rest of the thawed cells, since i only make some streaks on the agar plate?

Thanks.

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hi

well i use a tissue "wetted" by detergent and gently wash the tube.



I don't do that and as you thaw cells on ice and put the vial back to -80° just after picked your cells i don't think that allows growth contamination. Moreover, stripping bacterias on plate will reduce contamination if there is one. But you must add selection pressure in your culture. And there is less risk of contamination in the vial than in the culture, isn't it? Moreover, i made two aliquots in the first time. One that i never open before getting problem with the working-vial.


That's better but not completely necessary
but please correct me if i'm wrong


back to -80°C!

hope i've clarified situation

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As for washing the tube, I do not really see the point if you are working properly. The Only tubes that may cause problems are the tubes with the cap screw protruding out of the cap, that may be contaminated. But I would avoid at all cost to stock my bacteria in this kind of tube.

Coming to the point of streaking, I strongly disagree with fred_33, avoid as much as possible to thaw your frozen stock! Each cycle of freezing/thawing create new ice crystals that kills your bacteria, so a frozen stock should be kept as cold as possible. Some people I know throw away the frozen stock after each use, but it is probably too much.
A good way to store frequently used frozen culture is to prepare a "reference" stock, and from that stock some aliquots that will be the "working" stocks. And when you run out of aliquots prepare a new batch of them from the "reference". You thus limit the risk of contamination of the refernec and give it a longer shelf life by limiting freezing and thawing cycles.

HTH

Patrick

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hi
well i want to precise my answer.
i make a reference aliquot (1ml of culture) and several mini aliquots (200µl of culture) i thaw bacterias of minialiquots no more than 2-3times after that i discard the aliquot.
but i always strike bactrias on a plate before making a new prep. that ensures good viability of bacterias !

i apologize for not having been precise in my first answer.

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Sorry I was also a bit too strong in my disagreeing. No apologies are necessary.
And you are right, if possible it is always better to streak and restart from isolated colony rather than to start a liquid culture. Not only too improve viability, but also too reduce the risk of contamination (you may see different colony morphologies on the plate in this case..)

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