10 replies to this topic

[arnabde2000]

I understand glycoerol stocks are to be stored at -80C. Can they be stored at -20C. Does glycerol stocks even freeze at -20C?
One more qs: Is it necessary to streak a glycerol stock into a plate or can they be grown directly in 5 ml LB (with antibiotic)? Finally, does one thaw the glycerol stocks first and then use it for LB culture? After thawing can one freeze the cells again?

Sorry for the long drawn question. I look forward for some educated help!

[phage434]

Glycerol stocks are not stable for long periods at -20. Store them at -80 for long term storage. They should ideally never be thawed. Instead, scrape a small amount of ice from the top and either streak on a plate (preferred) or directly inoculate a culture. The plate approach is more conservative, and lets you evaluate the antibiotic resistance of the cells and check the colony morphology, as well as selecting a clonal copy. Since you ideally grew the original culture from a single colony, this step can be omitted if you are in a hurry and willing to risk possible problems.

[perneseblue]

I have not tried it. But I don't think it is a good idea to keep glycerol stocks at -80C. Since these stocks are for long term storage, I think it is not a good idea to store them at a higher temperature where the cells may lose viability faster.

It is better to streak cells from the frozen stocks onto LB plates first. The cells are not too happy/healthy after surviving the "frozen" experience and don't grow well in liquid media (probably due to a lack of O2 in the flask). And sometimes the stock may be contaminated by something else, and it is a law of Nature that this foreign contamination will grow faster, better and stronger then the bug you want.

As for the frozen stock, even in use, keep it frozen (with dry ice if possible). Just use a sterile loop and scrape some of the frozen mass off and plate it. Cells don't like being frozen and several freeze thaw cycles will be a killer. Resulting in your frozen stock losing viability and becoming useless. Not a useful thing if that vial is for long term storage.

[arnabde2000]

Thank you Phage 434 and pernesblue for your reply!
Since I got the original bacterial stock from a single colony, I omitted the streaking step and innoculated a 5ml LB culture. Now my problems were as follows:
My bacterial stock apparently did not stay frozen at all at -20C. It was a liquid. I used 5ul of this for the 5ml LB culture. After growing for 14 hrs and harvesting the cells, I got the cell pellets that I mini-prepped but did not find DNA. My question is is it possible that the bacteria was dead (lost viability) as it was kept at -20C for around a week. But then why did I get a good mass (as I normally get) if cell pellets after harvesting (this tells me that the bacteria must have grown in the 5ml LB with antibiotic). After this, I froze the liquid original bacterial stock at -80C. Would you opine that this freeze-thawing (although the thawing comes in as it did not freeze at all at -20C) would surely have killed the bacteria?

[phage434]

Did you grow the cells with the correct antibiotic? Plasmids will be lost if cells are grown without selection. It's easy to see if you have killed cells -- just streak a plate from your (now frozen) glycerol stock.

[fishdoc]

arnabde2000, on Oct 25 2009, 07:12 PM, said:

Thank you Phage 434 and pernesblue for your reply!
Since I got the original bacterial stock from a single colony, I omitted the streaking step and innoculated a 5ml LB culture. Now my problems were as follows:
My bacterial stock apparently did not stay frozen at all at -20C. It was a liquid. I used 5ul of this for the 5ml LB culture. After growing for 14 hrs and harvesting the cells, I got the cell pellets that I mini-prepped but did not find DNA. My question is is it possible that the bacteria was dead (lost viability) as it was kept at -20C for around a week. But then why did I get a good mass (as I normally get) if cell pellets after harvesting (this tells me that the bacteria must have grown in the 5ml LB with antibiotic). After this, I froze the liquid original bacterial stock at -80C. Would you opine that this freeze-thawing (although the thawing comes in as it did not freeze at all at -20C) would surely have killed the bacteria?

Maybe too little inoculum? I know for cultures I do, I usually inoculate 1/100 and grow for 12-16 hrs, and they grow well. I've tried doing 1/1000 for some things (mostly minimal media growth curves) and the growth was pretty poor. Maybe that was because of the minimal media I was using, however. Regardless, even if I'm doing growth curves in regular media, I usually use a 1/100 inoculum. Perhaps the culture did not have enough time to grow in a 1/1000 inoculum? Particularly if it was coming out of a stressful environment.

[arnabde2000]

Thanks for your replies. It helps. Two conceptual qs: Can someone explain why is it a law of Nature that this foreign contamination will grow faster, better and stronger then the bug I want.
And why does the cell reject the plasmid when grown in a media without antibiotic. It seems counterintuitive, right?

[phage434]

Several reasons. Plasmid replication and antibiotic resistance gene products consume energy and materials which could otherwise be used for cell growth. Cells without plasmid will grow faster. Also, many antibiotic resistance genes are mildly toxic to cells (especially the Tet resistance genes). In both cases, slower growth with the plasmid will select for cells without the plasmid, and over several generations, these cells will become completely dominant. Without selection, you also have a good chance of growing cells from contaminants that can be found in the environment, especially if you are not using good microbiological technique.

[arnabde2000]

So I scraped some ice from the glycerol stock and streaked it out in a plate. The cells only grew where its concentration was really high and not in the 'streaks' (I normally have fewer cells in the diluted streaks, in this case I have none). This makes me think that most of the cells in the glycerol stock is dead, so diluting it (meant no cells at all in the diluted streaks).
Am I correct?
I sit better to store DNA in TE or as glycerol stocks?

[phage434]

Store DNA in TE at -20 or -80 for long periods of time. Avoid freeze/thaw cycles if possible.

You are probably right about the health of your cells. On the good side, you only need a single viable cell to recreate the strain.

[stock-man]

glycerol stocks remain viable 'for ever'[0].
*However* there is a risk of plasmid rearrangement, even at -80, as I
have experienced - for stocks that were 3 years old or less.