Dear Alejandro,
I've been working with bacterial cell cultures druing several years and I must say that all my glycerol stocks are made with overnight cultures (recently reached stationary phase) with a OD ~ 1.4. After years of storage the glycerol stocks grow perfectly when you inoculate them in rich media.
Hi Alejandro
We usually growth the bacteria until stationary phase before storage in glycerol. In this way, we obtain enough cell biomass to save new batches of the strain and further analysis
Saludos
It depends on what you mean by 'optimal'. In my experience (E. coli, production of recombinant proteins under a variety of expression systems), using stationary phase bacteria to prepare glycerol stocks in academic/research labs is certainly more convenient, may save valuable time, and seldom comes back to bite your a**. It shouldn't be done -there's plenty of literature describing the changes taking place in stationary phase bugs- but it does work most of the time.
Industry, however, is another matter. I have witnessed repeatedly, and can confidently assure, that doing the same thing to prepare cell banks is guaranteed to result in inconsistent production runs down the line (I mean 300 L-plus fermentors, long seed trains, etc), provided your cell bank manages to somehow pass through QC (Another big if).
So, just in case, I always prepare my stocks from log-phase cultures.
for gram neg bacteria I am with Suma, overnight culture and then a short subculture into about half of the exponential phase. For gram pos sporeforming bacteria I would go for saturated phase where you can expect spores to have formed. They will be more robust in storage and thawing.
Bacterial growth depends on several characteristics
1) composition of growth media
2) bacterial host strain used
3) plasmid vector (high or low copy number)
4) growth conditions (temp, aeration, culture volume)
However, to answer your question the bacterial stock should be prepared when the cultures are exponentially growing. Also as mentioned earlier by Alejandro for protein expression it desirable to have the glycerol stock prepared from early stationary phase to mid-stationary phase. Hence it's always recommended to study the growth curve of bacterial culture in question.
Under optimal growth conditions at 37degC for 12-16hrs, culture can be used to prepare glycerol stock. The OD of culture would range from ~1:5 to ~2.5. It usually works fine.
I normally inoculate a fresh single colony in LB for 8-10hrs at 37degC, OD corresponding to 0.8-1.2, to prepare glycerol stocks. If you would like to grow cultures overnight (16hrs) I would grow cultures at 30degC.
Hope it helps.
Bacterial growth depends on several characteristics
1) composition of growth media
2) bacterial host strain used
3) plasmid vector (high or low copy number)
4) growth conditions (temp, aeration, culture volume)
However, to answer your question the bacterial stock should be prepared when the cultures are exponentially growing. Also as mentioned earlier by Alejandro for protein expression it desirable to have the glycerol stock prepared from early stationary phase to mid-stationary phase. Hence it's always recommended to study the growth curve of bacterial culture in question.
Under optimal growth conditions at 37degC for 12-16hrs, culture can be used to prepare glycerol stock. The OD of culture would range from ~1:5 to ~2.5. It usually works fine.
I normally inoculate a fresh single colony in LB for 8-10hrs at 37degC, OD corresponding to 0.8-1.2, to prepare glycerol stocks. If you would like to grow cultures overnight (16hrs) I would grow cultures at 30degC.
Hope it helps.
Bacterial growth depends on several characteristics
1) composition of growth media
2) bacterial host strain used
3) plasmid vector (high or low copy number)
4) growth conditions (temp, aeration, culture volume)
However, to answer your question the bacterial stock should be prepared when the cultures are exponentially growing. Also as mentioned earlier by Alejandro for protein expression it desirable to have the glycerol stock prepared from early stationary phase to mid-stationary phase. Hence it's always recommended to study the growth curve of bacterial culture in question.
Under optimal growth conditions at 37degC for 12-16hrs, culture can be used to prepare glycerol stock. The OD of culture would range from ~1:5 to ~2.5. It usually works fine.
I normally inoculate a fresh single colony in LB for 8-10hrs at 37degC, OD corresponding to 0.8-1.2, to prepare glycerol stocks. If you would like to grow cultures overnight (16hrs) I would grow cultures at 30degC.
Hope it helps.
Bacterial growth depends on several characteristics
1) composition of growth media
2) bacterial host strain used
3) plasmid vector (high or low copy number)
4) growth conditions (temp, aeration, culture volume)
However, to answer your question the bacterial stock should be prepared when the cultures are exponentially growing. Also as mentioned earlier by Alejandro for protein expression it desirable to have the glycerol stock prepared from early stationary phase to mid-stationary phase. Hence it's always recommended to study the growth curve of bacterial culture in question.
Under optimal growth conditions at 37degC for 12-16hrs, culture can be used to prepare glycerol stock. The OD of culture would range from ~1:5 to ~2.5. It usually works fine.
I normally inoculate a fresh single colony in LB for 8-10hrs at 37degC, OD corresponding to 0.8-1.2, to prepare glycerol stocks. If you would like to grow cultures overnight (16hrs) I would grow cultures at 30degC.
Hope it helps.
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