I would like to prepare glycerol stock from Photobacterium and Morganella. But I m not sure which phase is more appropriate to make a glycerol stock. Could someone explain me please?
If you have pure culture then grow it for 16 hrs in LB broth and then make glycerol stock
It depends on how fast the bacteria grows. For conventional microorganisms leading to visible colonies after 24 hours some 16-18 hours is correct. For bacteria growing more solowly, such as Legionella or Brucella, you may need 24-36 hours.
I am agree with Ercan Yatmaz's answer. So you can do it when your culture on log phase or late log phase according to your bacterial growth pattern.
If solid media, then pick the colonies when they are visible and complete,
If Liquid media: If you see any tubidity or a good change in OD.
Its always better to pick up isolated colonies from overnight incubated (18 hours) enriched media- for glycerol stock.
We make glycerol stocks in a different way. We use this for making glycerol stocks of E. coli strains, M. smegmatis and B. subtillis. This method gives a thick glycerol stocks that last for yeas.
Streak the bacteria on an entire agar plate that is supplemented with appropriate antibiotics and nutrients. Incubate till you see a lawn. Scrape the lawn with sterile loop and transfer all the cells from one plate into a freezing vial. Add 500 ul of media and 500 ul of 50% Glycerol. Mix gently till the cell lumps suspend uniformly. Store at -80C.
Besides the correct advice to use the log phase, I would recommend to freeze the cell suspension containing 20-30% glycerol (final concentration) in liquid nitrogen and then immediately store at -80 °C. If you do not have this possibility, store the glycerol stock at -20 °C for a few hours and finally store at -80 °C.
Depends upon your selected isolate's growth behavior, you can prepare, but i would like to prefer late log phase (Optical Density at 600 around 0.6) because in these phase cells are healthy, active metabolically. You can prepare glycerol stock in the range of 15 to 20%.
Many thanks for valuable suggestions. Can I take 500 microliter of culture directly from the broth when I see around 0.6 OD600. What is the best way?
bests
Yes, you can but prefer 1 ml that is best fit for your cuvette.
Before taking O.D, vortex your culture in a such way that it should be homogeneous, so you obtain good results. Any querry, you welcome
For aerobic bacteria like E. coli or S.aureus, I usually make stocks from overnight cultures (liquid media) grown with maximal aeration (shaking). Those cultures are in the late log phase or the begining of stationary phase. Those bacterial stocks remain viable for years.
In general, for preservation purpose, it is good to use early/mid -log phase growth.
Take the cells at log phase; mix 0.3 ml of cell culture with 0.7 ml of glycerol gently; put it in liquid nitrogen immediately; store at -80C and finally enjoy it :)
In general, use late log phage for glycerol stock. This will give you more viable cell numbers. But if you are preserving the stocks for DNA related studies, suggested to use mid-log phage culture and save more tubes of stocks.
Most preferable is log phase culture.
for preservation cn try any concentration of glycerol from 10-50%
It is always advisable to prepare the stock culture of cells when they are in their log phase. Cells in their log phase content maximum genomic content .So, grow the culture as per their log phase time and then separate out them from medium content through spinning. Active cells mass can be mixed with sterile 40% glycerol and preserve it.
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