I like to preserve some bacteria culture using stab culture technique but I like to know if I need to add more sodium chloride to my normal LB broth which contains 5g/L of sodium chloride
I worked with E.coli strains and made stab cultures from NZY broth. I did not change anything. Just made sure that the liquid cultures were in lag phase. They were viable after 3-4 months.
Obtain an LB agar plate with appropriate antibiotic.
Label the bottom of the plate with the plasmid name and the date. It is also a good idea to add the antibiotic resistance and your initials.
Keep your lab bench area sterile by working near a flame or bunsen burner.
Using a sterile toothpick or pipette tip, touch the bacteria growing within the punctured area of the stab culture.
Run this tip lightly over a section of the plate to spread the bacteria over approximately one-third of the surface area of the plate.
Using a fresh sterile pipette tip, pass through streak #1 and spread the bacteria over the next one-third section of the plate, to create streak #2.
Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last one-third section of the plate, to create streak #3.
Incubate the plate overnight (12-18 hrs) at the designated growth temperature.
Check for single colonies. A single colony should appear as a white dot growing on the solid medium.
Once you have single colonies, proceed to Inoculating an Overnight Liquid Culture.
Hi,
We usually use LB agar to make plates and we don't put more NaCl, only the resistance antibiotic. We preserve bacterial stocks in -80ºC freezer with 50% o/n liquid bacterial suspension in LB and 50% glicerol. These preparations are viable for more than a year.
I agree with Ms.Alejandra,, NaCl has inhibiting property especially at high concentration, TSA plus glycerol is OK n less than 16 hrs incubation before freezer will preserve bacterial cultures. And expect some variation in the characteristics of the organism.
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