Hi,
I am doing my first protocol. But I am confused on how to grow my bacterial cell cultures. Is there a protocol that lays out how to grow the overnight culture and how to store it later? Also how do I calculate the volumes that seem appropriate for the LB medium?
Type of bacterial cell: E. coli K12 TB1 cells
Medium: Liquid LB broth
Thank you :)
You want to grow your bacteria into broth/liquid culture.
first step is inoculate your bacteria cell from solid agar medium (example: LA) using ose and transfer to LB and incubate overnight. this culture is your starter. You need to quantify your cell using haemocytometee or spectrophotometer.
then, transfer your bacteria cells from this starter culture into your LB medium which later this will be used for your experiment.
how many mL your starter needed to transfer into LB medium is depend on how many the volume of your LB medium.
for E.coli overnight incubation at 37 degree Celsius with shaking at 120 rpm.
after that you can harvest your cells by centfugation and remove the supernatant and your pellet Cells you can add gliserol and store at -80 degree Celsius
It very much depends upon what experiment you are doing, that would dictate the volume you need. Generally for small volumes (1-10ml range), you could just pick a single colony from a plate as described by Wiwiek Harsonowati and inoculate a tube or shake flask. Be sure you aerate the culture well for maximal growth. If you need a much larger volume, you can use the small culture to innoculate a larger culture. Normally a 1:100 dilution is fine but there is much flexibility in that, I've used from 1:10 to 1:10,000 depending upon what I needed and how long I wanted the growth to take.
Firstly you have cultural your Bacteria on agar media then you have prepared LB broth and sunpended loopfull colony into the LB broth, after 24 hrs of incubation you have to quantify the live cell through spectrophotometer or microscope by cell counter chamber...the amount of cell will be your starter culture then you can use this for your experiment....
The expression 'overnight' is ambiguous. I will prefer to say it as it is; 18 h old culture. The volume of culture you desire depends on what you want to do with it and the total amount your experiment will consume
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