Dear All,
I am using following Minimal media composition:-
M9 salts (5X) 20 mL
Glucose (20%;)a 2 mL
MgSO4 (1 M) 200 μL
CaCl2 (1M) 10μL
H2O 78 ml
Glucose is sterilized by filter sterilization and rest all components are sterilized by autoclaving for 15 mins at 15lbs.
Media is prepared fresh at the time of use. (And not stored like routine LB or NB broths)
I had inoculated wild type E coli, but its overnight density is also very low as compared to that of one which we get usually in nutritionally rich media like LB broth. The colonies which I am using to inoculate into minimal media are maintained onto plain LB agar.
I had to grow these cultures for 24 hours, 8 hours in 37C 160 rpm shaker and static in overnight time.
After that i tried to make secondary culture for my experiment, but there also even using 1:50 dilution of the primary culture, E coli took more than 3 hours to reach OD of 0.2 when kept at 37C 160 rpm incubator.
Can anyone suggest me why such slow growth ? If at all I am lacking any component which needs to be added, please let me know.
Its clear that, the cells did not like the medium content. Why dont you use any standard agar medium?
Try using this! Instead of maintaining on LB agar maintain your culture on m9 agar!
Why are you using Minimal medium? In case you can tell me that then we can answer the question right!
: ) Most of the times it is the "pH" (from your water quality!) adjust it and make it physiological even for the bacteria!
Thanks,
Well, there is a high possibility of repression or probably your plasmid loss its antibiotic resistance.
I experienced similar problem, what I normally do is to grow my cultures in LB broth have it washed and grow my final culture in the minimal media supplemented with glucose.
Good luck!
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