Dear all,
I would like to count the number of cells from my OD600 values.
The OD600 value for B. Substilis and S. epidermis are 0.5.
Could somebody tell me how to convert these values to number of cells.
For E. coli, the OD600 of 1.0 = 8e+8 cells/ml
Is it same for the above two bacteria also?
Can I say my both samples (Bacillus and Staphylococcus) contains 4e+8 cells/mL ?
Hoping some positive suggestions
Best regards
Bikash
To my understanding, even you presented reference for E. coli is not absolute value as depends on media and many other factors. I would suggest, just try to make your own calibration curve from the CFU vs OD600. Then next evrey time, you can use same media and conditions and easily can get a relative value (viable cells in this case) against OD600. To make this one time calibration curve, you can use: CFU, automated cell counter etc and of course your OD counter (e.g., spectrophotometer). Next time, you will just use OD600 to save time and money. Thanks
Yes. OD600 is the a gross estimation of opacity. For your own culture media and bacterial strain you need to count the CFU in agar plate.
Dear Dr. De,
Thank you very much for your suggestions. Using serial dilution technique on agar plate, I could count the exact number of cells. However, as per my study is concern, I am trying to estimate the number of cells in an approximation. In such cases, could we correlate the OD600 value to an approximate number of cells.
Thanking you
Yours sincerely
Bikash
Dear Dr. Biswas,
Thank you so much for these information. I am interested in finding an approximate quantification for my growth sample. I can understand, the absolute value can only be measure from serial dilution in plate. However, is it right to approximate the OD600 of bacillus and staphylococcus in reference to E coli?
Thanks and regards
Bikash
The OD600 usually depends on the size of the cell. For instance, if B. subtilis cells are bigger than E. coli cells I would expect the OD600 of 8 x 108 cells be higher than 1.0.
You could use the E. coli value as a reference and do only one or 2 dilutions for CFU counting. I think around 200 µL diluted to 10-6 is a nice starting point.
Hi, Bikash. I'm doing spore count for bacillus subtilis. I'm facing the same problem as you. In my case, i'm counting spore which is smaller in size. I had use haemocytometer count, but the number of spore were so abundance, hard to count and very time consuming.
Recently， i'm thinking to use OD600 to measure the concentration of spore. Got one Dr taught me about in OD600 , value 10 is equivalent to 10^10 spore for bacillus subtilis. So i planned to count my sample according this ratio. I wish it's not significance different with my haemocytometer count result.
I hope we can exchange opinion in this matter. I'm also not sure about my method are right or not.
Dilute your inoculum differently into several tubes with sterile water. Thereafter, plate out specific volume of each inoculum sample unto individual plates and incubate appropriately. Generate a standard curve from the CFUs obtained. All the best!
The best recommended method to use in such case is Mc Farland standard solution and reference should be taken from International microbiological standards.
eg: suppose 0.5 Mc Farland equavalent of Bacillus subtilis has n*10e8 CFU/ml, then measure it and multiply
You will need to generate a standard curve by following your bacterial cultures over time and measuring both their OD600 and cell density at several time points.
Measuring optical density cannot, however, distinguish between live and dead cells since both will scatter light. A direct cell count without a viability stain cannot distinguish between live and dead cells either.
Note that different mutants or strains of the same species may also differ in their OD600 relative to cell densities. Hence, standard curves may need to be developed for every strain you work with if there are significant differences in cell size, cell arrangement, or cell morphology.
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