12/21/17

Dear Ruchir,

The researchers who described the procedure in the quote above have probably done this many times. After 6 hr @ 37C, they probably measure the turbidity of the culture on a spectrophotometer. From past experiments, they know that a certain absorbance is equivalent to a specific # of CFU/mL (as determined by doing plate counts). Knowing this, all that is necessary is to spin down a predetermined # of mL of culture, wash the cell pellet, then resuspend the washed cells in a volume of PBS that will give them the desired CFU/mL for their experiments.

I hope this information helps you.

Bill Colonna Center for Crops Utilization Research, Iowa State University, Ames, Iowa USA [email protected]

Dear W.J Colonna,

Thanks for your answer. Can you tell me how is it possible to determine the CFU/ml with the help of spectrophotometer absorbance?

12/21/17

Dear Ruchir,

To do this, you will need to do a growth curve of your organism. The procedure can be found in any good microbiology lab manual. Start early in the day, as doing a growth curve takes a long time.

Start early in the day, as doing a growth curve takes a long time. Grow the organism in the medium that you normally use. At time zero, measure the turbidity of the medium on a spectrophotometer at an appropriate wavelength. (Some researchers use 600 nm; others use 620, 650 nm. ) After you measure the absorbance, aseptically withdraw a small sample of broth, dilute it w/ sterile water or saline, then plate it out on agar and incubate at a suitable temperature. Incubate the flask w/ the broth culture at a suitable temperature and shaking rate. Periodically (e.g., every 1 or 2 hrs), remove the flask and repeat the process: measure Absorbance and plate a sample of the cells. Continue the process AT LEAST until the cells have passed from the lag phase COMPLETELY through the logarithmic phase and into the stationary phase.

Grow the organism in a broth culture in a shake flake. At time zero (T-0), immediately after inoculating the broth, measure and record the absorbance of the broth w/ cells. (You can use a wavelength of 600, 620 or 650 nm. Other wavelengths are also suitable.) After this, aseptically remove a sample of broth. Dilute it with sterile water or saline and plate the dilution on agar. Incubate the plates at a suitable temperature. Also incubate the flask with the broth culture. Periodically, at 1-2 hr intervals, repeat the absorbance reading and plating steps as above. Be sure to keep track of the time points. Continue doing this AT LEAST until the cells have progressed from the lag phase, COMPLETELY through the logarithmic phase and into the stationary phase. (This can take all day or even longer, depending on the organism.)

In a day or two when the plates have grown, count the colonies. This will give you the # cfu/mL at each time point. You can then plot cfu/mL AND the absorbance values vs. time. This will give you a growth curve with the # cfu's/mL that correspond with the absorbance values. The next time you grow the cells, you need only measure the absorbance. By reference to your growth curve, you will know the corresponding # of cfu/mL.

I hope this information helps you.

Bill Colonna Center for Crops Utilization Research (CCUR), Iowa State University, Ames, Iowa USA [email protected]

Dear W. J. Colonna,

Thank you very much for such an elaborated and informative response. It made the procedure clear without any obscurities. Thanks again for your time.

Dear W.J. Colonna,

you can use the MC farland scale too. You buy this scale which determines the turbidity of the tube with a concentration of bacteria. In Brazil this scale is very cheap.

This scale is more easy to work because you don´t need the spectrophotometer to read you sample.

I hope to help you,

Michele