Dear Pariya Soltani-Nezhad ,

the classical way to do this is to establish a calibration curve displaying the correlation between OD600 and biomass concentration. From the calibration curve you can obtain the trendline equation, which you then use to convert absorbance to biomass concentration. If the trendline is linear, it will look like this: y = mx + n

where y is OD600, x the biomass concentration and n the intercept with the y-axis.

For biomass determination you can consult following textbooks:

Methods for General and Molecular Microbiology, Third Edition (Article Methods for General and Molecular Microbiology. Edited by C....

Brock Biology of Microorganisms (https://www.pearson.com/us/higher-education/program/Madigan-Brock-Biology-of-Microorganisms-Plus-Mastering-Microbiology-with-Pearson-e-Text-Access-Card-Package-15th-Edition/PGM116784.html)

Microbiology: A laboratory manual (https://www.pearson.com/us/higher-education/program/Cappuccino-Microbiology-A-Laboratory-Manual-11th-Edition/PGM96372.html)

Good luck!

Thank you very much for your reply

But I didn't understand well about the curve drawing...

What is the correct formula to determine Bacteria concentration?

Is it " Concentration (µg/ml) = (A600 reading) × dilution factor × 50µg/ml " ?

What is dilution factor?

Dear Pariya Soltani-Nezhad ,

have a look at following discussion in which a figure was uploaded showing the correlation between OD and dry biomass: https://www.researchgate.net/post/Determination_of_dry_mass_concentration_in_fermentation_broth.

The figure also contains the linear trendline I mentioned. My advice is to conduct an experiment to establish this calibration curve. This can be done by following your protocol to grow Bacillus. While you grow your Bacillus you take samples at suitable intervals (preferably during log phase), measure the OD and determine the dry biomass in your sample. You could use centrifugation or filtration method. For centrifugation method transfer 1.5 mL of your sample into a dry, preweighed Eppendorf centrifuge tube. Centrifuge at 10'000 rpm for 5 min, carefully discard the supernatent without disturbing the pellet at the bottom of the Eppendorf, and dry the pellet with Eppendorf at 105C until constant weight. You may want to include a negative control (1.5 mL sterile growth medium in Eppendorf) to account for weight loss of Eppendorf and presence of salts from growth medium).

Let's say you have done all the above and you established a trendline with equation (concentration c in g/L):

OD600 = 1.0749 * c - 0.6108

You have carried out your experiment, taken a sample and measured an OD600 = 0.6. The concentration can be calculated as follows:

c = (OD600 + 0.6108) / 1.0749 = (0.6 + 0.6108) / 1.0708

c = 1.13 g/L

If the OD reading is from a diluted sample (e.g. 10x), and the calibration curve is based on undiluted samples, you need to determine the OD of the undiluted sample. This is done by multiplying the OD of the diluted sample with dilution factor. If we assume an OD600 = 0.3 for 10x diluted sample the OD of undiluted sample is 0.3 x 10 = 3.0. The dry biomass is now 2.54 g/L.

Does that make sense? If not speak to seniors in your research group, or if you are from an engineering faculty go to science faculty and look for biology department and find postgrad students who grow cell cultures. The literature I provided in my previous reply is highly recommended and easy to understand.

Regards, Rob.

The answer was complete,

Thank you so much.

Can we use this equation for all bacteria measured in optical density

Simply u can do 4.5/ your sample O.D u will get approximate value

If i am given only with OD, time and dilution factor. Could i get cell dry mass from it?