Hello all,
I am confused about calculating the CFU/mL for my serial dilutions, for context I am conducting an experiment that involves creating a standard curve of absorbency (measured via spectrophotometry) against CFU/mL of each dilution. I have conducted the spectrophotometry and plated each of my serial dilutions but my confusion occured when I began calculating the CFU/mL and found that it increased as my samples got more dilute which logically is the opposite of what I expected. Furthermore, I am unsure if it possible to calculate the viable cells in each dilution as the calculation CFU/mL=(number of colonies*dilution factor)/volume plated, seems to be for calculating the number of viable cells in the origional sample.
Any advice would be greatly appreciated.
Hello, I think when you are calculating CFU/ml using serial dilutions, you are indeed determining the concentration of viable cells in the original sample. The dilution factor accounts for the dilution effect and allows you to extrapolate back to the original concentration based on the colonies counted at each dilution level.
Thank you for your contribution Akram Khanmohammadi, if you happen to know any formula for calculating the CFU/mL of individual dilutiutions that would be very helpful although your previous answer will be very useful to me.
My field is not about bacteria and I only have experience with these, and I wanted to share with you something which may help you.
Spectrophotometry is used based on optical density and doesn’t differentiate between viable and dead bacteria, so it’s not an exact method to determine the concentration of bacteria in a sample solution, but it is a fast way to estimate the count of bacteria.
Therefore, the spectrophotometry method is completely different to the colony-counter, which is counting viable bacteria on agar plates.
I think a colony count has specific conditions. You dilute your solution and then transfer to the agar plate until you find the best plate. The best plate is that it has colonies between 30–300 colonies per standard agar Petri dish of 8 cm. If the number of colonies is more than 300, it may easily result in an underestimation of individual colonies and estimate less than the real number. If the number of colonies be less than 30 per plate, it can result in large standard deviations.
you do serial dilution your origin sample to find best petri dish (30-300 colony) and then use this furmola for calculate the amount colony in the original solution:
CFU/ml – (Number of colonies*dilution factor) / volume of culture plate.
Thank you for this information, however I have already done lots of research into the uses of spectrophotometry in microbiology. Unfortunately there is not alternate method that I could possibly use given the nature of my experiment and the rules/regulations of that lab it will be conducted in; meaning a standard curve is necessary.
Then I think it’s better you plot absorbance versus CFU/ml (without dilution factor).
Remember that the dilution factor accounts for the dilution performed during the serial dilution process. For example, if you counted 150 colonies on a plate with a dilution factor of 1:100, you can calculate the CFU/mL for the original sample using the above steps1
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