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Bacterial calibration curve using OD and CFU/ml

Method · February 2018

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 Protocol: Making of Bacterial calibration curve using OD and CFU/ml
1. Streak the pure culture of a bacterium species on an agar plate and incubate 24 hrs or you can also
make broth culture.
2. Pick up colonies from the plate and make suspension whose optical density (OD) using
spectrophotometer within the range between of 1-1.5
3. Dilute the suspension with distilled water and record different OD values like 0.2, 0.4. 0.56, 0.72, 0.9
etc. using spectrophotometer. You should measure values in a very short time. As the generation time of
bacteria is very short, so you could cover the test tube/ microtube on ice to reduce the temperature to stop
the growth of bacteria.
4. Again make a serial dilution of each diluted suspension from 10-3-10-7 as you need.
5. Take 100ul diluted suspension and streak on the agar plate and then incubate 24 hrs at an ambient
temperature required for the growth of the specific bacterium.
6. If you find colonies more than 300, then dilute more. If get colonies nearly 600, in such a case dilute 2
times more.
7. Count CFU and make a standard curve against OD. You will get a linear curve and the equation is
y=mx+c.
8. By establishing this curve you can assume the bacterium density/ number/ cfu by measuring just only
the OD value.

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