Actually i was measuring absorbance of Mycobacterium smegmatis at 580 nm but i got different results.. i have taken 20
µl culture and diluted with 20 µl, 40 µl, 60 µl and 80 µl media and further analysed the absorbance at 580 nm using media as a blank. but unfortunately, i got increasing value of absorbance instead of expected decreasing absorbance... further same type of unexpected results were also obtained using bacillus subtilis culture..please suggest the remedy if any..
Have you ever test to measure the absorbance using 0.8% NaCl physiological as replace of your media?
Hope it will give you a good results as you’re expected.
i hope so it will work but why do you think so in the place of media, 0.8% NaCl will work because ideally if we have to measure the absorbance of microorganism in media ..media should be taken as blank..
In your case, you mention that after dilution of your samples 1x, 2x, 3x, 4x by using media, you obtain increasing value of absorbance instead of expected decreasing absorbance.
As in Beer-Lambert law, absorbance and concentration is directly proportional.
I can suggest you several things:
a. Try to use 0.8% NaCl for every steps of dilution and also use as a blank during measurement.
If you use media for dilute your samples, I just aware that your samples have fast doubling time (M. smegmatis about 3 - 4 hours while B. subtilis about 120 minute). Your media contain rich nutrient for the optimal growth of your bacteria during your measurement. That’s why you got increasing value of absorbance eventhough you already make dilution.
b. Make sure to well homogenized your samples using vortex either before pick up 20 ul of samples or after dilution or remove to cuvette for measuring.
c. As we know that absorbance measurement is just based on turbidity. That’s why make sure that your cuvettes are clean enough. You can rinse them using dH2O or 70% EtoH before or after use.
Hope it’s useful.
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