Today when I was trying to measure OD of a bacterial solution I obtained very strange readings. The OD was always same for every diluted solution and even for the same solution as control solution. In order to be sure about calibration and proper working of our spectrophotometer we tried with another set that the same results showed. Then we suspected our solution. By checking the ingredients we found that we wrongly used insoluble yeast extract (Y4000- Sigma) which is not suitable for growth media. May the problem be for using the insoluble yeast extract in water?
I recommend that you try calibration with Mcfarlad standars solution to be compared with the results
Yes, that is the answer. The spectrophotometer cannot tell the difference between bacteria and particles of any sort. The original MacFarland standard is nothing more than an inorganic barium sulphate particle suspension.
Any particle in the solution will scatter the light from the photometer and mimic an extinction. In order to be sure about your bacteria density you have to use a clear media.
Yes. Your OD will be exaggerated. One potential way to solve this problem is to use a bit of the insoluble yeast extract in your blank.
@Rasha: This will only work, if you get exactly the same amount of particles into the solution. Otherwise the blank will substract too much (or too little) from your extinction.
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