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When measuring absorbance using a plate reader, is it necessary (or better) to resuspend the culture if the plate has been sitting still for 1-2 days?

While this could be a good physics question, I am more interested in the correct biological assay technique.

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The two options are:

1) growing microbes in a plate inside a static incubator and then put the plate in a plate reader without agitating the liquid

2) growing microbes in a plate inside a static incubator and then put the plate in a plate reader after agitating the liquid

In option 2, the agitation could be achieved by gently pipeting the liquid up and down a few times...

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Definitely (2). Clumps of cells will cause a lot of noise in your absorbance measurements (depending on whether the light beam hits a clump or some empty space).

I don't know what your microbe is or what growth medium you're assaying on, but typically it's very important to maintain the culture well-mixed at all times, not just during measurement. If your cells clump to the bottom, you will not get the same aeration as you will if they're well-mixed, and accessibility of other nutrients could potentially be limited as well. Generally the most controlled condition is to maintain good mixing at all times, but certainly this is organism- and medium-dependent.

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  • $\begingroup$ +1 If a liquid culture had been standing for a few days with no shaking, there must be many dead cells. Shake or no shake, the OD will be misleading. $\endgroup$ – Nick Alexander Nov 19 '16 at 19:17

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