Question about measuring bacterial OD - requensing inputs on measurement volume (Jul/06/2011 )
When you say you usually measure 1ml- is that in a cuvette in a spectro? If yes, then you aren't actually measuring the whole 1ml are you? Just the absorbance of light that passes through that small bit of the cuvette in the middle somewhere. Volume is irrelevant- its all about path length.
My guess is that the micro plate reader has been set up in such a way as to calculate the OD600 accurately for a 200ul volume. So when you add a different volume, you stuff the readings up. Again, as for spectro the volume is almost irrelevant- the 200ul is used so that the path length is known, and that is all.
So adding 100ul, or 300ul and you change the path length, making your readings useless.
(If I am understanding what you are doing correctly)