I'm growing Pichia pastoris in BMGY medium currently and I'm trying to follow, how it grows, so I measure OD600. But as I expected the yeast to grow (and hence increase OD600), I diluted it more (5x instead of 2x) and the values were completely off.
I've added tables to show, what I mean. The top row shows the dilution. Then there is time at which I measured it and the actual readings of OD600. In the lower table, there are the "actual" values, calculated from the measured ones and dilution.
As you can see, the more I dilute it, the lower the value, but if I keep the dilution the same, it is somewhat reliable.
I have two suspects:
1) I added antifoam Structol SB2121 at 1%, which is quite heterogenous, so maybe it depends on how much I take it (although the trends remain, so the readings are not completely random).
2) when I let the flasks stand for a while (like preparing the tubes and pipetting), the Pichia settles down and evethough I try to mix it again, maybe it's not complete?
To make sure that it's not effect of time or different samples (although it is quite consistent across samples, as I wrote above), I diluted one sample and measured OD600. This time I used water as blank, as I was lazy to make so many blanks, but I suppose, the results would not be so much different.
The undiluted sample is obviously outlier, because of the spectrophotomere limitations, but otherwise obviously, the more it is diluted, the lower OD600 is measured. How?
Dear Phil,
please, see the second table that I attached. It shows that the *calculated* (not measured) OD600 decreases the more I dilute the sample.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction