My project looks at the joint toxicity in yeast cells (Saccharomyces cerevisiae), I am using lipid derived electrophiles (LDEs) such as acrolein (ACR), crotonaldehyde (CRO), methyl vinyl ketone (MVK), and hexenal (HEX). I treated 1 million yeast cells in 24 well plates with the LDEs individually, as well as combining them in concentrations that didn't cause significant toxicity when treated alone, but produces a significant toxicity when mixed.

I used the following concentrations:

ACR- 0.05 mM

CRO- 0.3 mM

MVK- 0.02 mM

HEX- 0.6 mM

In triplicates, I treated 50 microliters of LDEs in a well containing 900 microliters of YPD media and 50 microliters of live culture.

For the mixture I added 50 microliters of each of these four LDEs in a well containing 750 microliters YPD and 50 microliters live culture.

I then incubated the plate in a spectrophotometer at 30 C for 18 hours with a 5 second shake ever 30 minutes.

After processing the results, the curves were as expected, however during the stationary phase towards the end, the wells that were treated with the toxicants showed an increased absorbance compared to the control, even the mixture that showed the most toxicity had the highest absorbance compared to other curves, which I find counterintuitive.

I have attached the graph.

So can anyone help me with what is happening here?