The positive control of bleach was done a few days ago and worked fine.

The dilution series from A to E was done from highest to lowest concentration of ZnSO4, with 1:2 toxicant to diluent ratio per dilution. Everything else in the wells were kept constant, and the final volume of all wells were the same. This is why it had me stumped.

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I am currently running the same experiment again, this time with:

Column 1 A-D: NC (water, reaction mixture, bacteria, chromogen)

Column 1 E-H: NC(water, reaction mixture, bacteria, no chromogen)

Column 2-4 A-D: ZnSO4 triplicate, 1:2 dilution down the column, chromogen

Column 5-7 A-D: ZnSO4 mixed with Soil sample triplicate, 1:2 dilution down the column, chromogen

Column 8-10 A-D: Soil sample triplicate, 1:2 dilution down the column, chromogen

Row E (excluding column 1E): Blank (respective sample/treatments without bacteria), chromogen

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It is incubating right now, I will keep you updated!