Have you been able to solve the problem with reading the endpoint of your assay? If not, here is another suggestion:.
The endpoint on this test kit depends on the methylene blue turning from blue to colorless when the sodium thiosulfate reduces all of the methylene blue in the sample and this occurs when all of the iodine in the sample is converted to iodide.
The traditional method for doing the Winkler dissolved oxygen test method used starch solution as an indicator. The end point is a dramatic blue-black color of the starch-iodine complex changing to colorless as the iodine is reduced to iodide. http://serc.carleton.edu/microbelife/re ... xygen.htmlhttp://cchdo.ucsd.edu/manuals/pdf/91_1/culber2.pdf
So, you might want to repeat the assay, substituting a 1% starch solution for the methylene blue. You can make the starch solution by using any starch, such as corn starch. Try adding 1 tsp cornstarch to 8 ounces of water and boil it or microwave it while stirring frequently until the solution changes from cloudy to clear.
When you titrate with the sodium thiosulfate (reagent D), add this reagent until the solution turns to a pale straw color, Then add 1 ml of the starch solution so a blue color forms and continue the titration until the sample turns clear. This endpoint should be clear and easy to read.
This will not solve the problem, if there is something in your sample that it interfering with the assay, or if you a mixing the sample to much and adding oxygen back into the sample, but it may help with troubleshooting.
Please let me know the answers to the previous questions and if you decide to try using the starch to read the endpoint.