Hi Wasabi - thanks - yes, we will be using the 9-9.99 pH eggwhites for the real attempt in July, but since these farm fresh eggs cost a fortune here I wanted to do my practice sorts on the regular store-bought eggs, which are slightly lower in pH. When I checked, they are running close to 8.5 pH.
I also wanted to update you on my trial MSU attempts this past weekend and maybe you can get some helpful hints out of this. For the first attempt, DH drank red bull about 1 hour before that and also had 1 tablet of Musinex. He DTDed into a cup and I then transferred his sample into a narrow glass test tube with a long pipette. Since I did not want to lose any spermies, I introduced a lot of air bubbles into the sample, so here is my first advice - do not try to squeeze the last drop out of the pipette, so as not to blow air into the sample. It will be hard to get rid of those bubbles later on. I then put the same amount of room temperature egg-whites on top and I was lucky because the EW remained floating on top of the semen. I did however again introduce bubbles, so same advice. Do not squeeze the last drop of EW out. We then put the test tube into the home-made incubator (used a styrofoam cooler for that) at a 45 degree angle for 1 hour. We tried to keep the temp a steady 99 degrees but the thermostat was not that sensitive, so we had to keep an eye on it the whole time and we were adjusting the lid on the incubator to regulate the temp. After 1 hour, we sucked up 20% from the top and put it on the glass slide piece and covered it with a thin square glass plate to flatten it out. When we looked inder the microscope, all the spermies appeared to be dead and not moving. We took some more samples and those too were all not moving. We thought they all died from being in the incubator. Our possible guesses "for the cause of death" were that the lightbuld in the incubator was too bright for them, that there was no humidity and possibly because the temp varied slightly from time to time. So for the next attempt we changed and improved on a bunch of things. First of all we put a tall pot filled with 99 degree water into the incubator to stabilize the temperature, since it's easier to keep water at a constant temp rather than the air. This also resolved the issue with the light shining straight onto the test tube and also added some humidity. We could not finish the attempt all the way to the end, though, since the EW sank this time (DH did not consume red bull this time), BUT we did look under the scope to see if the spermies are alive in his sample and guess what - all the sperm was dead even before we did anything with it! We then realized that the sperm were not moving because of the thin glass piece that we were placing on top of it. Once we stopped doing that and were simply placing a drop of the sample straight onto the slide all the sperm was happy and alive and moving. So, second piece of advice, when you look at your sample under the scope - DO NOT cover that sample with the thin glass piece to flatten it out.
We will be trying again tomorrow and plan to improve again on the test tube to see how we can minimize the danger of the EW sinking. I did not like that even when the EW did not sink in the first trial run, there is not a clear cut definitile line between the semen and the EW. Maybe some nesting test tubes with some sort of filter on the end will help with creating a more defined border between the two and will help with keeping the EW on top. Has anyone seen anything like that for same in some medical lab?