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Would you give us directions for a trichrome method of staining Protozoa that works well using PVA with zinc fixative? Since giving up the mercury fixative, we have trouble getting good nuclear staining.
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First of all, you need to realize that zinc fixatives do not fix organisms as well as mercury, so some of the morphology differences are unavoidable. The trichrome stain is used the same way with the following exceptions:
1. You do not need to use the iodine dish to remove the mercury prior to staining since no mercury is found in the fixative. After the slides are dry and ready for staining, they can be given one 70% alcohol rinse first or the dry smear can be immersed directly into the trichrome stain.
2. Since each batch of stain may vary a bit, if the nuclei tend to be somewhat pale, you can stain the smears for a longer period of time. Trichrome is quite variable and staining times can vary from 5-10 min to 30 min. Also, make sure you do not make the original PVA smears too thick. Remember to absorb out some of the PVA onto a paper towel (5-10 min), then prepare the smears from the stool remaining on the towel.
3. Another factor involves the dehydration reagents (this may be your problem). The best approach is to use 100% ethanol for the absolute; if you are using lab 100% which is a combination of 95/5 alcohol, this will not dehydrate the smears as well, so change the dishes more often.
4. Also remember than xylene substitutes do not dehydrate as well as xylene, so again, make smears thin rather than thick and change dishes about every 50 slides, particularly if you are using Coplin jars rather than staining dishes.
5. Also, xylene substitutes will not dry as quickly as xylene.
These tips should help produce a better smear. However, remember that if the original fixation was not sufficient (poor mixing, wrong ratio of fixative to stool (3:1) - nothing you can do in staining will improve the quality.
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