Approximately 5 to 10 min before you want to examine the slide, place a drop of immersion oil on the dry fecal film. Allow the oil to sink into the film for a minimum of 10 to 15 min. If the smear appears to be very refractile on examination, you have not waited long enough for the oil to sink into the film or you need to add a bit more oil onto the film.
C. Once you are ready to examine the slide, place a no. 1 (22- by 22-mm) coverslip onto the oiled smear, add another drop of immersion oil to the top of the coverslip (as you would normally do for any slide with a coverslip), and examine with the oil immersion lens (100× objective).
D. Do not eliminate adding the coverslip; the dry fecal material on the slide often becomes very brittle after dehydration. Without the addition of the protective coverslip, you might scratch the surface of your oil immersion lens. Coverslips are much cheaper than oil immersion objectives!
17. Examine the smear microscopically with the 100× objective. Examine at least 200 to 300 oil immersion fields before reporting a negative result.
*Slides may be held for up to 24 h in these solutions without harming the quality of the smear or stainability of organisms.
Modified Iron Hematoxylin Stain (Incorporating the Carbol Fuchsin Step)
The following combination staining method for SAF-preserved fecal specimens was developed to allow the microscopist to screen for acid-fast organisms in addition to other intestinal parasites (Fig. 3.21). For laboratories using iron hematoxylin stains in combination with SAF-fixed material and modified acid-fast stains for Cryptosporidium, Cyclospora, and Cystoisospora, this modification represents another staining option (49). This combination stain provides a saving in both time and personnel use. However, some laboratories prefer to use these two stains as single methods; the overall result may be better than the combination approach. The selection of one approach or the other would be up to the laboratory and would be based on personal preferences.
Figure 3.21 Iron hematoxylin stain incorporating the carbol fuchsin step. Note the modified acid-fast Cryptosporidium oocysts (4–6 µm) and the Giardia lamblia cyst. doi:10.1128/9781555819002.ch3.f21
Any fecal specimen submitted in SAF, other non-mercury single-vial-system preservatives, or the Universal Fixative can be used. Fresh fecal specimens after fixation in SAF for 30 min can also be used. This combination stain approach is not recommended for specimens preserved in Schaudinn’s fixative using a mercuric chloride base (with or without PVA).
Mayer’s Albumin
Add an equal quantity of glycerin to a fresh egg white. Mix gently and thoroughly. Store at 4°C, and indicate an expiration date of 3 months. Mayer’s albumin from commercial suppliers can normally be stored at 25°C for 1 year.
Stock Solution of Hematoxylin Stain
1. Mix well until dissolved.
2. Store in a clear-glass bottle in a light area. Allow to ripen for 14 days before use.
3. Store at room temperature with an expiration date of 1 year.
Mordant
Ferrous ammonium sulfate
Ferric ammonium sulfate
Working Solution of Hematoxylin Stain
1. Mix equal quantities of stock solution of stain and mordant.
2. Allow the mixture to cool thoroughly before use (prepare at least 2 h prior to use). The working solution should be made fresh every week.
Picric Acid
Mix equal quantities of distilled water and an aqueous saturated solution of picric acid to make a 50% saturated solution.
Add enough ammonia to bring the pH to approximately 8.0.
Carbol Fuchsin
1. To make basic fuchsin (solution A), dissolve 0.3 g of basic fuchsin in 10 ml of 95% ethanol.
2. To make phenol (solution B), dissolve 5 g of phenol crystals in 100 ml of distilled water. (Gentle heat may be needed.)
3. Mix solution A with solution B.
4. Store at room temperature. The solution is stable for 1 year.
Procedure for Modified Iron Hematoxylin Stain (Carbol Fuchsin Step)
1. Prepare slide.
A. Place 1 drop of Mayer’s albumin on a labeled slide.
B. Thoroughly mix the sediment from the fecal concentration with an applicator stick.
C. Add approximately 1 drop of the fecal concentrate to the albumin, and spread the mixture over the slide.
2. Allow the slide to air dry at room temperature (the smear appears opaque when dry).
3. Place the slide in 70% alcohol for 5 min.
4. Wash the slide in a container of tap water (not under running water) for 2 min.
5. Place the slide in Kinyoun’s stain for 5 min.
6. Wash the slide in running tap water (constant stream of water into container) for 1 min.
7. Place the slide in acid-alcohol decolorizer for 4 min.*
8. Wash the slide in running tap water (constant stream of water into container) for 1 min.
9. Place the slide in iron hematoxylin working solution for 8 min.
10. Wash the slide in distilled water (in container) for 1 min.
11. Place the slide in picric acid solution for 3 to 5 min.
12. Wash the slide in running tap water (constant stream of water into container) for 10 min.
13. Place the slide in 70% alcohol plus ammonia for 3 min.
14. Place the slide in 95% alcohol for 5 min.
15. Place the slide in 100% alcohol for 5 min.
16. Place the slide in two changes of xylene for 5 min.
*This step can also be performed as follows.
A. Place the slide in acid-alcohol decolorizer for 2 min.
B.
