Figure 3.17 Guess the identity of these intestinal protozoa! See p. 76 for the answers. doi:10.1128/9781555819002.ch3.f17
*Slides may be held for up to 24 h in these solutions without harming the quality of the smear or stainability of organisms.
Procedure for Trichrome Stain with Non-Mercury-Based Fixatives (Iodine-Alcohol Step and Alcohol Rinse Not Required) (Fig. 3.16)
1. Prepare the slide for staining as described above.
2. Place the slide in 70% ethanol for 5 min.*
3. Place it in the trichrome stain for 10 min. Some people prefer to place the dry smear directly into the stain and eliminate step 2 from the protocol.
4. Place it in 90% ethanol plus acetic acid for 1 to 3 s. Immediately drain the rack (see Procedure Notes), and proceed to the next step. Do not allow slides to remain in this solution.
5. Dip the slide several times in 100% ethanol. Use this step as a rinse.
6. Place it in two changes of 100% ethanol for 3 min each.*
7. Place it in xylene or xylene substitute for 5 to 10 min.*
8. Place it in a second container of xylene or xylene substitute for 5 to 10 min.*
9. Mount with a coverslip (no. 1 thickness), using mounting medium (e.g., Permount).
10. Allow the smear to dry overnight or after 1 h at 37°C.
11. 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.
Results and Patient Reports from the Trichrome Staining Method
Protozoan trophozoites and cysts are readily seen, although helminth eggs and larvae may not be easily identified because of excess stain retention (wet smears from the concentration procedure[s] are recommended for detection of these organisms) (Fig. 3.18 and 3.19). Yeasts (single and budding cells and pseudohyphae) and human cells (macrophages, PMNs, and RBCs) can be identified. The following quantitation chart can be used for examination of permanent stained smears with the oil immersion lens (100× objective; total magnification, ×1,000).
Figure 3.18 Intestinal protozoa (stained with Wheatley trichrome stain). Rows, from top to bottom (left and right), show Iodamoeba bütschlii trophozoite and I. bütschlii cyst; Entamoeba histolytica trophozoite (note the ingested RBCs) and Entamoeba histolytica/E. dispar cyst; Giardia lamblia (G. duodenalis, G. intestinalis) trophozoite and G. lamblia cyst; Entamoeba coli trophozoite and E. coli cyst; and Endolimax nana trophozoite and E. nana cyst. doi:10.1128/9781555819002.ch3.f18
Figure 3.19 Intestinal protozoa (preserved with TOTAL-FIX, stained with Wheatley trichrome stain). From top to bottom and left to right. Top row: Giardia lamblia (G. duodenalis, G. intestinalis), 2 trophozoites, 2 cysts. Second row: Giardia lamblia (G. duodenalis, G. intestinalis), 3 trophozoites, 1 cyst. Third row: Endolimax nana, 3 trophozoites, 1 cyst. Note the nuclear variation in the three trophozoites (very common). Fourth row: Entamoeba coli, 3 trophozoites and 1 cyst. Fifth row: Dientamoeba fragilis (3 trophozoites) and Entamoeba hartmanni (1 trophozoite). doi:10.1128/9781555819002.ch3.f19
| Quantitation of parasites, cells, yeasts, and artifacts | |
| Quantity | No. per 10 oil immersion fields (×1,000) |
| Few Moderate Many | ≤2 3–9 ≥10 |
1. Report the organism and stage (do not use abbreviations).
Examples: Entamoeba histolytica/E. dispar trophozoites
Giardia lamblia (G. duodenalis, G. intestinalis) trophozoites
2. Quantitate the number of Blastocystis spp. organisms seen (rare, few, moderate, many). Do not quantitate other protozoa.
Example: Moderate Blastocystis spp.
3. Note and quantitate the presence of human cells.
Example: Moderate WBCs, many RBCs, few macrophages, rare Charcot-Leyden crystals
4. Report and quantitate yeast cells.
Example: Moderate budding yeast cells and few pseudohyphae
Note Because yeast can continue to grow if the stool is not immediately preserved, some laboratories do not report yeast, since the report can be misleading. They elect to call the physician and discuss the findings. Another option is to add a report comment indicating that reports of yeast (budding and/or pseudohyphae) might be misleading due to a lag time between stool passage and specimen fixation.
5. Save positive slides for future reference. Label prior to storage (name, patient number, organisms present). Most laboratories discard their negative permanent stained smears after several weeks (rotate storage boxes and discard when full).
Procedure Notes for the Trichrome Staining Method
1. The single most important step in the preparation of a well-stained fecal smear is good fixation. If this has not been done, the protozoa may be distorted or shrunk, may not be stained, or may exhibit an overall pink or red color with poor internal morphology.
2. Slides should always be drained between solutions. Touch the end of the slide to a paper towel for 2 s to remove excess fluid before proceeding to the next step. This will maintain the staining solutions for a longer period. The slide can also be touched against the staining dish to drain off excess fluid before moving on to the next dish.
3. Incomplete removal of mercuric chloride (Schaudinn’s fixative with a mercuric chloride base [with or without PVA]) may cause the smear to contain highly refractive crystals or granules, which may prevent finding or identifying any organisms present. Since the 70% ethanol-iodine solution removes the mercury complex, it should be changed at least weekly to maintain the strong-tea color. A few minutes are usually sufficient to keep the slides in the iodine-alcohol; too long a time in this solution may also adversely affect the staining of the organisms.
4. When using non-mercury-based fixatives, the iodine-alcohol step (used for the removal of mercury) and the subsequent alcohol rinse (removal of the iodine) can be eliminated from the procedure. The smears for staining can be prerinsed with 70% alcohol and then placed in the trichrome stain, or they can be placed directly into the trichrome stain as the first step in the staining protocol (Fig.
