(Lower left) Microsporidial spores in a urine sediment; (lower right) spores from nasopharyngeal aspirate (both images stained using calcofluor white). doi:10.1128/9781555819002.ch6.f12
1. Place corneal scrapings on slides, and air dry them.
2. Fix the slides in absolute methyl alcohol for 3 to 5 min.
3. Add several drops of solution (0.1% calcofluor white and 0.1% Evans blue dissolved in distilled water); leave on for 5 min.
4. Turn the slide on its side, and allow excess stain to run off onto paper towels.
5. Add a coverslip, and examine under the fluorescence microscope for pale blue chemofluorescence of the amebic cysts (will not stain trophozoite). The microsporidial spores usually stain brighter; however, fluorescence can vary between 1+ and 3+.
Note Select UV irradiation with an exciter filter which transmits the 365-nm group of intense mercury spectral emission lines (Zeiss UGI or G365). View through a barrier filter which removes UV while emitting visible blue light and longer wavelengths (Zeiss no. 41 or LP420).
Leishmaniasis
Material containing intracellular Leishmania organisms must be aspirated from below the ulcer bed through the uninvolved skin, not from the surface of the ulcer (Fig. 6.13). It is very important that the surface of the ulcer be thoroughly cleaned before specimens are taken; any contamination of the material with bacteria or fungi may prevent recovery of organisms from culture. Cutaneous ulcers can be seen in Fig. 6.14.
Figure 6.13 Proper way to aspirate material from below the ulcer bed (Leishmania spp.); sterile saline (0.5 to 1.0 ml) can be injected under the ulcer prior to aspiration. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch6.f13
Figure 6.14 Leishmania cutaneous lesions. doi:10.1128/9781555819002.ch6.f14
Biopsy specimens are recommended for the diagnosis of tissue parasites. The following procedures may be used for this purpose in addition to standard histologic preparations: impression smears and teased and squash preparations of biopsy tissue from the skin, muscle, corneas, intestines, liver, lungs, and brain. Tissue to be examined by permanent sections or electron microscopy should be fixed as specified by the laboratories that will process the tissue. In certain cases, examination of a biopsy specimen may be the only means of confirming a suspected parasitic infection. Specimens that are going to be examined as fresh material rather than as tissue sections should be kept moist in saline and submitted to the laboratory immediately.
Importance of Biopsy Specimens
Success in detection of parasites in tissue depends in part on specimen collection and on the presence of sufficient material to perform the recommended diagnostic procedures. Biopsy specimens are usually quite small and may not be representative of the diseased tissue. Examination of multiple tissue samples often improves diagnostic results. To optimize the yield from any tissue specimen, examine all areas and use as many procedures as possible. Tissues are obtained by invasive procedures, many of which are very expensive and lengthy; consequently, these specimens deserve the most comprehensive procedures possible. It is also important to remember that if the tissue specimen is not sufficient for all diagnostic procedures requested, the procedures should be prioritized after consultation with the physician.
Submission of Specimens
Tissue submitted on a sterile sponge dampened with saline in a sterile container may be used for cultures of protozoa after mounts for direct examination or impression smears for staining have been prepared. If cultures for parasites will be included, sterile slides should be used for smear and mount preparation.
1. Use sterile slides for impression smears; slides can be either autoclaved or prepared by an alternative method in which they are soaked in 95% ethyl alcohol and flamed prior to use.
2. Use sterile (autoclaved or flamed) forceps for handling tissue.
3. Place tissue in sterile petri dish to examine macroscopically and select a sample for microscopic evaluation. Provided that it is kept sterile, minced tissue can be used.
A. If biopsy tissue is several millimeters to a centimeter in size, select the specimen from an abnormal area (granulomatous lung or ulcerated area of intestinal tissue). Generally, the tissue fragments are too small to permit such a judgment to be made.
B. If several small pieces of tissue that look alike are submitted, use one piece. However, if they look different, use one of each type for microscopic examination. Again, sometimes this assessment to make.
4. Prepare impression smears.
A. Blot the tissue sample on sterile toweling. If the sample size is sufficient, cut the tissue with a scalpel (very sharp cut) and use the cut surface to touch the slide.
B. Press tissue against the sterile slide, lift, and press again. Turn the sample over, and press the other end against the slide to make two more impressions. Keep impressions close together to speed the screening process with the microscope.
C. Air dry and fix smears in absolute methanol for 1 min for subsequent blood stain, methenamine-silver nitrate, modified acid-fast, and modified trichrome staining. If the amount of tissue is sufficient, multiple smears should be prepared for each stain (Fig. 6.15).
Figure 6.15 (Upper) Leishmania donovani amastigotes in a cell; the one on the right is stained with Giemsa stain. (Lower left) Leishmania amastigote; note the nucleus and bar (primitive flagella). (Lower right) Cryptosporidium spp. oocysts stained with modified acid-fast stain. Note that the sporozoites are visible within some of the oocysts. doi:10.1128/9781555819002.ch6.f15
D. Place wet slide in Schaudinn’s fixative for subsequent trichrome staining.
E. Fix the slide as specified by the manufacturer for immunospecific staining.
5. Teased preparations are made as follows.
A. Place sample in the bottom of a plastic petri dish, and cover with 2 to 4 drops of saline.
B. Gently tease tissue apart with needles, or hold the tissue with forceps while pulling apart with a scalpel or needles.
C. Put a cover on dish, and leave at room temperature for 30 min.
6. Squash preparations are made as follows.
A. Using a scalpel, cut selected tissue portions into very fine pieces.
B. Place a piece of tissue on a 1- by 3-in. slide, add 1 drop of saline, cover with a second 1- by 3-in. slide, and hold together with membrane clips (from a surgical supply company). If these are not available, paper clips can be used but are not as efficient. You can also press the slides together with your fingers, but always wear gloves and be careful not to contaminate the microscope stage with tissue fluids.
7. Skin scrapings should be submitted in a small vial.
8. Prepare cultures to demonstrate the following organisms (see chapter 8): E. histolytica, Acanthamoeba and Naegleria spp., and Leishmania spp.
9. Mouse passage for Toxoplasma gondii:
A. Grind tissue in 0.85% NaCl into a fine suspension.
B. Inject 0.2 to 0.4 ml of suspension