style="font-size:15px;"> 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 intraperitoneally into three to five mice of any laboratory strain weighing ∼20 g.
C. Maintain mice in isolation.
D. Every day, check for signs of central nervous system dysfunction; if symptoms are detected, perform an autopsy on the animal.
Examination of Specimens
1. Examination of impression smears is summarized in Table 6.1.
2. Examine skin snips in teased preparations for detection of microfilariae of Onchocerca volvulus and Mansonella streptocerca as follows.
A. Tease the small bit of tissue apart in a few drops of saline to release the microfilariae.
B. Remove drops of saline to a 1- by 3-in. glass slide, cover with a no. 1 coverslip, and examine under low light for microfilariae.
C. For a permanent preparation, place 100% methanol under the coverslip to fix filariae, partially dry, remove the coverslip, and stain with Giemsa or one of the other blood stains (including the rapid stains).
3. Examine squash preparations for detection of Trichinella spp. in muscle microscopically at low power (×100) and under low light.
4. Examine scrapings of skin for Sarcoptes scabiei (scabies) microscopically at low power (×100) and under low light. Confirmation at high dry power (×400) may be necessary (Fig. 6.16). See chapter 35 on arthropods for specific/detailed skin-scraping protocol for scabies.
Figure 6.16 Sarcoptes scabiei mites from skin scrapings. (Upper) Note the two mites to the left of center. (Lower) Note the two eggs, the small nymph, and the adult mite (photographed at a higher magnification). If the light is too strong when examining the scrapings in saline, the mites will probably not be seen. doi:10.1128/9781555819002.ch6.f16
5. Inoculate cultures with ground tissue suspensions (to release organisms from the cells).
A. Place a small tissue sample in a sterile tissue grinder (Ten Broeck or Dounce) in 0.5 ml of sterile saline, and grind until tissue is broken up but not totally liquefied.
B. Add several drops of ground tissue to culture medium as follows.
a. NNN medium (see chapter 8): add drops of tissue to the bottom of the slant, where they will “pool” with condensed moisture. Incubate at room temperature (isolation of Leishmania spp.).
b. TYSGM-9 medium (see chapter 8): add drops of tissue to the liquid medium, add 3 drops of the starch suspension, and incubate at a 45 to 50° angle at 35°C for 48 h (isolation of E. histolytica).
c.
