With a Pasteur pipette, remove from the bottom of each tube the entire sediment and inoculate the sediment into fresh tubes containing rice starch and antibiotics.
9. Incubate as above for another 48 h.
10. Examine the tubes as before and discard the tubes if amebae are still not seen. Report patient results as negative.
Note The patient results should not be reported unless the quality control organisms and cultures are growing, thus indicating the culture system is performing according to expected results.
11. If amebae are present in small numbers, chill the tube in ice-water for 5 min and centrifuge the tube for 5 min at 250 × g. Aspirate and discard the supernatant, and inoculate the sediment into a fresh tube as before.
12. If amebae are present in large numbers, let the tube stand upright for 30 min and remove about 0.2 ml of sediment from the bottom. Inoculate the sediment into fresh tubes as before.
13. If you do not have an inverted microscope, stand the tubes upright for about 30 min at 35°C. With a sterile pipette, remove about 0.5 ml of sediment from the bottom of the tube and place a couple of drops onto each of two slides. Add 2 drops of methylene blue solution to one of the slides. Cover both with coverslips, and examine the slides under the microscope for amebae. Amebae may appear rounded or with pseudopodial extrusions. The nuclei may be clearly seen in the methylene blue preparation. Proceed to steps 7 through 12.
Axenic Culture
Axenic culture is used for research when strains of organisms are necessary for work requiring a culture system free from bacterial contaminants. If the axenic culture tubes become contaminated, 1,000 U of penicillin per ml and 1,000 µg of streptomycin or 50 µg of gentamicin per ml can be added to each tube. If, however, the contaminant happens to be Pseudomonas spp., it is probably better to discard the tube and use an uncontaminated tube for subculture purposes (3, 15).
1. Remove tubes containing TYI-S-33 medium from 4°C, and incubate at 35°C for 1 to 2 h.
2. With an inverted microscope, examine stock culture tubes of E. histolytica for any signs of bacterial contamination (no longer acceptable for use). Select one or several tubes showing good growth of amebae. Since the tubes are incubated in a slanted position, usually at an angle of 5 to 10°, a thick button of amebae will be seen at the bottom of the tube. Gently invert the tubes once or twice to disperse the amebae uniformly, and examine the tubes again. A majority of the amebae should be attached to the tube walls and show pseudopodial motility. If you do not have an inverted microscope, examine organisms from the bottom of the tube (as a wet smear). If you can see pseudopodial motility, proceed to step 3.
3. Immerse the tubes in a bucket of ice-cold water for about 5 to 10 min to dislodge the amebae from the tube walls. Invert the tubes several times to distribute the amebae.
4. Remove, with a Pasteur pipette, about 1.0 to 1.5 ml of culture medium; inoculate 0.5 to 1 ml into a fresh tube. Inoculate the rest of the fluid into nutrient agar, brain heart infusion, and thioglycolate broth for routine monitoring of bacterial contamination. Inoculate several tubes this way, and incubate the cultures slanted at 5 to 10° at 35°C as before.
5. If amebic growth is not good but some amebae are attached to the tube walls, remove about 10 ml of medium from the bottom with a serologic pipette and add 10 ml of fresh medium.
6. If amebic growth is not good and only few amebae are present along with a lot of debris, centrifuge the tube at 250 × g for 10 min, aspirate the supernatant fluid, and transfer the sediment to a fresh tube and incubate as before.
Quality Control for Intestinal Protozoan Cultures
The following control strains should be available when using these cultures for clinical specimens (3): ATCC 30925 (Entamoeba histolytica HU-1:CDC), ATCC 30015 (Entamoeba histolytica HK-9), and ATCC 30042 (Entamoeba histolytica-like, Laredo strain, culture at 25°C) (17).
1. Check all reagents and media (Balamuth’s aqueous egg yolk infusion medium, Boeck and Drbohlav’s LES medium, PBS solution no. 8, rice starch suspension, Tween 80 solution, and TYSGM-9 and TYI-S-33 media) each time they are used or periodically (once a week). The media and all solutions should be free of any signs of precipitation and bacterial and/or fungal contamination.
2. Maintain stock cultures of E. histolytica at 35°C (ATCC 30925 [strain HU-1:CDC] and ATCC 30015 [strain HK-9]). Maintain E. histolytica-like Laredo strain (ATCC 30042) at 25°C.
A. Transfer stock culture (ATCC 30925) every other day with TYSGM-9 medium.
B. Transfer stock culture (ATCC 30015) once every 3 days with TYI-S-33 medium.
C. Transfer stock culture (ATCC 30042) once a month with TYSGM-9 medium.
D. E. histolytica trophozoites measure 10 to 60 µm and demonstrate directional motility by extruding hyaline, finger-like pseudopodia from the cytoplasm. Cysts are not usually found in cultures.
E. Trophozoites are uninucleate and characterized by finely granular, uniform, evenly distributed peripheral chromatin. The nucleolus is small and usually centrally located but may be eccentric.
3. Depending on its use, the microscope(s) may need to be calibrated (within the last 12 months), and the original optics used for the calibration should be in place on the microscope(s). The calibration factors for all objectives should be posted on the microscope for easy access.
Note If the tubes containing fecal material are positive for amebae after 48 h of incubation, confirm the identification with the permanent stained smear. If the tubes do not show any amebae, subculture the contents of the tubes as described above and incubate for an additional 48 h. If the tubes are still negative for amebae, report the specimen as negative and discard the tubes. Even when the culture system is within quality control guidelines, a negative culture is still not definitive in ruling out the presence of E. histolytica. Xenic cultures of E. histolytica serve only as a supplemental procedure and never replace the primary diagnosis by microscopic examination of concentration sediments and permanent stained smears. Axenic culture is used to maintain quality control strains and for research purposes.
Specimens would include cerebrospinal fluid (CSF), biopsy tissue, and autopsy tissue of the brain; for Acanthamoeba spp., corneal scrapings or biopsy material, contact lenses and contact lens paraphernalia such as lens cases and solutions, skin abscess material, ear discharge, or feces can also be used. All clinical specimens should be processed within 24 h; 2 to 3 h is recommended. However, eye-related specimens can be shipped by mail with apparently few problems (18–21). The procedure for growing Naegleria and Acanthamoeba from clinical specimens involves the use of a nonnutrient agar spread with E. coli or some other nonmucoid bacteria. Amebas begin feeding on bacteria and soon grow to cover the agar surface in 1 to 2 days at 37°C. The presence of the protozoa can be confirmed by examining the agar surface using an inverted microscope or with a conventional microscope by inverting the plate on the stage and focusing through the agar with a 10× objective (Fig.
