Triple-Centrifugation Method for Trypanosomes
The triple-centrifugation procedure may be valuable in demonstrating the presence of trypanosomes in the peripheral blood when the parasitemia is light (4, 9) (Fig. 7.16).
1. Centrifuge anticoagulated blood for 10 min at 300 × g.
2. Remove the supernatant fluid, and transfer it to another centrifuge tube.
3. Centrifuge this fluid for 10 min at 500 × g.
4. Again, remove the supernatant fluid to another centrifuge tube.
5. Centrifuge this fluid for 10 min at 900 × g.
6. Decant the supernatant fluid, and examine the sediment as a wet preparation.
7. The sediment may be used to prepare thin films that can then be stained with any of the blood stains.
Note Remember, you are saving the supernatant fluid in step 2 for subsequent centrifugation; do not accidentally pour this fluid off for disposal. The final sediment after the third centrifugation step is used to prepare stained films for examination.
Special Stain for Microfilarial Sheath
Some of the material that is obtained from the concentration procedures can be allowed to dry as thick and thin films and then stained with Delafield’s hematoxylin, which demonstrates greater nuclear detail as well as the microfilarial sheath, if present. In addition, fresh thick films of blood containing microfilariae can be stained by this hematoxylin technique (Fig. 7.17 and 7.18) (4, 9).
Figure 7.17 Wuchereria bancrofti microfilaria on a thick film stained with Delafield’s hematoxylin. Note the presence of the sheath. (Image courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch7.f17
Figure 7.18 Wuchereria bancrofti microfilaria. (Upper) Microfilaria stained with Giemsa stain. Note that the sheath is not visible. (Lower) Microfilaria stained with Delafield’s hematoxylin stain. Note that the sheath is visible. doi:10.1128/9781555819002.ch7.f18
Preparation of Stain
Dissolve 180 g of aluminum ammonium sulfate in 1 liter of distilled water (saturated solution). Heat until dissolved. The cooled supernatant fluid is saturated aluminum alum.
Hematoxylin Solution
Dissolve the hematoxylin in alcohol, and add it to the alum solution. Expose the solution to sunlight and air for ripening in a clear, cotton-plugged bottle for approximately 1 week; then filter and add the following:
Age for 1 month or longer in sunlight, and then run test smears to determine whether the solution is properly aged. Nuclei should stain blue, and cytoplasm should stain different shades of red.
Procedure
1. Prepare thick films from the concentration material or from fresh blood.
2. Allow the films to air dry.
3. Lake the films in 0.85% sodium chloride or distilled water for 15 min (not necessary for films prepared from the Knott sediment). Allow to air dry.
4. Fix all films in absolute methanol for 5 min. Allow to air dry.
5. Stain in undiluted Delafield’s hematoxylin for 10 to 15 min.
6. Wash off excess stain in tap water.
7. Intensify the blue color by placing the films into tap water containing several drops of ammonia (NH4OH) for several minutes.
8. Rinse again in running tap water for 5 min.
9. Air dry.
10. Mount in Permount or some other mounting medium; use a no. 1 coverglass.
References
1. Code of Federal Regulations. 1991. Occupational exposure to bloodborne pathogens. Fed Regist 29CFR1910.1030.
2. Field JW, Sandosham AA, Fong YL. 1963. The Microscopical Diagnosis of Human Malaria 1. A Morphological Study of the Erythrocytic Parasites in Thick Blood Films. Institute for Medical Research, Kuala Lumpur, Malaya.
3. Clinical and Laboratory Standards Institute. 2000. Laboratory Diagnosis of Blood-Borne Parasitic Diseases. Approved guideline M15-A. Clinical and Laboratory Standards Institute, Villanova, PA.
4. Isenberg HD (ed). 2004. Clinical Microbiology Procedures Handbook, 2nd ed., vol. 1, 2, and 3. ASM Press, Washington, D.C.
5. Isenberg HD (ed). 1995. Essential Procedures for Clinical Microbiology. American Society for Microbiology, Washington, D.C.
6. Garcia LS (ed). 2010. Clinical Microbiology Procedures Handbook, 3rd ed, vol 1, 2, and 3. ASM Press, Washington D.C.
7. Kokoskin E. 2001. The Malaria Manual. McGill University for Tropical Diseases, Montreal, Canada.
8. Spudick JM, Garcia LS, Graham DM, Haake DA. 2005. Diagnostic and therapeutic pitfalls associated with primaquine-tolerant Plasmodium vivax. J Clin Microbiol 43:978–981. PMID 15695723
9. Garcia LS. 2009. Practical Guide to Diagnostic Parasitology, 2nd ed. ASM Press, Washington, DC.
10. Shute PG. 1966. The staining of malaria parasites. Trans R Soc Trop Med Hyg 60:412–416. PMID 4162149
11. Shute PG, Maryon ME. 1966. Laboratory Technique for the Study of Malaria, 2nd ed. J. & A. Churchill, Ltd., London, United Kingdom.
12. Hollander DH. 1963. An oil-soluble antioxidant in resinous mounting media to inhibit fading of Romanowsky stains. Stain Technol 38:288–289.
13. Bruckner DA, Garcia LS, Shimizu RY, Goldstein EJC, Murray PM, Lazar GL. 1985. Babesiosis: problems in diagnosis using autoanalyzers. Am J Clin Pathol 83:520–521. PMID 2984920
14. Garcia LS, Shimizu RY, Bruckner DA. 1986. Blood parasites: problems in diagnosis
