Name That Bug: An Overview of Techniques for Identification of Parasitic Protozoa
IAAAM 1994
Sarah L. Poynton, BSc, PhD
Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC

Many protozoa are serious pathogens of aquatic animals. Over 2,000 species have been described from fish; yet in contrast relatively few protozoan species have been described from invertebrate, pinnipeds and cetaceans hosts. Because protozoa require different techniques for their identification than do the larger and more readily identified helminth parasites, there are many gaps in our knowledge. Lack of detailed identifications has hampered our understanding of such aspects as host-parasite specificity and susceptibility, the pathogenicity of individual species, and geographic ranges.

For an exact, reliable determination of protozoans, a thorough record of the life stages must be made. In most cases, this includes the use of both fresh smears and stained material. In vivo observations are indispensable for identifying members of most groups, since fixation induces an uncontrollable degree of shrinkage, distorts the shape of most protozoa, and makes some important diagnostic characters unrecognizable. Measurement of myxosporean and microsporidian spores, as well as coccidian oocysts, should always be done using fresh material. Certain protozoa, such as flagellates and ciliates, have characteristic motility, which is readily observed and can be recorded on video tape, or in digital format.

Observations of nuclear, pellicular, and infraciliary structures necessitate the use of stained preparations. For flagellates, ciliates and opalinids, Protargol (silver protein) staining is essential to visualize the arrangements of flagella, cilia, and nuclei. In the mobile peritrich ciliates, (the trichodinids), a Klein's silver nitrate stain is used to demonstrate the components of the adhesive disc. Only in a few groups such as the hemogregarines, does species identification depend exclusively upon stained preparations. In histological sections, special stains can be used to demonstrate special features of certain protozoan groups (Table 1).

Electron microscopy is a valuable tool for characterization of protozoa, with scanning EM being used to visualize surface features, and transmission EM being used to visualize internal ultrastructural features. In some taxa, such as the microsporidia, TEM is essential for determination of genus; in many taxa, such as the diplomonad flagellates, EM is essential to distinguish species within a genus.

Cultured and cryopreserved organisms offer a readily available source of standard organisms that can be used for such purposes as comparative studies, and experimental infections.

Descriptions of new taxa should follow the most recent specific guidelines (e.g. for coccidia see Bandoni & Dusynski, 1988). Naming of new taxa should follow the International Code of Zoological Nomenclature (Ride et al., 1985). Representative specimens of new taxa should be deposited in a museum, such as the International Protozoan Type Slide Collection at the Natural History Museum at the Smithsonian Institution in Washington, D.C., and cultures may be deposited at the American Type Culture Collection (ATCC) Rockville, MD.

Table 1. Special stains for protozoa in tissue section (from Gardiner, Fayer & Dubey, 1988; Murchelano & MacLean, 1990)





Gomori's methenamine










mature oocysts of





Azure eosin/Giemsa

polar capsules



polar capsules &

spore walls




Ziehl Nielsen acid fast

polar capsules &

spore walls




Giemsa Colophonium



Good pastures aniline-



carbol fuchsine






Ziehl-Nielsen acid fast



Periodic Acid Schiff (PAS)

polar cap

Recommended Literature


1.  Bandoni, S.M. & Duszynski, D.W. 1988. A plea for improved presentation of type material for coccidia. Journal of Parasitology 74 (4) 519 - 523.

2.  Lee, J.J. & Soldo, A.T. 1992. Protocols in Protozoology. Society of Protozoologists, Lawrence, Kansas.

Hosts and Parasites

1.  Dierauf, L. A. 1990. CRC Handbook of Marine Mammal Medicine. CRC Press Inc., Boca Raton, FL.

2.  Howard, E.B. 1983. Pathobiology of Marine Mammal Diseases. Volume 1. CRC Press Inc., Boca Raton, FL.

3.  Howard, E.B. 1983. Pathobiology of Marine Mammal Diseases. Volume II. CRC Press Inc., Boca Raton, FL.

4.  Kinne, 0. 1983. Diseases of Marine Animals. Volume 11. Introduction, Bivalvia to Scaphopoda. Biologische Anstalt Helgoland, Hamburg, FRG.

5.  Kinne, 0. 1984. Diseases of Marine Animals. Volume IV. Part 1. Introduction and Pisces. Biologische Anstalt Helgoland, Hamburg, FRG.

6.  Kinne, 0. 1985. Disease of Marine Animals. Volume IV, Part 2. Introduction, Reptilia, Aves, Mammalia. Biologische Anstalt Helgoland, Hamburg, FRG.

7.  Kinne, 0. 1990. Diseases of Marine Animals. Volume 111. Introduction, Cephalopoda, Annelida, Crustacea, Chaetognatha, Echinodermata, Urochordata. Biologische Anstalt Helgoland, Hamburg, FRG.

8.  Lee, J.J., Hutner, S.H. & Bovee, E.C. 1985. An Illustrated Guide to the Protozoa. Society of Protozoologists, Lawrence, Kansas.

9.  Lom, J. & Dykova, 1. 1992. Protozoan Parasites of Fishes. Elsevier.

10. Sindermann, C.J. 1990. Principal Diseases of Marine Fish and Shellfish. Vol. 1. Diseases of Marine Fish. Second Edition. Academic Press.

Parasite in Tissue Sections

1.  Cosgrove, G.E. 1975. Parasites in tissue sections: recognition and reaction. In Ribelin, W.E. & Migaki, G. The Pathology of Fishes. University of Wisconsin Press, Madison.

2.  Gardiner, C.H., Fayer, R. & Dubey, J.P. 1988. Atlas of Protozoan Parasites in Animal Tissues. U.S. Department of Agriculture.

3.  Murchelano, R.A. & MacLean, S.A. 1990. Histopathology Atlas of the Registry of Marine Pathology. U.S. Department of Commerce, National Oceanic and Atmospheric Administration.

4.  Ultrastructure Scholtyseck, E. 1979. Fine Structure of Parasitic Protozoa. Springer-Verlag, Berlin, Heidelberg, New York. Zoological Nomenclature Ride, W.D.L., Sabrosky, C.W., Bernardi, G, & Melville, R.V. 1985. International Code of Zoological Nomenclature. University of California Press, Berkeley and Los Angeles. Address Dr. Tom Nerad, Protistology Department, American Type Culture Collection, 12301 Parklawn Drive, Rockville, NM 20852 - 1776. Ph: 301 231 5516 Fx: 301 770 1848.

Speaker Information
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Sarah L. Poynton, BSc, PhD
Division of Comparative Medicine
Johns Hopkins University School of Medicine
Baltimore, MD, USA

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