Selection of the proper procedure is paramount in the successful diagnosis of protozoan infections in man and animals. A understanding of the type of sample needed, the proper procedure called for and complicating factors that might hinder parasite recovery are all important considerations when attempting to identify parasitic protozoan organisms.
Sample collection and storage.
Feces
Samples should be collected in a clean, dry container fitted with a tight-fitting lid. Samples should be about 50 gr. of fresh feces that is free of contamination with blood, urine and litter. Microscopic examination should be accomplished as soon as is possible. Diarrhea samples should be examined within 30 minutes of collection and should be stored at room temperature. Normal stools can be stored under refrigeration or the feces can be mixed with a preservative such as polyvinyl alcohol , 10% formalin, or merthiolate-iodine-formaline solution.
Blood
Blood films can be made with fresh blood or blood collected in EDTA. Care should be taken when handling samples since hemolysis of erythrocytes will cause destruction of intraerythrocytic parasites.
Tissue impressions.
Tissue preparations are made from any biopsy or postmortem material. The cut edge of the tissue should be blotted to remove excess blood and the specimen touched to a clean microscope slide. This preparation can then be fixed and stained as for thin blood films.
Aspirates
Needle aspirates from many tissues often contain motile trophozoites so should be examined immediately. Other material can be smeared on a microscope slide and treated as tissue impressions.
Examination of material
Feces
Direct smear.
The direct smear technique is used to detect motile trophozoites. This is most common for diarrheic or mucoid samples. This procedure involves placing a drop of saline on a clean slide and applying a coverslip to the material. The consistency of the sample should be such that newsprint can be read through it. The light on the microscope should be adjusted down since the unstained organisms appear transparent.
Fecal flotation
Fecal flotation procedures are used to concentrate parasitic stages from fecal material. The various flotation solutions are made so that their specific gravity is greater that that of the parasitic stages, thus the stages float to the surface and the debree settles to the bottom. Many different concentration solutions are used, each of which has advantages and disadvantages. This procedure is most useful in concentrating helminth eggs and protozoan cysts. Various modifications such as sedimentation and centrifugation techniques are also used for this same purpose.
Blood
Thin blood film.
This procedure is used to identify both interrythrocytic parasites and those free in the serum. A small drop of blood is placed at one end of a clean microscope slide. The end of another slide, held at a 30 degree angle is placed in the middle of the blood drop. The blood is allowed to spread along the width of the spreader slide and then in a rapid even motion, the spreader slide is pushed across the length of the original slide. The film is now air dried at room temperature. The preparation must now be fixed in methyl alcohol and stained with a Giemsa-type stain. This preparation, when dried and stained, can be examined microscopically under oil immersion.
Other procedures
Various culture procedures are used to increase the chances of detecting parasitic protozoan parasites. Many culture media exist and various liquid and cell cultures are available for some organisms. These procedures are most useful in identifying low level infections that might not be sufficient in number to be detected under routine procedures.
Animal inoculation is also useful for this same reason. Specifics about animal susceptibility, route of inoculation, prepatient time, etc. should be determined prior to using this procedure.
Immunodiagnostic procedures.
Serologic testing for many parasitic organisms (detection of either antibody against the parasite or the presence of parasite antigen) are now available. Specifics about the type of sample needed, collection procedures, and test sensitivity should be determined before considering this procedure.