Parasitology Exam Technique

Study Notes

Designed to facilitate recovery of protozoan cysts, coccidian oocysts, microsporidial spores, and helminth eggs and larvae.
Two types: flotation methods and sedimentation methods.
Both methods use differences in specific gravity and centrifugation to separate parasites from fecal debris and increase their recovery.

Parasites are less dense than the solutions used and float to the surface during centrifugation.
Produce a cleaner preparation.
Three types:
- Brine Flotation Method (by Willis)
- Zinc Sulfate Flotation Technique
- Sheather's Sugar Flotation Technique

Uses saturated table salt solution (brine solution).
Advantages: low cost and simple.
Disadvantages: not useful for operculated eggs like Clonorchis, Opistorchis, and Heterophyids, and helminth eggs of Hookworm and Schistosoma become badly shrunken.

Original procedure developed by Faust in 1938 for recovery of both helminth eggs and larvae and protozoan cysts.
Main reagent is 33% Zinc Sulfate Solution (most widely used reagent).
Specific gravity: 1.18 for fresh stool and 1.20 for preserved stool.
May not detect operculated or heavy eggs (Clonorchis eggs or unfertilized Ascaris eggs).

Parasites are concentrated in the sediment of the tube following centrifugation and the sediment is examined microscopically.
Best technique for recovery of:
- Schistosoma (heavy spine)
- Operculated eggs
- Trematode eggs
- Cestode eggs
- T.trichiura eggs
- C.philippinensis
Four types:
- Simple Gravity Sedimentation Technique
- Acid Ether Concentration technique (AECT)
- Formalin Ether/Ethyl Acetate Concentration Technique (FECT/FEACT)
- Merthiolate Iodine Formalin Ether Concentration Technique (MIFECT/MIFCT)

PROCEDURE: Stool + tap water à mix à allow parasites to settle down by gravity.
Glycerinated water (0.5% solution of glycerin in tap water) enhances sedimentation or settling of parasites.
Advantages:
- Minimal use of glassware, reagents, and equipment
- Useful in cases of Schistosoma and Strongyloides infections
- Sediments are useful for study and future references
- Allows settling of cysts, eggs, and larvae
Disadvantages:
- Considerable amount of fecal debris is incorporated into sediment
- Time-consuming

Principle: Hydrochloric acid is used to dissolve albuminous material.
Recommended for the recovery of Trichuris, Capillaria, and Schistosoma.
Method of choice if stool material comes from animals like cats and dogs.
Disadvantages:
- Loss of parasite to the plug of debris
- Possible destruction of protozoan cysts
- Ether is flammable and explosive – replaced with ethyl acetate (less efficient in the extraction of fat or mucoidal material from stool)

Most widely used sedimentation technique.
Main Reagents:
- Ether or ethyl acetate – dissolve neutral fats/lipids and carbohydrates in stool
- 40% Hydrochloric acid### Formalin Ether/Ethyl Acetate Concentration Technique
Formalin is an all-purpose fixative, and Ether/Ethyl Acetate is used to dissolve neutral fats/lipids and carbohydrates in stool, but Ether is explosive and flammable, so Ethyl Acetate is used as an alternative.
To prepare a fecal suspension, a small portion of stool (about the size of a thumb) is comminuted in 10 mL of 10% formalin and left to stand for 15-30 minutes for fixation to occur.
The suspension is then strained through one layer of wet gauze and received in a centrifuge tube.
Ether (3 mL) is added, and the mixture is shaken vigorously for 1 minute, then centrifuged at 1500 rpm for 2 minutes.
The acid-ether layers are poured off, and the sediment is loosened with an applicator stick.
The sediment is then shaken and transferred to a glass slide with a pipette, covered with a coverslip, and examined for parasites.

More parasites can be recovered from formalin-preserved samples, and morphology is better preserved in formalin than in PVA.
Sediments from FECT can be stored for a long period of time.
Disadvantages of FECT include loss of parasites to the plug of debris, possible destruction of protozoan cysts, and the flammability and explosiveness of ether.

This technique is recommended for the better identification of protozoan cysts.
Formalin is used as a fixative, Ether is used to dissolve neutral fats/lipids and carbohydrates in stool, and Merthiolate and Iodine are used as stains.
The stock Merthiolate Formaldehyde Solution is prepared by mixing distilled water, Tincture of Merthiolate, Glycerin, and Formaldehyde.
In the procedure, the stock MIF solution is mixed with Lugol's iodine solution and fecal specimen, and then shaken and centrifuged to separate the layers.
The sediment is then pipetted onto a glass slide, covered with a coverslip, and examined for parasites under the microscope.