Ova and Parasite (O&P) Examinations

Introduction | Macroscopic | Microscopic |

Microscopic Examination - Ova and Parasite Examination

Introduction | Direct Wet Smear | Concentration | Permanent Stained Smear

Concentration

Procedures

Transfer 1/2 teaspoon (about 4 g) of fresh stool into 10 ml of 5 or 10% formalin in a round-bottom tube (container may be modified to suit individual laboratory preferences). Mix the stool and formalin thoroughly and let the mixture stand for a minimum of 30 min for fixation. If the specimen is already in 5 or 10% formalin (or SAF), restir the stool-formalin mixture.
Depending on the amount and viscosity of the specimen, strain a sufficient quantity through wet gauze (no more than two layers of gauze and one layer if the new "pressed'' gauze [e.g., Johnson & Johnson nonsterile three-ply gauze, product no. 7636] is used) into a conical 15-ml centrifuge tube to give the desired amount of sediment (0.5 to 1 ml) in step 3 below. Usually 8 ml of the stool-formalin mixture prepared in step 1 will be sufficient. If the specimen is received in vials of preservative (5 or 10% formalin or SAF), then approximately 3 to 4 ml will be sufficient unless the specimen has very little stool in the vial. If the specimen contains a lot of mucus, do not strain through gauze but immediately fix in 5 or 10% formalin for 30 min and centrifuge for 10 min at 500 x g. Then proceed directly to step 10.
Add 0.85% NaCl or 5 or 10% formalin (some workers prefer to use formalin for all rinses) almost to the top of the tube and centrifuge for 10 min at 500 x g. The amount of sediment obtained should be approximately 0.5 to 1 ml.
Decant the supernatant fluid and resuspend the sediment in saline or formalin; add saline or formalin almost to the top of the tube and centrifuge again for 10 min at 500 x g. This second wash may be eliminated if the supernatant fluid after the first wash is light tan or clear. Some prefer to limit the wash step to one (regardless of the clarity or color of the supernatant fluid after centrifugation) to eliminate additional manipulation of the specimen prior to centrifugation. Each time the specimen is washed, some organisms may be lost through manipulation.
Decant the supernatant fluid and resuspend the sediment on the bottom of the tube in 5 or 10% formalin. Fill the tube half full only. If the amount of sediment left in the bottom of the tube is very small or the original specimen contained a lot of mucus, do not add ethyl acetate in step 6; merely add the formalin, spin, decant, and examine the remaining sediment.
Add 4 to 5 ml of ethyl acetate. Stopper the tube and shake vigorously for at least 30 s. Hold the tube so the stopper is directed away from your face.
After a 15- to 30-s wait, carefully remove the stopper.
Centrifuge for 10 min at 500 x g. Four layers should result: a small amount of sediment (containing the parasites) in the bottom of the tube; a layer of formalin; a plug of fecal debris on top of the formalin layer; and a layer of ethyl acetate at the top.
Free the plug of debris by ringing the plug with an applicator stick; decant all of the supernatant fluid. After proper decanting, a drop or two of fluid remaining on the side of the tube may run down into the sediment. Mix this fluid with the sediment.
If the sediment is still somewhat solid, add 1 or 2 drops of saline or formalin to the sediment, mix, add a small amount of material to a slide, add a coverslip (22 by 22 mm, no. 1), and examine.
Systematically scan with the 10 x objective. The entire coverslip area should be examined under low power (total magnification of x 100).
If something suspicious is seen, the 40 x objective can be used for more detailed study. At least one-third to one-half of the coverslip should be examined under high dry power (total magnification of x 100) even if nothing suspicious has been seen. As in the direct wet smear, iodine can be added to enhance morphological detail, and you can tap on the coverslip in order to see objects move and turn over.