Ova and Parasite (O&P) Examinations

Microscopic Examination - Ova and Parasite Examination

The gauze should never be more than one (pressed gauze) or two (woven gauze) layers thick; more gauze may trap mucus (containing Cryptosporidium oocysts and/or microsporidial spores).
Tap water may be substituted for 0.85% NaCl throughout this procedure, although the addition of water to fresh stool may cause Blastocystis hominis cysts (central body forms) to rupture and is not recommended. In addition to the original 5 or 10% formalin fixation, some workers prefer to use 5 or 10% formalin for all rinses throughout the procedure.
Ethyl acetate is widely recommended as a substitute for ether. It can be used in the same way in the procedure and is much safer. Hemo-De can also be used and is felt to be safer than ethyl acetate.

After the plug of debris is rimmed and excess fluid is decanted, while the tube is still upside down, the sides of the tube can be swabbed with a cotton-tipped applicator stick to remove excess ethyl acetate. This is particularly important to do if you are working with plastic centrifuge tubes or the plastic commercial concentrators. If the sediment is too dry after swabbing the tube, then add several drops of saline before preparing the wet smear for examination.
If there is excess ethyl acetate in the smear of the sediment prepared for examination, there will be bubbles present which will obscure the material that you are trying to see.

If fecal specimens are received in SAF, then begin the procedure at step 2.
If specimens are received in PVA, the first two steps of the procedure should be modified as follows.

Immediately after stirring the stool-PVA mixture with applicator sticks, pour approximately one-half of the mixture into a tube (container optional) and add 0.85% NaCl (or 5 or 10% formalin) almost to the top of the tube.
Filter the stool-PVA-saline (or formalin) mixture through wet gauze into a 15-ml centrifuge tube. The standard procedure is followed from here to completion, beginning with step 3.

Too much or too little sediment will result in an ineffective concentration.
The centrifuge should reach the recommended speed before the centrifugation time is monitored. However, since most laboratories have their centrifuges on automatic timers, the centrifugation time in this protocol takes into account the fact that some time will be spent coming up to speed prior to full-speed centrifugation. If the centrifugation time at the proper speed is reduced, some of the organisms (Cryptosporidium oocysts or microsporidian spores) may not be recovered in the sediment.