Ova and Parasite (O&P) Examinations

Introduction | Macroscopic | Microscopic |

Microscopic Examination - Ova and Parasite Examination

Introduction | Direct Wet Smear | Concentration | Permanent Stained Smear

Permanent Stained Smear

Introduction | Trichrome Stain | Iron Hematoxylin Stain | Modified Iron Hematoxylin Stain | Procedure Review

Trichrome Stain

Procedures

Mercury-Based Fixatives

Note: In all staining procedures for fecal and gastrointestinal tract specimens, the term xylene will be used in the generic sense. Xylene substitutes are recommended for the safety of all personnel performing these procedures.

Prepare slide for staining as described above.
Remove slide from Schaudinn's fixative and place slide in 70% ethanol for 5 min.
Place slide in 70% ethanol plus iodine (strong tea color) for 1 min for fresh specimens or 5 to 10 min for PVA air-dried smears.
Place slide in 70% ethanol for 5 min.
Place in second 70% ethanol for 3 min.*
Place in trichrome stain for 10 min.
Place in 90% ethanol plus acetic acid for 1 to 3 s. Immediately drain the rack (see Procedure Notes) and proceed to the next step. Do not allow slides to remain in this solution.
Dip several times in 100% ethanol. Use this step as a rinse.
Place in two changes of 100% ethanol for 3 min each.*
Place in xylene for 5 to 10 min.*
Place in second xylene for 5 to 10 min.*
Mount with coverslip (no. 1 thickness), using mounting medium (e.g., Permount). Note An alternative method to using mounting medium is as follows:

Remove the slide from the last xylene, place it on a paper towel (flat position), and allow it to air dry. Remember that some of the xylene substitutes may take a bit longer to dry.
Approximately 5 to 10 min before you want to examine the slide, place a drop of immersion oil on the dry fecal film. Allow the oil to sink into the fecal film for a minimum of 10 10 to 15 min. If the smear appears to be very refractile on examination, you have not waited long enough for the oil to sink into the film or you need to add a bit more oil onto the film.
Once you are ready to examine the slide, place a no. 1 (22- by 22-mm) coverslip onto the oiled smear, add another drop of immersion oil onto the top of the coverslip (as you would normally do for any coverslipped slide), and examine with the oil immersion lens (100 x objective).
Make sure that you do not eliminate adding the coverslip; the dry fecal material on the slide often becomes very brittle after dehydration. Without the addition of the protective coverslip, you might scratch the surface of the oil immersion lens. Coverslips are much cheaper than oil immersion objectives!

Allow the smear to dry overnight or after 1 h at 37°C.
Examine the smear microscopically with the 100 x objective. Examine at least 200 to 300 oil immersion fields before reporting a negative result.

*Slides may be held up to 24 h in these solutions without harming the quality of the smear or stainability of organisms.

Non-Mercury-Based Fixatives

Prepare slide for staining as described above.
Place slide in 70% ethanol for 5 min.*
Place in trichrome stain for 10 min. Some people prefer to place the dry smear directly into the stain and eliminate step 2 from the protocol.
Place in 90% ethanol plus acetic acid for 1 to 3 s. Immediately drain the rack (see Procedure Notes) and proceed to the next step. Do not allow slides to remain in this solution.
Dip several times in 100% ethanol. Use this step as a rinse.
Place in two changes of 100% ethanol for 3 min each.*
Place in xylene for 5 to 10 min.*
Place in second xylene for 5 to 10 min.*
Mount with coverslip (no. 1 thickness), using mounting medium (e.g., Permount). Note: An alternative method to using mounting medium is as follows:

Remove the slide from the last xylene, place it on a paper towel (flat position), and allow it to air dry. Remember that some of the xylene substitutes may take a bit longer to dry.
Approximately 5 to 10 min before you want to examine the slide, place a drop of immersion oil on the dry fecal film. Allow the oil to sink into the fecal film for a minimum of 10 to 15 min. If the smear appears to be very refractile on examination, you have not waited long enough for the oil to sink into the film or you need to add a bit more oil onto the film.
Once you are ready to examine the slide, place a no. 1 (22- by 22-mm) coverslip onto the oiled smear, add another drop of immersion oil onto the top of the coverslip (as you would normally do for any coverslipped slide), and examine with the oil immersion lens (100 x objective).
Make sure that you do not eliminate adding the coverslip; the dry fecal material on the slide often becomes very brittle after dehydration. Without the addition of the protective coverslip, you might scratch the surface of the oil immersion lens. Coverslips are much cheaper than oil immersion objectives!

Allow the smear to dry overnight or after 1 h at 37°C.
Examine the smear microscopically with the 100 x objective. Examine at least 200 to 300 oil immersion fields before reporting a negative result.

*Slides may be held up to 24 h in these solutions without harming the quality of the smear or stainability of organisms.