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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


The detection and correct identification of many intestinal protozoa frequently depend on the examination of the permanent stained smear with the oil immersion lens (100 x objective). These slides not only provide the microscopist with a permanent record of protozoan organisms identified but also may be used for consultations with specialists when unusual morphological characteristics are found. Considering the morphological variations that are possible, organisms may be found that are very difficult to identify and do not fit the pattern for any one species.

Although an experienced microscopist can occasionally identify certain organisms on a wet preparation, most identifications should be considered tentative until confirmed by the permanent stained slide. The smaller protozoan organisms are frequently seen on the stained smear when they are easily missed with only the direct smear and concentration methods. For these reasons, the permanent stain is recommended for every stool sample submitted for a routine parasite examination (mandatory part of the O&P examination).

There are a number of staining techniques available; selection of a particular method may depend on the degree of difficulty of the procedure and the amount of time necessary to complete the stain. The older classical method is the long Heidenhain iron hematoxylin method; however, for routine diagnostic work, most laboratories select one of the shorter procedures, such as the trichrome method or one of the modified methods using iron hematoxylin.

Most problems encountered in the staining of protozoan trophozoites and cysts in fecal smears occur because the specimen is too old, the smears are too dense, the smears are allowed to dry before fixation, or fixation is inadequate. There is variability in fixation in that immature cysts fix more easily than mature cysts, and E. coli cysts require a longer fixation time than do those of other species.

Preparation of Material for Staining

Fresh Material

  1. When the specimen arrives, prepare one or two slides with applicator sticks or brushes and immediately (without drying) place them in Schaudinn's fixative. Allow the slides to fix for a minimum of 30 min; overnight fixation is acceptable. The amount of fecal material smeared on the slide should be thin enough that newsprint can be read through the smear. Smears preserved in liquid Schaudinn's fixative should be placed in 70% alcohol to remove the excess fixative prior to placement in iodine-alcohol (used for mercury-based fixatives).
  2. If the fresh specimen is liquid, place 3 or 4 drops of PVA on the slide, mix several drops of fecal material with the PVA, spread the mixture, and allow it to dry for several hours in a 37° C incubator or overnight at room temperature. This method should NOT be used with semi-formed or formed stool; there will be insufficient mixing between the stool and fixative.
  3. Proceed with the trichrome staining procedure by placing the slides in iodine-alcohol.

PVA-Preserved Material

  1. Stool specimens that are preserved in PVA should be allowed to fix for at least 30 min. Thoroughly mix the contents of the PVA bottle with two applicator sticks.
  2. Pour some of the well-mixed PVA-stool mixture onto a paper towel and allow it to stand for 3 min to absorb out PVA. Do not eliminate this step.
  3. With an applicator stick (or brush), apply some of the stool material from the paper towel to two slides and allow them to dry for several hours in a 37°C incubator or overnight at room temperature. Note The PVA-stool mixture should be spread to the edges of the glass slide; this will cause the film to adhere to the slide during staining. It is also important to thoroughly dry the slides to prevent the material from washing off during staining.
  4. The dry slides may then be placed into iodine-alcohol. There is no need to give them a 70% alcohol rinse prior to the iodine-alcohol, because the PVA smears are already dry (unlike the wet smears coming out of the Schaudinn's fixative).

SAF-Preserved Material

  1. The SAF-stool mixture is thoroughly mixed and strained through gauze into a 15-ml centrifuge tube.
  2. After centrifugation (1 min at 500 x g), the supernatant fluid is decanted. Although stains for C. parvum are not recommended from PVA-preserved material, remember that the centrifuge speed indicated here is probably not sufficient to recover the oocysts. Centrifugation time and speed is recommended as 10 min at 500 X g. The final sediment should be about 0.5 to 1.0 ml. If necessary, adjust by repeating step 1 or by resuspending the sediment in saline (0.85% NaCl) and removing part of the suspension.
  3. Prepare a smear from the sediment for later staining by placing 1 drop of Mayer's albumin on the slide, to which is added 1 drop of SAF-preserved fecal sediment. Allow the smear to air dry at room temperature for 30 min prior to staining. The SAF stool smear can also be postfixed in Schaudinn's fixative prior to staining (begin the trichrome stain protocol with the 70% alcohol rinse prior to iodine-alcohol).
  4. After drying, the smear can be placed directly into 70% alcohol (step 4) of the staining procedure (the iodine-alcohol step can be eliminated).