Faecal smears are made for the following reasons:

• Provide information on the exudate present. (Romanowsky stains)
• Helpful in accurately identifying flagellates. (Romanowsky stains, Iron haematoxylin)
• When parasites cannot be detected in either the direct wet preparation or concentrated deposit a permanent stain of a fresh faecal smear can reveal the presence of intestinal parasites. (Romanowsky stains, Trichrome stain, modified Ziehl-Neelsen)
• Useful in demonstrating the nuclear patterns of cysts thus facilitating identification. (Iron haematoxylin, Trichrome stain)

 1. Romanowsky Stains

a. Modified Rapid Field's Stain²

This is a modification of Field's stain enabling rapid staining of fixed thin films of various clinical samples. This particular staining method is very useful for staining faecal smears, faecal exudate and duodenal aspirates.

Method.

a. Make a thin film of faeces/exudate. Allow to air dry.

b. Fix in methanol for 1 minute.

c. Flood slide with 1 mL of Field's stain B (diluted 1:4 with distilled water)

d. Immediately add an equal volume of undiluted Field's stain A, mix well and allow to stain for 1 minute.

e. Rinse well in tap water and drain dry.

• The exudate present.
• The presence of flagellate trophozoites eg Giardia lamblia.
• The presence of protozoa which are not readily detected or difficult to detect in the wet preparation eg. Dientamoeba fragilis and Blastocystis hominis .

 2. Modified Ziehl-Neelsen³

Use of the modified Ziehl-Neelsen stain for faecal smears has already been established for coccidian protozoa, in particular, oocysts of Cryptosporidium species, but it is also useful to confirm the presence of oocysts of Isospora belli and Cyclospora cayetanensis.

Method

a. Faecal smears are made either directly from the stool sample or from the concentration deposit.

b. Allow to air dry.

c. Fix in methanol for 3 minutes.

d. Stain with strong carbol fuchsin for 15-20 minutes.

e. Rinse thoroughly in tap water.

f. Decolourise in acid alcohol (1% HCl in methanol) for 15-20 seconds.

g. Rinse thoroughly in tap water.

h. Counterstain with 0.4% malachite green (or methylene blue) for 30-60 seconds.

i. Rinse thoroughly and air dry.

j. Examine using x40 and x100 objectives.

3. Phenol- auramine stain³

This stain can be used as an alternative to the modified Ziehl-Neelsen stain for staining oocysts of Cryptosporidium parvum.

Method

a. Make faecal smears as for ZN and fix in methanol.

a. Stain with phenol-auramine (Lemperts) for 10-15 minutes.

b. Rinse thoroughly in tap water.

c. Decolourise in acid alcohol. (as for ZN)

d. Rinse thoroughly in tap water.

e. Counterstain with 0.1% potassium permanganate for 30 sec.

f. Rinse thoroughly in tap water, allow to air dry.

Do not blot dry, many brands of blotting paper will fluoresce!

g. Observe the films with blue light under an incident light fluorescent microscope and low power followed by oil immersion x100 objective if oocysts are suspected.

The oocysts of Cryptosporidium parvum appear as bright yellow discs against a dark background.

4. Trichrome Stain

The trichrome method for staining protozoa is especially recommended for identifying features of amoebic cysts and trophozoites

Solution A: Schaudinn's fixative or SAF.

• Solution B: Iodine alcohol.

To prepare a stock solution, add enough iodine crystals to 70% ethanol to make a dark concentrated solution.

To prepare working solution, dilute stock solution by adding 70% ethanol (solution. should resemble weak tea!) The exact concentration is unimportant.

Background and yeasts stain green

 5. Modified Trichrome Stain⁴

A modified version of the trichrome stain is recommended, on thin faecal smears, for the detection of species of Microsporidia in immunocompromised patients with diarrhoea. Thus ill patients may not need to be subjected to invasive biopsy techniques in order to look for the organism.

 Reagents

Chromotrope 2R (Gurr) 6.0 g

Fast Green     0.15 g

Phosphotungstic acid 0.7 g

Gloves must be worn when weighing the reagents.

 The spores of Microsporidia are very small - 1.0 x 0.5 µm, oval in appearance and frequently exhibit a central band like structure flanked on either side by non-staining areas. Microsporidia spores stain pinkish – red against a greenish background.

6. Funguqual (Uvitex 2B or Calcofluor)

This stain can be used as an alternative to the modified trichrome stain for the detection of spores of Microsporidia species

Method

a. Stain methanol fixed thin smears in 1% Uvitex 2B (Ciba Geigy) for 15 minutes or 0.5% Calcofluor (Sigma) for 5 minutes.

b. Rinse in PBS.

c. Dip momentarily in 0.5% Evans Blue (Sigma)

d. Wash in PBS.

e. Mount.

f. Examine under blue light fluorescence (360-370nm)

These stains bind to the chitin in the endospore layer of the spore wall and fluoresce brilliant blue-white. As other structures contain chitin, e.g. fungal spores, some experience is needed to differentiate these from Microsporidia - the small size of microsporidian spores helps and a modified Trichrome method will distinguish them from fungal spores. Immature spores with little chitin fluoresce faintly but mature spores fluoresce brightly.

 7. Iron Haematoxylin

Staining with iron haematoxylin is useful to demonstrate the nuclear chromatin pattern and cytoplasmic inclusions of protozoan cysts .

 Reagent1

Schaudinn's fixative or SAF