Iron hematoxylin - solution A and solution B
Preparation Of Working Iron Haematoxylin Solution -

Method
1. Mix equal volumes of the two solutions and filter.
2. Allow to stand at least two hours (preferably overnight) before use in order that the chemical reaction is complete. Staining is optimum 3-5 days after preparation.

If the Iron Haematoxylin Solution is used immediately after preparation the parasites may be stained an intense blue with little nuclear detail differentiation.
When the Iron Haematoxylin stain is mature (usually 3-5 days after preparation) the background should stain grey with the protozoa light blue, and the nuclei blue-black. The background staining of the slide depends on the composition of the specimen.
The stain is normally useable for a week. Shelf life can be extended by storage in a stoppered bottle in the dark after each use.

Trichrome for microsporidia
Method
1. Make smears from unconcentrated stool specimens in 10% formalin (1:3 ratio).
NOTE: ensure that the smears are extremely thin.
2. Fix in methanol for 5 mins.
3. Stain in modified Trichrome stain (code 1489) for 90 mins.
4. Rinse in acid alcohol for 10 secs.
5. Rinse briefly in 95% alcohol.
6. Place in 95% alcohol for 5 mins.
7. Place in 100% alcohol for 10 mins.
8. Clear in Xylene for 10 mins.
9. Examine under x 100 oil immersion objective, using Immersion Oil.

Interpretation
Microsporidial spores are ovoid and refractile and the spore wall stains bright pinkish-red. Occasionally the cellular content of some spores does not stain and appears transparent, others show a pinkish-red stained belt girding the spores either diagonally of equatorially.The spores are approximately 1.5 by 0.9µ. The background debris and bacteria are counterstained faint green.

Trichrome for protozoa
May be used to stain fresh faeces, prefixed faeces (only certain fixatives) or cultured organisms. The method varies slightly depending on the sample preparation used

Carbol fuchsin for modified z/n stain (cold kinyoun)
Suggested Method
1. Flood the fixed smears with Carbol Fuchsin (Kinyoun) and stain for 2 minutes without heating.
2. Wash with tap water and decolourise with acid-alcohol until the dyeno longer runs off the slide.
3. Wash with tap water and counterstain for 10-30 seconds with Methylene Blue of Malachite Green.
4. Wash, blot dry and examine.

Acid fast organisms stain red, the background and other organisms stain blue.

Malachite green
Malachite Green is a green counterstain used to differentiate bacteria

Methylene blue
Methylene is a blue counterstain used to differentiate bacteria.

Modified z/n stain pack
Suggested Method

1. Flood the heat fixed smears with Carbol Fuchsin (ZN) and steam gently for 5 minutes.
Add more stain if necessary to prevent drying.
2. Wash with water and decolourise with acid-alcohol until the dye no longer runs off the slide.
3. Wash with water and counterstain for 10-30 seconds with Methylene Blue of Malachite Green.
4. Wash, blot dry and examine.

Acid fast organisms stain red, the background and other organisms stain blue