Staining procedure

1 Place a drop of formol ethyl acetate deposit on a microscope slide and allow to airdry for at least 1h. With unconcentrated material make a thin smear on a slide. Process a positive control at the same time as the clinical specimens.

2 Fix the slide in methanol in a staining jar for 10min.

3 Place the slide on a staining rack and cover with carbol fuchsin. Stain for 20min.

4 Rinse well under running tap water.

5 Decolourize with acid alcohol in a staining jar until the red colour of the smear has disappeared.

6 Rinse under running tap water.

7 Cover the slide with malachite green stain for 30s.

8 Rinse under running tap water.

9 Air-dry the preparation.

10 Mount under a coverslip with DPX. Allow to dry.

Examine the whole preparation under the light microscope. Use the 40* objective for screening and 100* oil immersion for confirmation. Measure any oocysts.

Cryptosporidium oocysts; round, 4-6^m in diameter, stained in varying shades of pale pink to dark red against a green background. Completely unstained oocysts may be present. Identifying sporozoites inside oocysts is diagnostic for Cryptosporidium.

Cyclospora oocysts; 8-10^m in diameter and similar in colouring to Cryptosporidium but without a definite inner structure. Completely uncoloured oocysts with a glassy appearance are often seen. Isospora oocysts; 20-30*10-19^m containing either a granular zygote or two sporoblasts. They are coloured in different shades of red; some oocysts are unstained.

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