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MODIFIED ZIEHL-NEELSEN METHOD
FOR CRYPTOSPORIDIUM OOCYST
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Ziehl-Neelsen Method for Cryptosporidiosis. |
FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Paraffin sections cut at 5 μm. Smears and touch preparations may
be done. These are fixed in 10% buffered neutral formalin for 15
minutes.
SOLUTIONS:
Carbol Fuchsin Solution
Pararosanilin, C.I. 42500 or Basic fuchsin, C.I. 42510 -------------------------- 0.8 gm
Isopropyl alcohol ---------------------------------------------------------------- 14.0 ml
Phenol -------------------------------------------------------------------------- 7.5 ml
Distilled water ------------------------------------------------------------------- 140.0 ml
Dissolve the pararosanilin or basic fuchsin in the isopropyl alcohol
and the phenol in the distilled water. Mix the two solutions. Filter
through Whatman #1 filter paper before use.
0. 1% Hydrochloric Acid
Alcohol, 70% -------------------------------------------------------------------- 100.0 ml
Hydrochloric acid, concentrated ------------------------------------------------- 0.1 ml
Fast Green. Stock Solution
Fast green FCF, C.I. 42053 ---------------------------------------------------- 0.2 gm
Distilled water ------------------------------------------------------------------- 100.0 ml
Acetic acid, glacial -------------------------------------------------------------- 0.2 ml
Fast Green, Working Solution
Fast green, stock solution ------------------------------------------------------ 10.0 ml
Distilled water ------------------------------------------------------------------- 35.0 ml
l . Deparalfinize and hydrate to distilled water.
2. Stain in carbol-fuchsin solution for 15 minutes.
3. Rinse with three changes of distilled water.
4. Decolorize in the acid-alcohol solution until no more color comes off
(approximately 30 - 60 seconds.)
5. Wash in running tap water for 30 seconds and rinse in two changes of distilled water.
6. Counterstain with working fast green solution for 30 seconds.
7. Rinse in three changes of distilled water.
8. Dehydrate in graded alcohols.
9. Clear in three or four changes of xylene.
10. Mount with synthetic resin.
Cryptosporidium oocysts ------------------------------------------- red
Background --------------------------------------------------------- green
Henriksen, S.A. and Pohleuz, J.F.L.: Staining of cryptosporidia by modified ZichlNeelsen technique. Acta. Vet. Scand. 22:594-596, 1981.
Human Cryptosporidiosis: New Engl. J. Med. 309:1325-1327, 1983.
Image Examples:
Diagnostic Application: