Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Chlamydia (Castaneda's method)
How do you carry out the Castaneda stain for Chlamydia?
Castaneda's stain for elementary bodies and Rikettsiae (1930)
Castaneda's staining solution
Potassium dihydrogen phosphate, anhydrous
Disodium hydrogen phosphate
Dissolve the potassium dihydrogen phosphate
in 100 ml distilled water and the disodium hydrogen phosphate
in 900 ml distilled water. Mix the two solutions to give a
buffer pH 7.5, and add formaldehyde as a preservative.
Methylene blue 1 g
Solution A 20
Solution B 0.15 ml
Safranine (0.2% aqueous solution)
Acetic acid (0.1% aqueous solution)
* Prepare films from infected tissue and dry in air
* Apply the stain for 3 min.
* Drain, do not wash
* Counterstain for a 1-2 seconds in safranine-acetic acid
* Wash in running water, blot dry.
Rickettsiae, elementary bodies of psittacosis: blue. Cell nuclei and cytoplasm: red.
Reference: "Biological stains and staining methods." BDH leaflet, 1966.
Several modifications of Castaneda's original technique are given in: Langeron, M.:"Precis de Microscopie", 1934 and 1948.