Histology FAQ

Staining, Histochemistry and Histotechnology

(Frequently Asked Questions)

Dr. John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada

FAQ Home > Staining Methods, Histochemistry

Stain for Chlamydia (Castaneda's method)

Question.

How do you carry out the Castaneda stain for Chlamydia?

Answer.

Castaneda's stain for elementary bodies and Rikettsiae (1930)

    Castaneda's staining solution

    Solution A

       Potassium dihydrogen phosphate, anhydrous    1 g
       Disodium hydrogen phosphate                        25 g
       Distilled water                                          1000 ml
       Formalin                                                     1 ml

       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.

    Solution B

       Methylene blue    1 g
       Methanol        100 ml

    Staining solution

       Solution A      20 ml
       Solution B    0.15 ml
       Formalin         1 ml

    Safranine-acetic acid

       Safranine (0.2% aqueous solution)     1 part
       Acetic acid (0.1% aqueous solution)   3 parts

Procedure.

   * 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.

Yvan Lindekens
(yvan.lindekens[AT]rug.ac.be)