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)