Applications for SPI-Chem™ Methylene Blue
Use in Castaneda's stain for elementary bodies and Riketttsiae
This is often times referred to as the "Chlamydia stain".
The procedure involves the mixing of two solutions, Solution A and Soluation
B, and the preparation of the Staining Solution by the mixing of A and B
with formaldehyde. A separate step involves the preparation of the
safranine-acetic acid solution.
Here are the details:
Reagent preparation protocol
Solution A
Potassium dihydrogen phosphate, anhydrous 1 g
Disodium hydrogen phosphate 25 g
Distilled water 1000 ml
Formaldehyde 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 1g
Methanol 100ml
Staining solution
Solution A 20ml
Solution B 0.15ml
Formaldehyde 1ml
______________________________________________________
Safranine-acetic acid
Safranine (0.2% aqueous solution) 1 part
Acetic acid (0.1% aqueous solution) 3 parts
______________________________________________________
Use Protocol
* 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.
Staining results:
Rickettsiae, elementary bodies of psittacosis
stain blue; cell nuclei and cytoplasm stain red.
Information contained on this page has been taken from Biological stains
and staining methods, BDH leaflet, 1966.
Several modifications of Castaneda's original technique in:
Langeron, M., "Précis de Microscopie", 1934 and 1948.
Other stains for Rickettsia, Coxiella and Chlamydiae (Macchiavello
and Gimenez): "Staining Procedures", p. 412-416.
Special thanks to Yvan Lindekens for the posting on the Histonet
Listserver on July 22, 1998 highlighting the details of this valuable
procedure.
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Monday May 21, 2012
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