Pinkerton's Method for Rickettsia

EMS26131

Components:

Methylene Blue Solution 1%

Basic Fuchsin 0.25% Aqueous

Citric Acid 0.5% Aqueous Solution

Gram's Iodine Solution

Lugol's Iodine

Sodium Thiosulfate 5% Aqueous

Fixation:

Zenker’s, Buffered Neutral Formalin, 10% or Moller’s.

Sections:

Paraffin at 6µm sections

Staining:

1. Deparaffinise and hydrate to distilled water.
2. If Zenker fixed, remove “Zenker’s crystals” by placing in Gram’s Iodine or Lugol’s Iodine for 15 minutes. Rinse in water and clear in Sodium Thiosulfate, 5% for three minutes. Wash in water for 10 minutes or longer.
3. Stain overnight in Methylene Blue Solution. Rinse in 95% alcohol for 5 seconds to prevent loss of blue colour. Rinse quickly in distilled water for 2-3 seconds.
4. Stain for 30 minutes in Basic Fuchsin, 0.25%.
5.  Decolourise rapidly in Citric Acid, 0.5% for 1-2 seconds, never more than 3 seconds.
6.  Differentiate further in absolute alcohol until nuclei stand out blue and rickettsia clumps red.
7.  Clear with 2 changes of Xylene.
8. Mount with Permount or SHUR/Mount.

Stain Results:

References:

Simons, J.S. and Gentzkow, C.J., - Laboratory Methods of the United State Army, 5th ed,. Lee & Febiger, Philadelphia, p. 572, 1944.

Luna, L.G., (ed) Manual of Histologic Staining Methods of the AFIP, 3rd edition, McGraw-Hill, New York, p. 237, c. 1968.