Wolbach’s Giemsa Method

EMS26132, EMS26773

Components:

Giemsa Stock Solution

Giemsa Working Solution

Rosin Alcoholic Stock Solution

Rosin Alcoholic Working Solution

Lugol's Iodine

Gram's Iodine Solution

Sodium Thiosulfate 5% Aqueous

Fixation:

Zenker’s or other well fixed tissue fixatives.

Sections:

Paraffin @ 6µm

Staining Procedures:

1. Deparaffinise and hydrate to distilled water.
2. Remove “Zenker Crystals” by placing in Lugol’s Iodine or Gram’s Iodine for 15 minutes. Rinse in water, place in Sodium Thiosulfate, 5% for 3 minutes and wash in running water for 15 minutes.
3. Rinse in distilled water and stain in Working Giemsa Solution* overnight.
   1. Working Giemsa Solution may be made from the stock solution by mixing:
      Giemsa Stock - 1.25ml
      Methanol - 1.50ml
      Distilled water - 50.0ml
       NOTE: Giemsa stain colours more effectively in tissue at an acid pH. If this has not occurred in the preparation or decalcifications steps, wash in an acid alcohol, and begin stain.
4. Differentiate in Rosin Alcohol Working until the sections are purplish-pink color. Check under microscope.
5. Dehydrate in two changes of absolute alcohol and clear in two changes of Xylene.
6. Mount with Permount.

Stain Results:

References:

Wolbach, S.B., Todd, J.L., and Paltrey, F.W., The Etiology of Pathology Typhus. Harvard University, University Press, Cambridge, MA, p. 13-14, c. 1922..

Luna, L.G., (ed). Manual of Histologic Staining Methods of the AFIP 3rd edition. McGraw-Hill, NY, p. 119, c. 1968.