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U.S. Department of Health and Human Services
Health Resources and Services Administration

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Staining Procedure

Materials:

DEPARAFFINIZATION SOLUTION

Xylene ----------------------------------------------------------- 2 parts
Peanut or mineral oil ---------------------------------------- 1 part

1% ACID ALCOHOL

Hydrochloric Acid, Conc. ----------------------------------- 1 cc

Alcohol, 70% ------------------------------------------------- 99 cc

CARBOL FUCHSIN SOLUTION

 Phenol Crystals (melted) ------------------------------- 2.5 cc

Absolute Alcohol ------------------------------------------- 5.0 cc

 Basic Fuchsin ---------------------------------------------- 0.5 gm

Add distilled water to make 50 cc.

Store at room temperature.

WORKING METHYLENE BLUE SOLUTION

Methylene blue --------------------------------------------- 0.5 gm

Glacial Acetic Acid ---------------------------------------- 0.5 cc

Tap Water --------------------------------------------------- 100 cc

Procedure:

This procedure must be followed exactly for best results.

  1. Deparaffinize sections with xylene-Peanut oil or xylene-mineral oil solution: 2 changes, 6 minutes each.
  2. Rinse with running tap water for 1 minute. Drain slides for about 45 seconds. DO NOT REMOVE ALL OF THE OIL . There should be a thin film of oil on the slide at all times during the procedure. The oil hastens acid-fast staining without the use of heat. Do not blot the slides.
  3. Stain with Carbol Fuchsin for 20 minutes at room temperature.
  4. Wash in running tap water for 3 minutes.
  5. Decolorize in 1% Acid Alcohol until the slide is a faint pink. This takes about one minute for most slides and slightly longer for larger sections. The Acid Alcohol should be changed regularly and never used when it is more than a pink color. THIS IS THE MOST CRITICAL STEP.
  6. Wash in tap water for 5 minutes.
  7. Counterstain with methylene blue for 1-2 minutes according to desired background.
  8. Wash in tap water for 20 seconds.
  9. Allow to air dry. After the slide has air-dried thoroughly, wipe excess stain and oil off the slide with a clean cloth dampened with water. Do not blot or wipe tissue sections.
  10. Mount in permount or other suitable adhesive. The slide should be mounted as soon as possible and never allowed to remain open to the air overnight.

This procedure is a slight modification of that of G. L. Fite, et al. in the Archives of Pathology, Vol. 43 :624-625, 1947.