Histology Hardball

The following procedures complement the February print issue’s cover story, Histology Hardball.

Procedure for Determining the Endpoint of Decalcification1


Mineralized bone specimens may be received in the histology lab that require the specimens to be decalcified, such that they can be routinely processed and embedded in paraffin.Whether employing rapid or slow decalcification techniques, it is important to determine the end point of the decalcification procedure.


Bone specimens that have undergone decalcification


5 percent ammonium hydroxide:

  • ammonium hydroxide, concentrated–5 mL
  • distilled water–95 mL

5 percent ammonium oxalate:

  • ammonium oxalate–5 gm
  • distilled water–100 mL

Caution: Ammonium hydroxide is corrosive. Wear safety glasses, chemical resistant gloves and an apron. Work under a hood. Always add acid last.


  1. Draw 5 mL of decalcifying solution, from the bottom of the container, which has been in contact with the tissue for 6-12 hours, and add to an empty beaker.
  2. Add 5 mL each of 5 percent ammonium hydroxide and 5 percent ammonium oxalate.
  3. Mix and let stand 15 minutes.
  4. A cloudy solution caused by calcium oxalate indicates that the specimen is not thoroughly decalcified. Such a result indicates the necessity of changing the decalcifying solution and continuing the decalcification process.
  5. When a milky solution is no longer obtained from such a mixture, the specimen is completely decalcified. The test can be performed as frequently as necessary.

Procedure for Preparing Nail Specimens2


Toenail or fingernail specimens may be received in the laboratory with a differential diagnosis that may include fungal infection or melanoma, arising in the nail bed. This procedure describes how to fix the tissue and soften it such that it can be cut and stained with routine hematoxylin and eosin (H&E) and other special stains such as periodic acid Schiff’s, Fontana-Masson and immunoperoxidase methods.


Fixation. Nail specimens fixed in 10% neutral buffered formalin.


Nail softener solution

  • Tween 85 (Sigma catalog # P4634)–5 mL
  • 10 percent formaldehyde–95 mL

Caution: Formaldehyde is toxic by inhalation and if swallowed; irritating to the eyes, respiratory system and skin; may cause sensitization by inhalation or skin contact; risk of serious damage to eyes. May cause cancer; repeated or prolonged exposure increases the risk. Wear safety glasses, chemical resistant gloves, and an apron. Work under a hood.


  1. Confirm that the specimen is a nail either visually or from the requisition.
  2. If the specimen is too large to fit into a cassette, cut into appropriate sizes and submit in multiple cassettes if necessary, such that all tissue is submitted.
  3. Place the entire cassette(s) into the nail softening solution and allow to stand overnight.
  4. Remove from the nail softening solution and place into formalin for later loading onto the tissue processor.
  5. Process as usual, with at least an eight hour processing time.
  6. Embed as usual with the nail pieces up on edge in the mold.
  7. Once the sections are cut, they should be picked up on gelatin coated slides and baked in a conventional oven at 65 C for a minimum of 45 minutes. This will insure that the sections remain on the slide, regardless of the staining method.

Procedure for Preparing Gelatin Coated Slides1,3,4


Certain specimens are received in the histology lab that require the sections to be picked up on specially coated slides. Decalcified bone and nail specimens should be picked up on gelatin coated slides.


Routinely processed, paraffin embedded blocks of decalcified bone and nail tissues.


0.5 percent Gelatin solution

  • Gelatin; (Fisher catalog # G8-500)–1.0 gm
  • Distilled water–200 mL


  1. Add 1.0 gm of gelatin to 200 ml distilled water in a glass beaker that has been previously warmed on a hot plate to 60 C.
  2. Allow to dissolve.
  3. Transfer to a 250 ml plastic slide holder.
  4. Load clean microscope slides into the vertical slide holder.
  5. Immerse the slide racks containing the slides into the gelatin solution. Allow to soak for one minute.
  6. Remove and allow to dry in the fume hood.
  7. Place slides into storage boxes and keep in the refrigerator. This will inhibit bacterial growth. Use a one year expiration date.


  1. Luna L. ed. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed., McGraw-Hill, New York, 1968:10.
  2. Rosai J. Ackerman’s Surgical Pathology. 7th ed. CV Mosby, Washington DC, 1989.
  3. Bancroft JD, ed. Theory and Practice of Histological Techniques. Churchill and Livingstone, 3rd edition, 1990.
  4. Chapman CM. A Guide for the Histologist. Available at www.medi-sci.net. Last accessed Feb. 15, 2012.

Clifford M. Chapman is technical director, Strata Pathology Services Inc., Lexington, MA.

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