Acid Orcein and Giemsa Stain

(Pinkus and Hunter)

Fixation

Formalin or alcohol.  Fixatives containing chromate or mercury are not suitable.

Solutions

A.                 Dissolve 0.2 gm of Orcein Synthetic Harleco (Harleco, Inc., Philadelphia) in 100 ml 70 percent ethyl alcohol; add 0.6 ml concentrated hydrochloric acid.  Solution is ready for use immediately and improves on standing.  It has a long shelf life but becomes exhausted by frequent use.

B.                 Dilute Giemsa solution: one drop of any good Giemsa stock solution for each 20 ml of distilled water.  A phosphate buffer solution adjusted to pH 7.0 may be used if distilled water is too acid.

C.                 A few drops of 1 percent alcoholic solution of eosin yellow are added to 95 percent alcohol for decolourisation of excessively blue sections.

Staining Procedure

1.      Deparaffinize sections in xylene and bring through absolute and 95 percent alcohol into 70 percent alcohol.

2.      Immerse in solution A for 30 to 60 minutes.  This new synthetic orcein stains elastic fibres specifically and produces very little background staining.  The background may be completely decolourised by short immersion of the stained sections in absolute alcohol or 0.1 percent acid alcohol.  This is rarely necessary.

3.      Wash in running tap water for 10 minutes.

4.      Stain for 12 to 15 hours (preferably overnight) in solution B.  Do not hurry this step.  The various tissue components pick the various shades out of a very dilute solution if given enough time.  One may speed up this step by having the sections stained for 1 hour in 1 percent Giemsa solution in an oven at 60C.

5.      Wipe excess fluid off slides.  Generally the sections are now stained blue.  The excess blue is removed by dehydrating the sections in 95 percent alcohol to which a few drops of solution C have been added.  How much eosin should be used depends on the degree of overstaining and is learned by experience.  If the sections have almost the desired rose-pink colour of the collagen when they come out of the Giemsa solution, omit eosin altogether in this step.

6.      Remove sections when the collagen of the skin begins to turn pink from blue. Finish dehydration and decolourisation in two changes of absolute alcohol.

7.      Two changes of xylene.

8.      Mount in Permount or other suitable medium.