ROUTINE HEMATOXYLIN AND EOSIN.
DRAFT COPY ONLY.
(Procedure 49).
http://www.netautopsy.org/axsop/axsop049.htm


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United States Government Work, uncopyrighted, public-domain, DRAFT COPY ONLY. This document does not necessarily represent the views or policies of any United States Government agency. This document is provided "as is", without warranty of any kind, express or implied, including but not limited to the warranties of merchantability, fitness for a particular purpose and non-infringement. In no event shall the authors be liable for any claim, damages or other liability, whether in an action of contract, tort or otherwise, arising from, out of, or in connection with the document or the use or other dealings made with the document.

See also: Tissue Stains: 51, 52, 55, 56, 69, 72, 81.



PRINCIPLE OF THE TEST.
      To stain paraffinized tissue sections for hematoxylin and eosin, required in the routine microscopic diagnosis of all specimens received in anatomic pathology that are examined microscopically.



SPECIMEN REQUIRED.

All human tissue excised at surgery, outpatient clinics, and postmortems, fresh or in fixative, along with a filled-out Tissue Examination Form (U. S. Standard Form 515, USSF515).



REAGENTS, INSTRUMENTATION.

      Hematoxylin solution. Eosin solution. Eosin Y or B. Phloxin. Ethyl alcohol 80%. 0.5% acid alcohol. Lithium Carbonate (LiCO3). 80% ETOH-substitute. 95% alcohol. Absolute alcohol. Clearing. Permount.



STEP-BY-STEP DESCRIPTION.
1. Deparaffinize to distilled water.

2. Stain in hematoxylin 5 minutes.

3. Rinse in tap water.

4. Differentiate in 0.5% acid alcohol 8 - 10 dips.

5. Wash in running tap water.

6. Blue in 1% ammonia water or Lithium Carbonate (LiCO3).

7. Wash well in running tap water 5 minutes.

8. 80% ETOH-substitute eosin 1 minute.

9. Three changes 95% alcohol.

10. Two changes absolute alcohol.

11. Three changes of clearing solution.

12. Mount in Permount.




RESULTS:
 
 Nuclei, blue.
 Cytoplasm, pink.
 Eosin preparation.
 Eosin Y or B         4 gm.
 Phloxin              1 gm.
 Ethyl alcohol 80%    2,000 ml.




REFERENCES.

1. Gamble M, Wilson I.
The Hematoxylins and Eosin. Chapter 8, pp. 125-138.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:125-138.
ISBN 0-443-06435-0, 796 pages.

2. Carazzi D.
Eine neue Hämatoxylinlösung. [German: A new hematoxylin solution].
Z f wissenschaftl Mikroskopie u mikroskop Technik. 1911;28:273.

3. Ehrlich P.
Fragekasten. [German: Query box.]
Z f wissenschaftl Mikroskopie u mikroskop Technik. 1886;3:150.

4. Gill GW, Frost JK, Miller KA.
A new formula for half-oxidized hematoxylin solution that neither overstains nor requires differentiation.
Acta Cytol. 1974;18:300.

5. Harris HF.
On the rapid conversion of hematoxylin into hematein in staining reactions.
J Appl Microsc Lab Methods. 1900;3;777.

6. Heidenhain M.
Noch einmal über die Darstellung der Centralkörper durch Eisenhämatoxylin nebst einigen allgemeinen Bemerkungen über die Hämatoxylinfarben. [German: Once again regarding the demonstration of nuclear bodies through staining with hematoxylin].
Z f wissenschaftl Mikroskopie u mikroskop Technik. 1896;13:186.

7. Lillie RD, Fulmer HM.
Histopathologic Technic and Practical Histochemistry. Fourth Edition.
New York: McGraw-Hill. 1976;:.

8. Mayer P.
Über Schleimfärbung. [German: On staining mucus].
Mitteil zoolog Station zu Neapel. 1896; 12:303.

9. Mayer P.
Notiz über Hämatin und Hämalaun. [German: Note regarding Hematin and Hemalaun].
Z f wissenschaftl Mikroskopie u mikroskop Technik. 1903;20:409.

10. Mallory FB.
On certain improvements in histological technique.
J Exptl Med. 1897;2:529.

11. Mallory FB.
A contribution to staining methods.
J Exptl Med. 1900;5:15.

12. McManus JFA.
Histological demonstration of mucin after periodic acid.
Nature (Lond). 1946;158:202.

13. McManus JFA.
Histological demonstration of mucin after periodic acid.
Nature (Lond). 1946;158:202.

14. McManus JFA, Mowry RW
Staining Methods, Histologic and Histochemical.
London: Harper & Row. 1964;:268.

15. Verhoeff FH.
Some new staining methods of wide applicability. Including a rapid differential stain for elastic tissue.
JAMA. 1908;50:876.

16. Weigert K.
Eine kleine Verbesserung der Hämatoxylin van-Gieson-methode. [German: A small improvement in the hematoxylin van-Gieson method].
Z f wissenschaftl Mikroskopie u mikroskop Technik. 1904;21:1.

17. Leong AS, ed.
Principles and Practice of Medical Laboratory Science. Volume 1: Basic Histotechnology. First Edition.
Philadelphia: W.B. Saunders Company. 1996;:.
ISBN: 0443053693, 171 pages.

18. Brown GG.
An introduction to histotechnology: A manual for the student, practicing technologist, and resident-in-pathology.
New York: Appleton-Century-Crofts. 1978;:.
ISBN: 0838543405, 453 pages.

19. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.

20. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.