Prepared by

ROY ELLIS

IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

Principle

The oxidation product of haematoxylin is haematin, and is the active ingredient in the staining solution.  Haematoxylin is not classified as a dye since the molecule possesses no chromophore.  The in situ oxidation of haematoxylin is effected by the addition of a strong oxidant to the stain, in this case sodium iodate.  Lillie’s variant of Mayer’s haemalum is discussed in Lynch et al. (pp 1032)

(please refer to Lillie Mayer)

Haematin exhibits indicator-like properties, being blue and less soluble in aqueous alkaline conditions, and red and more soluble in alcoholic acidic conditions.  In acidic conditions, haematin binds to lysine residues of nuclear histones by linkage via a metallic ion mordant, in this case aluminium.  To ensure saturation of chemical binding sites, the stain is applied longer than necessary, resulting in the overstaining of the tissues with much non-specific background colouration.  This undesirable colouration is selectively removed by controlled leaching in an alcoholic acidic solution, (acid alcohol), the process being termed "differentiation".  Differentiation is arrested by returning to an alkaline environment, whereupon the haematin takes on a blue hue, the process of "blueing-up".  The haematin demonstrates cell nuclei.

Full cellular detail is obtained by counterstaining with the eosin mixture.  There are three commonly used forms of eosin - eosin Yellowish (tetrabromofluorescein, disodium salt CI 45380), eosin Bluish (the dinitro- dibromo-derivative CI 45400), and eosin Alcohol Soluble (the ethyl derivative CI 45386), the former is preferred.  Colour enhancement is achieved by fortifying the stain with phloxine, a chemical member of the same family as eosin( halogenated fluorosceins).  The mechanism of their staining is not fully understood, but is believed to be of an electrostatic nature.  Visualisations most acceptable to the histologist are obtained by applying the dyes in acidic conditions, whereby more intense specific colourations are obtained, the more acidic tissue components taking up the dye to a greater intensity, hence the addition of acetic acid.

Technical Points

1.         (step 2) - The length of time necessary to over-stain the tissues will depend upon fixation and the type of alum haematoxylin employed. (Lillie Mayer's alum haematoxylin-formalin fixed tissues 5 mins).

Tissue Type	Haematoxylin	Acid alcohol 0.3%	Eosin	Comment
Routine tissues	4 minutes	See technical point 2	2 minutes
Renal biopsies	10 minutes	1-2 seconds	2-4 minutes	Check staining
Decals	10 minutes	1-2 seconds	30 seconds	Check staining after blueing. Hx step may need to be repeated if prolonged decal.

2.         (step 4) - Differentiation with acid alcohol requires some practical experience to ascertain the correct end-point, since the acid solution alters the colour of the tissue to red.  The correct end-point is when, after blueing up, the background is almost colourless.  For renal biopsy sections, two quick dips in 0.3% acid alcohol are all that is required

3.         (step 6) - If Scott's tap water substitute is employed, blueing up is achieved in a much shorter time.

4.         (step 8) - Eosin is highly soluble in water.  Over-staining is removed by washing in running water.

5.         Fixation - Not critical.  Acidic fixatives will give a more eosinophilic result.  Picric acid containing fixatives give an overall enhanced result.  Acidic decalcifying fluids give poor nuclear staining.

6.         Renals - 10% buffered formalin.  Sections cut at 2m

Method

1.      Bring sections to distilled water

2.      Stain nuclei with the alum haematoxylin (see note)

3.      Rinse in running tap water

4.      Differentiate with 0.3% acid alcohol (see note)

5.      Rinse in running tap water

6.      Rinse in Scott's tap water substitute (see note)

7.      Rinse in tap water

8.      Stain with eosin   2 mins

9.      Dehydrate, clear and mount.

Results

collagen............................................pale pink

                        muscle.............................................deep pink

                        acidophilic cytoplasm.............................red

                        basophilic cytoplasm..............................purple

                        nuclei...............................................blue

                        erythrocytes.......................................cherry red

Reagent Formulae

1.   Lillie Mayer alum haematoxylin
   aluminium ammonium sulphate ---- 200 g
   haematoxylin (CI 75290) ------------ 20 g
   ethanol -------------------------------- 40 ml
   sodium iodate ------------------------ 4 g
   acetic acid ---------------------------- 80 ml
   glycerol ------------------------------- 1200 ml
   distilled water ------------------------ 2800 ml

      In a 4L Ehrlenmeyer flask, to 1000 mls of the distilled water, add the aluminium ammonium sulphate.  Place the flask on a heater/stirrer, turn on the heater and allow to mix until the alum dissolves - this takes about 15 mins.  Remove the flask from the heater/mixer, allow to cool, then add the remaining 1800 mls distilled water - this will further cool the solution.  Add the haematoxylin powder to the  alcohol and  dissolve as much of the powder as possible by shaking for a few minutes.  Pour the strong alcoholic solution of haematoxylin into the cooled alum solution and stir to ensure all the Hx powder is dissolved, preferrably overnight.  Add the sodium iodate, acetic acid, and finally the glycerol.  Mix well, plug loosely and store.

It is appropriate to make up a batch of the required amount, dependant upon the usage rate.

2.   Acid alcohol 0.3% Acid Alcohol                    
   commercial grade ethanol ----- 2800 ml                                
   distilled water ------------------ 1200 ml                                
   conc hydrochloric acid --------- 12 ml                           
 

In a sufficiently large container, add the acid to the water, then add the alcohol and mix thoroughly.  The generation of fine bubbles is an indication that mixing is thorough.

3.   Scott's tap water substitute
   sodium hydrogen carbonate --- 10 gm
   magnesium sulphate ----------- 100 gm
   distilled water ------------------- 5 L
 

Dissolve the salts in the water.  Store stock solutions at room temperature. 

4.   alc acetified eosin/phloxine     TQEH
   1% eosin Y (CI 45380) ---------- 400 ml
   1% aq phloxine (CI 45405) ----- 40 ml
   95% alcohol ---------------------- 3100 ml 
   gl acetic acid -------------------- 16 ml       

                                             
Mix the above reagents together, and stir well.  The solution keeps well.

References

Mayer P,(1896),Mitt. zool. Stn. Neapel.,12,303

Lillie RD,(1965),Histopathologic Technic and Practical Histochemistry,3rd edition, McGraw-Hill Book Co., New York

Lynch MJ, Raphael SS, Mellor LD, Spare PD and Inwood MJ, (1969), Medical Laboratory Technology and Clinical Pathology, 2nd edition, WB Saunders Co., Philadelphia London Toronto

LG Luna, Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, third edition, McGraw Hill