hematoxylin staining procedures. history, types and procedures.

2. Hematoxylin & Staining
Presented by:
2nd Year M.D.S.
Dept. of Oral & Maxillofacial Pathology & Microbiology

3. INTRODUCTION
•Long history of use–Mayer 1904
•most widely used histological stain:
Comparative simplicity
Ability to demonstrate clearly an enormous number of different tissue
structures.
Hematoxylin stains cell nuclei blue black- shows good intranuclear detail.
stains cell cytoplasm and most connective tissue fibers in varying
shades and intensities of , orange, and red.

6. Hematoxlin - Greek word Haimato(blood) and Xylon(wood),
reffering to its dark red color in natural state and to its
origin(wood).
HEMATOXYLIN

7. Logwood Cut end of same logwood& hematoxylin
powder
Hematoxylin campechianum

8. The hematoxylin is extracted from logwood with hot water, and then
precipitated out fromthe aqueous solution using urea.

9. Historical Background
For years it was used
in textile industry
until WALDEYER
established its use in
histology in 1862.
Two years later
Bohmer combined
haematoxylin with
alumas a mordant
and obtainedmore
specificstaining.
Ehrlich(1886) overcame the
instabilityof hematoxylin
and alum by the additions
of glacial acetic acidand at
the same time producedhis
formulafor haematoxylin
as it is used today.
In 1891 Heidenhain
introducedhis
classical Iron alum-
haematoxylinmethod
whichtodayis still the
standard technique of
the cytologist.

10. • Hematoxylin has a deep blue-purple color and stains
nucleicacids by a complex, incompletely understood
reaction.
• Nuclei show varying cell-type- and cancer-type-specific
patterns of condensation of heterochromatin(hematoxylin
staining) that are diagnostically very important.
• Hematoxylin, generally without eosin, is useful as a
counterstain for many immunohistochemical or
hybridization procedures that use colorimetric substrates
(such as alkaline phosphatase or peroxidase).

11. Ripening
• Hematein- Ripening
• Oxidation (loss of electron) is demonstrated by the loss of hydrogen and its electron
fromthe Hematoxylin structure
Hematoxylin Hematein
• Carried out in 2 ways –
Natural oxidation
Chemical oxidation

12. NATURALLYRIPENEDHEMATOXYLINS
Ripening by exposure to light & air
Slow process (3-4 months)
Long shelf life, retain stability for a
long time
Example
 Ehrlich’s hematoxylin
 Delafield’s hematoxylin.
CHEMICALLYRIPENEDHEMATOXYLINS
Ripening by exposure to chemical oxidizing
agents.
Ripening instantaneous, ready to use immediately
after preparation
Shorter shelf life (because of continuing oxidation
process in air & light eventually destroys much of
the hematein converting it into a colourless
compound)
Example
 Sodium iodate in Mayer’s hematoxylin (SIM)
 Mercuric chloride in Harris’s hematoxylin (MCh)

13. • The process of over-oxidation of hematoxylin has established that
the production of oxyhaematein inhibits successful staining-
MARSHALL AND HOROBIN 1972.
• Glycerol has been incorporated in many formulas as it’s a stabiliser
to prevent over-oxidation and prevent evaporation.
Properties of chemically oxidized hematoxylin

14. Hematein
• Anionic- poor affinity for tissue - inadequate as a nuclear
stain.
• Mordant- A polyvalent metal ion which forms coordination complexes
with certain dyes.
• Net positive charge - dye-mordant complex
• Type of mordant- type of tissue component and its color.
• Most useful mordants - salts of aluminum, iron, and
tungsten.
• Lead as a mordant

15. •Biological staining – substance intermediate between dye and
tissue.
•Mordant forms a link between the “tissue and the stain”
•Incorporation of mordant
Incorporated into the hematoxylin
The tissue section can be pretreated
Mordant Dye LAKE

16. CLASSIFICATION
I. Based on the Oxidation Procedure
1. Natural oxidation – Ehrlich’s and Delafield’s
2. Chemical Oxidation - Mayer’s and Harris
II. Based on the Mordant Used
1.Alum hematoxylin
2. Iron hematoxylin
3. Tungsten hematoxylin
4. Lead hematoxylin
5. Molybedenum hematoxylin
6. Hematoxylin without mordant

17. •The mordant is aluminum in the form of
a) potashalum- aluminum potassium sulfate or
b) ammoniumalum-aluminum ammonium sulfate
• Stains the nuclie dark red- converted to familiar blue-
black- BLUING.
• Specific staining: –Nuclei; DNA in chromatin
• Non specific – Background staining: –Cytoplasm &
Goblet cells of G.I. tract; mucin- reduced or removed by
differentiation.

18. Bluing
• Alkaline solutions used for bluing
• Tap water is alkaline enough to produce this
colour change.
• Scott’s tap water substitute
• Saturated Lithium carbonate (disadvantage –
lithium has a tendency to form crystalline
deposits unless the slides are agitated in it
and well washed afterwards).
• Ammonia in distilled water (disadvantage –
ammonia is “hard” on delicate tissues and
will loosen sections from the slide).

19. Differentiation
• Provides a more controllable method in removing
excess stain from tissue component and glass slide.
• Traditional hcl/alcohol acts quickly and
indiscriminately, is more difficult to control, and
can result in light nuclear stain.
• 1ml of 5 – 10% solution of acetic acid in 99ml of 70
– 95% alcohol detaches dye molecules from the
cytoplasm/nucleoplasm while keeping nucleic acid
complexes intact.
• Exposure to air may oxidize and improve the
process.

20. Regressive Staining:
Stain - Regress - Continue
staining (Harris hematoxylin)
•Tissue is initially overstainedand then partially decolorized
(differentiated) until the proper endpoint is reached.
• Sharper degree of differentiation is obtained
•The differentiation is controlled visually by microscopic
examination.
•Faster and more convenient .
Progressive Staining:
Stain - Rinse- Continue
staining (Mayer’s, Gill’s I & II)
•Tissue is stained for a predeterminedtime for adequate staining of
the nuclei and leaves the backgroundtissue relatively unstained.
•Once the dye is taken up by the tissues, it is not removed.
•The tissue is left in the dye solution until it retains the desired
amount of coloration.
•The differentiationsolely relies on the selective affinity of dyes for
different tissue elements.

21. Classification of Alum Hematoxylins
•Ehrlich’s hematoxylin (Ehrlich 1886)
•Delafield’s hematoxylin (Delafield 1885)
•Mayer’s hematoxylin (Mayer 1903)
•Harris hematoxylin (Harris 1900)
•Cole’s hematoxylin (Cole 1943)
•Carazzi’s hematoxylin (Carazzi 1991)
•Gill’s hematoxylin (Gill et al 1974)

22. Ehrlich’s Hematoxylin (1886)
• Naturally ripened strong alum hematoxylin.
• Stains nuclei intensely and crisply
• Stains mucin in salivary glands, cartilage and cement lines of
bones
• Suitable for tissues subjected to acid decalcification, tissues
that have been stored for a long period in formalin fixatives.
• Suitable for Bouin’s fixed tissue.
• Not ideal for frozen sections.
Ehrlich’s Hematoxylin

23. Preparation :
Hematoxylin 2 g
Absolute alcohol 100 ml
Glycerol 100 ml
Distilled water 100 ml
Glacial acetic acid 10 ml
Potassium alum 15 g approx.
Ehrlich’s Hematoxylin

24. • The stain may be ripenednaturally by allowing to stand in a large flask,
looselystoppered with cotton wool – takes about 2 months.
• chemically ripened - sodium iodate - 50mg for every gram of hematoxylin.
• Filter before use.
Ehrlich’s Hematoxylin
Dissolve the hematoxylin in alcohol
Incorporate glycerol
Add potassium alumtill
saturation
• to slow down the oxidation
process and prolong shelf
time.

25. • Chemically ripened with sodium iodate.
• Used as both progressive and regressive stain.
• Used as a nuclear counterstainin the
demonstration of glycogen (PAS, mucicarmine)
in various enzyme histological techniques.
• Stain applied for a short period- 5-10 mins
until nuclei are stained and then blued without
any differentiation which might
destroy/decolor the stained cytoplasmic
components.
Mayer’s Hematoxylin (1903) Mayer’s Hematoxylin

26. Preparation
•Hematoxylin 1 g
•Distilled water 1000 ml
•Potassium or ammonium alum 50 g
•Sodium iodate 0.2 g
•Citric acid 1 g
•Chloral hydrate SLR 50 g
or
Chloral hydrate AR 30 g
Mayer’s Hematoxylin

27. • Chloral hydrate acts as a preservative
• Citric acid sharpens nuclear staining
boiled for 5 minutes
Mayer’s Hematoxylin
Hematoxylin + potassium alum + sodium iodate
Chloral hydrate and citric acid
cooled and filtered
• dissolved in distilled water by warming &
stirring

28. REFERENCES
References

29. •Nuclear stains. Kiernan JA. Cold Spring Harb
Protoc; doi: 10.1101/pdb.
•Hematoxylin and eosin staining of tissue and cell
sections. Fischer AH, Jacobson KA, Rose J, Zeller R.
CSH Protoc. 2008 May 1;2008:pdb.prot4986. doi:
10.1101/pdb.prot4986
References

31. Harris’s Hematoxylin (1900)
• Traditionally chemically ripened with mercuric oxide
• regressive stain - routine histology practice
• progressive stain - diagnostic exfoliative cytology
• Differentiation is required- acetic acid alcohol.
Harris’s Hematoxylin

32. Preparation
•Hematoxylin 2.5 g
•Absolute alcohol 25 ml
•Potassium alum 50 g
•Distilled water 500 ml
•Mercuric oxide 1.25 g
or
Sodium iodate 0.5 g
•Glacial acetic acid 20 ml
Harris’s Hematoxylin

33. Dissolve hematoxylinin alcohol
Add it to alum previously dissolvedin warmdistilledwater
The mixture is rapidly broughtto boil
Mercuric oxide is thenslowly and carefully added, whenthe solutionturns dark purple.
The stainis rapidlycooled under tap water.
Optional addition of glacial aceticacid.
Filter before use.
Harris’s Hematoxylin

34. Gill’s Hematoxylin (1974)
•Available in 3 concentrations –
o Gill’s I (normal)
o Gill’s II (double)
o Gill’s III (triple) most concentrated.
•More frequently used than Mayer’s hematoxylin for
routine H&E staining.
•More stable than Harris’s hematoxylin, as auto-oxidation
is inhibited to the extent.
Gill’s Hematoxylin

35. Preparation of solution
•Hematoxylin 2 g
•Sodium iodate 0.2 g
•Aluminum sulfate 17.6 g
•Distilled water 750 ml
•Ethylene glycol (ethandiol) 250 ml
•Glacial acetic acid 20 ml
Gill’s Hematoxylin

36. Advantages
• Fast in action.
• Stablefor at least months.
• Produce little or no surface
precipitate.
• Their preparation doesn’t involve
boiling the solution.
Disadvantages
• Staining of gelatin is adhesive and
even the glass itself.
• Some mucus may also stain
darkly, as compared to Harris’s
hematoxylin.
Gill’s Hematoxylin

37. •Alum hematoxylin, artificially ripened with an alcoholic iodine
solution.
•Has good keeping qualities and is suitable for use especially in
sequence with celestine blue unlike Ehrlich’s hematoxylin
Cole’s haematoxylin (1943) Cole’s Hematoxylin
Preparation
• Hematoxylin 1.5 g
• Saturated aqueous potassium alum 700 ml
• 1% iodine in 95% alcohol 50 ml
• Distilled water 250 ml

38. The hematoxylin is dissolved in warmdistilled water + iodine
solution.
The alumsolution is added and the boiled - then cooled
quickly.
The solution is ready for immediateuse, but mayneed on
occasion filtering after storage.
Filtered
Cole’s Hematoxylin

39. Carazzi’s hematoxylin (1911)
• Alum hematoxylin which is chemically ripened using potassiumiodate
• Used as a progressive nuclear counterstain.
• Largely confined to use with frozen sections
Carazzi’s Hematoxylin
Preparation
•Hematoxylin 5 g
•Glycerol 100 ml
•Potassium alum 25 g
•Distilled water 400 ml
•Potassium iodate 0.1 g

40. Hematoxylin is dissolvedin the glycerol.
Alumis dissolvedin most of the water overnight.
Potassiumiodate is dissolved in the rest of the water withgentle warming &
is then addedto the haematoxylin-alum-glycerol mixture.
The alumsolution is added slowlyto the hematoxylin solution.
Carazzi’s Hematoxylin
The final staining solution is mixedwell and is then ready for immediate use.

41. Delafield’s Hematoxylin (1885)
Preparation
• Hematoxylin 4 g
• 95% alcohol 125 ml
• Saturated aqueous ammonium alum 400 ml (15 g/100 ml)
• Glycerin 100 ml
Delafield’s Hematoxylin

42. Delafield’s Hematoxylin
The hematoxylin is dissolved in 25 ml of alcohol, and then addedto
the alumsolution.
This mixture is allowedto stand in light and air for 5 days and then filtered.
Allowthe stain to stand exposedto light and air for about 3–4 months or
until sufficiently dark in color.
Glycerin and a further 100 ml of 95% alcohol are addedto this mixture.
Filter before use.

43. Steps In Staining Procedure For Alum Hematoxylin
Progressive Staining Methodfor
paraffin sections
1. Bring sections to water with xylene and
ethanol.
2. Place into the staining solution for 10
minutes.
3. Rinse with water.
4. Blue.
5. Wash well with water.
6. Counterstain if desired.
7. Dehydrate with ethanol, clear with xylene
and mount with a resinous medium.
Regressive Staining Method for
paraffin sections
1. Bring sections to water with xylene and ethanol.
2. Place into the staining solution for 10 minutes.
3. Rinse well with water.
4. Differentiate with acid alcohol.
5. Rinse with water.
6. Blue.
7. Wash well with water.
8. Counterstain if desired.
9. Dehydrate with ethanol, clear with xylene and
mount with a resinous medium.
Staining

44. Staining times with alum hematoxylins
Staining time varies according to various factors:
• Type of hematoxylin used
• Age of stain
• Intensity of use of stain
• Whether the stain is used progressively or regressively
• Pre-treatment of tissues or sections
• Post-treatment of sections
• Personal preference
• General rule
Staining

45. Cole’s 20–45 min
Delafield’s
15–20 min
Ehrlich’s (progressive)
20–45 min
Mayer’s (progressive)
10–20 min
Mayer’s (regressive)
5–10min
Harris’s (progressive in cytology)
4–30 s
Harris’s (regressive)
5–15min
Carazzi’s (progressive)
1–2 min
Carazzi’s (frozen sections)
1 min
Gill’s I (regressive)
5–15min

46. CELESTINE BLUE
Preparation
•Celestine blue B 2.5 g
•Ferric ammonium sulfate 25 g
•Glycerin 70 ml
•Distilled water 500 ml

47. Ferric ammoniumsulphate is dissolved
in colddistilledwater with stirring.
The celestine blue B is addedto this solution and the
mixture is boiledfor fewminutes.
Filtered
Glycerine is added
Filter before use.

48. IRON HEMATOXYLINS
• In these hematoxylins, iron salts such as ferric chloride and ferric
ammonium sulfate are used both as the oxidizing agent and as mordant.
• Over-oxidation of the hematoxylin is a problem with these stains.
• Mordant/oxidant and hematoxylin solution are prepared separately and
mixed before use.
• Capable of demonstrating wider range of tissue structures compared to
alum haematoxylin.
• Techniques are more time consuming and needs microscopic control for
accuracy.

49. •An iron hematoxylin used as a nuclear stainin techniques where
acidic staining solutions are applied to the sections subsequently.
•e.g VanGiesonstain
• It is a useful stain, with eosin, for CNS tissues.
Preparation
Weigert’s Hematoxylin (1904)
a) Hematoxylin solution
• Hematoxylin 1 g
• Absolute alcohol 100 ml
This is allowed to ripen naturally for
4 weeks before use.
b) Iron solution
• 30% aqueous ferric chloride (anhydrous) 4 ml
• Hydrochloric acid (concentrated) 1 ml
• Distilled water 95 ml

50. • This iron hematoxylin uses ferric ammoniumsulfate as oxidant/mordant.
• It is a cytological stain used as the differentiating fluid used regressively.
• components are black or dark gray-black - removed progressively from
different tissue structures at different rates using the iron alum solution.
• Preparation
Heidenhain’s hematoxylin (1896)
a) Hematoxylinsolution
• Hematoxylin 0.5 g
• Absolute alcohol 10 ml
• Distilled water 90 ml
The hematoxylin is dissolved in the alcohol, and the
water is then added.
The solution is allowed to ripen naturally for 4 weeks
before use.
b) Ironsolution
• Ferric ammonium sulfate 5 g
• Distilled water 100 ml
• It is important that only the clear violet
crystals of ferric ammonium sulfate be
used.

51. Loyez’s hematoxylin
(1910)
•This iron hematoxylin uses ferric ammoniumsulfate as the
mordant.
•Differentiation is by Weigert’s differentiator (borax and
potassium ferricyanide).
•Used to demonstrate myelin.
•Can be applied to paraffin, frozen, or nitrocellulose sections.

52. Verhöeff’s hematoxylin (1908)
•This iron hematoxylin is used to demonstrate elasticfibers after all
routine fixative.
•Ferric chloride is included in the hematoxylin staining solution,
together with Lugol’s iodine, and 2% aqueous ferric chloride is used
as the differentiator.
•Coarse elastic fibres stain black, but the staining of fine fibers may be
less than satisfactory.
•The differentiation step is critical to the success of this method.

53. TUNGSTEN HEMATOXYLINS
• Widely used tungsten hematoxylin is original Mallory phosphotungstic acid
hematoxylin PTAH(Phosphotungstic acid hematoxylin Technique).
• Used to demonstrate fibrin, muscle striations, cilia and glial fibres.
• Myelin can also be demonstrated
• Widely used as a CNS stain.
Preparation
PTAH solution using haematin
Haematin 0.59 g
Phosphotungstic acid 5g
Distilled water 500ml
Stain is ready to use immediately, but
short-lived.
PTAH Solution (KMnO4)
Haematoxylin 0.59 g
Phosphotungstic acid 5g
Distilled water 500ml
0.25% Aqueous KMnO4 25 ml
Peak staining activity after 7 days

54. Results
•Muscle striations / neuroglia fibres / fibrin / amoeba – Dark Blue.
•Nuclei/cilia/RBC – Blue
•Myelin – Lighter blue
•Collagen /Osteoid / Cartilage / Elastic fibres – Deep brownish red.
•Cytoplasm – Pale pinkish brown.

55. MOLYBDENUM
HEMATOXYLINS
• Hematoxylin solution using molybdic acid as mordant- technique that gained
any acceptance was the Thomas (1941).
• Rare stain
• Used in demonstration of collagen, coarse reticulin.
• Also stains Argentaffin cell granules.
PreparationPhosphomolybdic acid hematoxylin stain (Thomas 1941)
Hematoxylin solution
Hematoxylin 2.5 g
Dioxane 49 ml
Hydrogen peroxide 1 ml
Phosphomolybdic acid solution
Phosphomolybdic acid 16.5 g
Distilled water 44 ml
Diethylene glycol 11 ml

56. Results
•Collagen and coarse reticulin violet to black
•Argentaffin cells black
•Nuclei pale blue
•Paneth cells orange

57. LEAD HEMATOXYLINS
•Used in the demonstration of the granules in the
endocrine cells of the alimentary tract and other regions.
•Most practical diagnostic application is in the
identification of endocrine cells in some tumors.
•Also used in research procedures such as in the
localization of gastrin-secreting cells in stomach
(Beltrami et al. 1975).

58. Hematoxylin without a mordant
•Freshly prepared hematoxylin is used to demonstrate various
minerals in tissue sections.
•These methods are now replaced with more specific
techniques.