Haematoxylin and its types.ppt

HAEMATOXYLIN AND ITS TYPES

INTRODUCTION

The word hematoxlin is drived from old Greek word Haimato(blood) and Xylon(wood), reffering to its dark red color in natural state and to its origin(wood).

A natural dye extracted from the log wood of tree Haematoxylon Campechianum .

It is the most important and most used dye in the histopathology, cytology and immunohistochemistry.

Basic in nature and stains acidic component of the tissue, nucleus, mitochondria etc.

It is one of the best nuclear stain.

■ Also use to stain metal ions e.g iron, lead etc.■ Stains tissue components in various shades of blue, pink,

red etc, depending on the nature of the mordant used and the second stain.

■ It is itself not an active dye, it is its oxidative product Haematein(natural dye).

■ Haematein is a weak dye have poor affinity for tissue and requires a mordant for adequate staining.

■ Mordant is a chemical substance that increases the affinity of dye for tissue and its staining efficiency by

forming link between the stain and the tissue.■ Mordants are the metallic salts e.g salts of aluminum

and iron etc.

Extraction:♣ It is extracted by boiling the

heart wood of Hematoxylon Campechianum in hot water.

♣ It is then precipitated out of theaqueous solution using urea or ether.

Oxidation:Two methods of oxidation:1. Natural oxidation: Exposed to air and sunlight. Takes 3-4 months to complete Resultant solutions have longer useful life.

Examples are Ehrlich’s and Delafield’s haematoxylin.

2. Chemical oxidation: Chemical oxidizing agents are used, e.g sodium

iodate, mercuric oxide, hydrogen peroxide, potassium permanganate etc.

Instant conversion of haematoxylin to haematein, immediately ready for use.

Resultant solutions have a shorter useful life. Examples are Mayer’s and Harris’s haematoxylins.

TYPES OF HAEMATOXYLIN

1. Alum haematoxylins

2. Iron haematoxylins

3. Tungsten haematoxylins

4. Molybdenum haematoxylins

5. Lead haematoxylins

6. Haematoxylin without mordants

ALUM HAEMATOXYLIN

Mordant used are alumminumsalts, either aluminum potassium sulphate (potash alum) or aluminum ammonium sulphate (ammonium alum).

Mainly used in routine H and E staining. Has different types but all of them stain the

nuclei blue-black. All types are commonly reffered as haemalums. Haemalums are used when counter stain does

not contain an acid. Can be used progressively or regressively.

TYPES OF ALUM HEMATOXYLIN

Ehrilch’s haematoxylin.

Mayer’s haematoxylin.

Harris’s haematoxylin.

Gill’s haematoxylin.

Cole’s haematoxylin.

Delafield’s haematoxylin.

Carazzi’s haematoxylin.

Stain Mordant Oxidation Applications Staining time

Life span

Ehrlich’s hematoxylin

Potash alum Natural Nuclear stain used with eosin

P= 20-45 min

More than a year

Mayer’s hematoxylin

Ammonium or potash alum

Chemical(Sodium iodate)

Nuclear stain used with eosin

P=5-10 minR=10-20 min

3-4 months

Hariss’s hematoxylin

Ammonium or sodium alum

Chemical(Mercuric oxide)


P= 4-30 sR=5-15 min

About 3 months

Gill’s hematoxylin

Aluminium sulphate

Chemical(Sodium iodate)


R=5-15 min About 3 months

Cole’s hematoxylin

Potash alum Chemical(Alcoholic iodine solution)


20-45 min 3 months

Delafield’s hematoxylin

Ammonium alum

Natural Nuclear stain used with eosin

15-20 min More than a year

Carazzi’s hematoxylin

Potash alum Chemical(Potassium iodate)


P= 1-2 minR= 10 min

6 months

COMPOSITIONS OF HAEMALUMS

Ehrlich’s hematoxylin

Hematoxylin 2g

Absolute alcohol 100ml

Distilled water 100ml

Glycerin 100ml

Glacial acetic acid 10ml

Potash alum 15g (approx)

Mayer’s hematoxylin

Hematoxylin 1g


Potash alum 50g

Sodium iodate 0.2g

Citric acid 1g

Chloral hydrate 50g

Hariss’s hematoxylin

Hematoxylin 2.5g

Absolute alcohol 25ml


Mercuric oxide 1.5g


Potash alum 50g

Gill’s hematoxylin

Hematoxylin 2g

Sodium iodate 0.2g

Aluminium sulfate 17.6g


Ethylene glycol 250ml


Cole’s hematoxylin

Hematoxylin 1.5g

Saturated aqueous potash alum 700ml

1% iodine in 95% alcohol 50ml


Delafield’s hematoxylin

Hematoxylin 4g

95% alcohol 125ml

Saturated aqueous potash alum 400ml

Glycerin 100ml

Carazzi’s hematoxylin

Hematoxylin 5g

Potash alum 25g

Potassium iodate 0.1g


glycerol 100ml

IRON HEMATOXYLIN

Iron salts are used as mordant, these are also oxidizing agents.

Commonly used iron salts are ferric chloride and ferric ammonium sulphate.

Overoxidation of the prepared and stored iron hematoxylins is the problem.

To avoid this problem hematoxylin and mordant solutions are either mixed just before use (as in weigert’s hemtoxylin) or used consecutively (as in heidenhain’s hematoxylin)

These are used when the counter stain is acidic (e.g Van Gieson and other Trichome stains).

Demonstrate a much wider range of tissue structure but is time consuming due to differentiation stage.

TYPES OF IRON HEMATOXYLINS

Weigert’s hematoxylin

Heidenhain’s hematoxylin

Verhoeff’s hematoxylin

Loyez hematoxylin

.

Stain Mordant Oxidation

Applications/ Results

Staining time


Ferric chloride Natural Nuclear stain with acid dyeStains the nucleus brown to black

15-30 min


Ferric ammonium sulphate

Natural Intracellular details (mitochondria, chromatin, nucleus, centrioles etc) and muscle fiber striationsStains cellular components black or dark gray-black

30-45 min at 60ºC12-24 Hours at room temp.


Ferric chloride Natural Stains elastic fibers black

25-60 min

Loyez hematoxylin

Ferric ammonium sulphate

Natural Myelin

COMPOSITIONS OF IRON HEMATOXYLINS


a) Hematoxylin solution b) Iron solution

Hematoxylin 1g 30% aqueous ferric chloride

4ml

Absolute alcohol 100ml Hydrochloric acid 1ml



a) Hematoxylin solution b) Iron solution

Hematoxylin 0.5g Ferric ammonium sulphate

5g

Absolute alcohol 10ml Distilled water 100ml



Stock solutions

a) 5% alcoholic hematoxylin 10ml

b)10% ferric chloride 4ml

c) Lugol’s iodine 4ml

TUNGSTUN HEMATOXYLIN

Mallory phosphotungstic acid hematoxylin (PTAH) is an example.

Phosphotungstic acid is used as mordant. Hematoxylin is oxidized naturally, can also be

oxidized using potassium permanganate. Can be prepared using hematein, no

oxidation required. Routine stain for nervous tissue, also used to

stain muscle striations and fibrin. Stains in shades of blue and red. Regressive stain with staining time of 1-16

hours at room temperature and 1-2 hours at 60ºC.

MOLYBDENUM HEMATOXYLIN

Molybdic acid is used as mordant. Very rare, most accepted molybdenum

hematoxylin is Thomas hematoxylin. Hydrogen per oxide is used for oxidation. Used to stain chollagen and granules in

endocrine cells. Stains:

i. Chollagen - violet to blackii. Argentaffin cells - blackiii. Nuclei - pale black

SOLCIA HEMATOXYLIN

Lead salts are used as mordant No oxidation Used to demonstrate granules in endocrine cells.

Mallory Hematoxylin No mordant required No oxidation Used to demonstrate various minerals in the

tissue e.g iron, copper, lead.

Haematoxylin and its types.ppt

Education

Transcript of Haematoxylin and its types.ppt