Basics of staining

1. STAINING
TECHNIQUES
-BY
DR.N.MANJULA
MODERATOR: DR.ABHINIZHA

2. STAINING
▪ Staining is the process of applying dyes
on paraffin cut sections to study the
architecturaland cellular pattern of the
tissues.

3. PURPOSE OF STAINING
▪ Outlines tissues and cellular
components
▪ Identification of tissue
▪ Establishes the presence or absence
of disease

4. TYPES
▪ ROUTINE: cells and tissues combinewith
active coloring agent. Eg:- H and E.
▪ SPECIAL: Eg:- impregnationof silver salts in
reticulin stain.
▪ VITAL: staining selective living tissue
components,demonstrating cytoplasmic
structures by phagocytosis of dye. Eg:-
Reticuloendothelial system withTRYPAN
BLUE.

5. ▪ SUPRAVITAL: stains living cells after
removal from body. Eg:-Retic stain.
▪ METACHROMATIC: certain dyes stain and
tissues in a color quite different from
fundamentalcolor of stain.This is
metachromasia. Preferable to use frozen
sections or fresh tissues.

6. BASED ON TISSUE AFFINITIES
▪ BASIC STAIN: active coloring substance combined
with a colourless inorganic acid radical.Any substance
stained by the basic dye – basophilic.In H and E
basophilicstructures appearBLUE.
▪ ACID STAIN: substance that is stained by an acid dye is
referred to as acidophilic. In H and E, acidophilic
substance due to eosin appear in various shades of pink.
▪ NEUTRAL STAIN: it is formedby mixing aqueous
solutions of certain acidic and basic stains. Eg:-
Romanowsky dyes.

7. BASED ON SOURCE
▪ NATURAL DYE: obtained from plants and
animals.
▪ Eg:- hematoxylin.
▪ SYNTHETIC DYE: derived from coal tar and
hydrocarbon benzene.
▪ Eg:- aniline, azo dyes.

8. METHODS OF STAINING
▪ DIRECTSTAINING:process of giving color to the
sections by using aqueous or alcoholic dye
solutions.
▪ Eg:- methyleneblue and eosin.
▪ INDIRECTSTAINING:process by which action of
dye is intensified by adding another agent
(MORDANT).
▪ Mordantis a polyvalentmetal ion which forms
coordinationcomplexes with certain dyes.
▪ Eg: Potassiumalum with Ehrlich hematoxylin,Iron
inWeigert hematoxylin.

9. Common staining methods
▪ Hematoxylinand Eosin staining in
histopathology
▪ Gram's stain
▪ Romanowsky staining in Haematology
▪ Papanicoloaustaining inCytology

10. Introduction
▪ Hematoxylin and eosin is the most widely
used stain because :
❖ It's simple
❖Ability to demonstrate clearly enormous
number of different tissue structures .
▪ Haematoxylin stains nucleus blue and gives
good intranuclear detail.
▪ Eosin stains cytoplasm pink.

11. Hematoxylin
▪ It is derived fromGreek word Haimato
meaning blood and Xylonmeaning wood.
▪ A natural dyeextracted from the core of tree
Haematoxylon campechianum.
▪ Hematoxylinis extracted from logwoodwith
hot water and then precipitated out from the
aqueous solution using urea.
▪ Major oxidization product of hematoxylin
HAMATEINwhich is responsiblefor the color
properties.

14. Ripening
▪ Process of oxidation of hematoxylin.
▪ Carried out in two ways:
▪ Natural
▪ Artificial

15. ▪ Natural - exposure to light and air, slow
process, 3 to 4 months, Eg:- Ehrlich's
hematoxylin.
▪ Artificial – by addition of oxygen or removal
of hydrogen.
▪ Adding chemicals mercuric oxide, sodium
update, potassium permanganate, instantly
prepared.

16. ▪ Haemateinis anionic, having poor affinity
for tissue, it is inadequate to stain nucleus
alone.
▪ Hence a mordant is required.
▪ Most useful are: salts of aluminium,iron.

17. TYPES OF HEMATOXYLIN
▪ ALUM HEMATOXYLIN
▪ IRON HEMATOXYLIN
▪ TUNGSTENHEMATOXYLIN
▪ MOLYBDENUMHEMATOXYLIN
▪ LEAD HEMATOXYLIN
▪ HEMATOXYLINWITHOUTMORDANT

18. Alum hematoxylin
▪ Used routinely to producegood nuclear staining.
▪ Mordant used are either aluminium potash sulfate
or aluminium ammonium sulfate is used in our dept.
▪ TYPES OF ALUM HEMATOXYLIN
▪ Ehrlich’s hematoxylin
▪ Mayer’s hematoxylin
▪ Harris hematoxylin is used in our dept.
▪ Delafield’s hematoxylin
▪ Cole’s hematoxylin
▪ Gill’s hematoxylin
▪ Carazzi’s hematoxylin

19. ▪ Major disadvantage is their instability to
resist acid solution and relative
ineffectiveness when used with fixatives
particularly of acid type.
▪ HenceWeigert’s is used which is resistant to
picric acid.
▪ Celestine blue B is successfully used as a
mordant for alum hematoxylin when used
with acid solutions.

20. ▪ PROGRESSIVESTAINING:sections are
stained for predetermined time so that nuclei
become well stained but background is
relatively understained.
▪ REGRESSIVE STAINING:sectionsare first
overstained then excess stain is removed in
acid alcohol.
❑Advantage:degree of stainingis controlledand
nuclei take good stainingwhere cytoplasmis
clear.

21. ALUM HEMATOXYLIN SPECIALCHARACTERS
HARRIS HEMATOXYLIN
EHRLICH HEMATOXYLIN
MAYER’S HEMATOXYLIN
GILL’S HEMATOXYLIN
DELAFIELD’S HEMATOXYLIN
COLE’S HEMATOXYLIN
CARAZZI’S HEMATOXYLIN
2-4 ml OFGLACIALACETIC ACID IS ADDED TO 100 mlOF
STAINTO INCREASE THE NUCLEAR STAIN PRECISION.
ALWAYS FILTER BEFORE USE.
IT IS A REGRESSIVE STAIN.
EXCELLENT NUCLEAR STAIN USED AS REGRESSIVE
STAIN.
USED AS BOTH PROGRESSIVEAND REGRESSIVE STAIN.
USED AS PROGRESSIVE NUCLEAR COUNTER STAIN FOR
GLYCOGEN.
THIS CAN BE READILY USED.
NATURALLY RIPENED ALUM HEMATOXYLIN.
ARTIFICIALLY RIPENED HEMATOXYLIN.
USED IN FROZEN SECTION.
PROGRESSIVE NUCLEAR STAINWITH SHORT STAINING
TIME

22. Iron hematoxylin
▪ Weigert’s hematoxylin
▪ Heidenhain’s hematoxylin
▪ Verhoeff’s hematoxylin
▪ Loyez hematoxylin

23. ▪ Iron salts are used as both oxidizing agent
and as mordants.
▪ Most commonlyused are ferric chloride
and ferric ammoniumsulfate.
▪ Not only used as nuclear stain but also to
demonstrate muscle striations, cellular
organelles – mitochondria.

24. IRON HEMATOXYLIN SPECIALCHARACTERS
WEIGERT’S HEMATOXYLIN
HEIDENHAIN’S HEMATOXYLIN
VERHOEFF HEMATOXYLIN
USED AS NUCLEAR STAIN.
CELESTINE BLUE ALUM HEMATOXYLIN HAS
REPLACED IT.
IT IS USEFUL WITH EOSIN FOR CNS TISSUES.
IT IS A PROGRESSIVE STAIN.
USED TO DIFFERENTIATE STRUCTURES BASED
ON DEGREE OF DIFFERENTIATION OF STAIN.
IT IS A REGRESSIVE STAIN.
TISSUE ELEMENTS DEMONSTRATED ARE
MITOCHONDRIA, STRIATIONS OF MUSCLE
DEVELOPED PRIMARILYTO STAIN ELASTIC
FIBRES.
COUNTER STAINUSED ISVANGIESONSTAIN.
IT GIVES HIGHCONTRAST WHICH CAN BE USED
FOR ELECTRON MICROSCOPY.
ELASTICFIBRES: BLUISH BLACK.
CYTOPLASM AND MUSCLE:YELLOW.
COLLAGEN: RED

25. TUNGSTEN HEMATOXYLIN
▪ Its use is applicable to bothCNS material
and general tissue structure.
▪ Stainingis more precise after sections have
been treated with acid dichromate solution.
▪ Good results are obtained with buffered
formalinfixative.
▪ Ripened both naturally and artificially.
▪ Time consuming process.

26. OTHERS
▪ MOLYBDENUM HEMATOXYLIN –
complexes of molybdenumand hematoxylin
are principallyused to stain collagen and
coarse chromatin.
▪ LEAD HEMATOXYLIN – used for
demonstrationof granules in neuroendocrine
system, keratohyalin granules and calcium
deposits.
▪ COPPER HEMATOXYLIN – used to stain
fatty acids, acidophils in pituitary, myelin
sheaths, mitochondria.

27. ▪ CHROMIUMALUM HEMATOXYLIN –
used to stain lipoproteins, myelin,
phospholipidsand cytoplasmicgranules in
beta cells of anterior pituitary and
pancreatic islets.
▪ HEMATOXYLINWITHOUTMORDANT:
free hematinstains collagen, elastin and
erythrocytes from yellow to orange brown.

28. EOSIN
▪ Most commonly used counterstain with
hematoxylin.
▪ Other counter stains are : orange G, phloxine,
Bordeaux red and Biebrich scarlet.
▪ But these give undesireable intense red colour
and fail to achieve subtle differentiation as in
eosin.
▪ They are fluorescent xanthene dyes.

29. ▪ Types: eosinY, ethyl eosin, eosin B.
▪ EosinY is widely used and is both water and
alcohol soluble.
▪ Glacial acetic acid may be added which
augments staining.
▪ Thymol added to prevent fungal growth.

30. STAIN PREPARATION
▪ REQUIREMENTS:
▪ XYLENE
▪ ACETONE
▪ HAEMATOXYLINE SOLUTION
▪ ACIDALCOHOL
▪ EOSIN SOLUTION
▪ DPX – distyrene, a plasticizer and xylene.

31. ▪ PREPARATION:
▪ Haematoxyline powder:2.5 g
▪ Absolute alcohol:50 ml
▪ Ammonium or potassium alum:50 g
▪ Distilled water:500 ml
▪ Mercuric oxide:1.5 g
▪ Glacial acetic acid: 10 ml

32. ▪ PREPARATION:
▪ Dissolve haematoxylinein alcohol seperately with
heat.
▪ Heat 500 ml of distilled water and add 50 g of
ammonium or potassiumalum till it dissolves.
▪ Then add Haematoxyline solutionin to the alum
water.
▪ After boiling add 1.5 gm mercuric oxideand mix it
well.
▪ Cool the solutionand add 10 ml of glacial acetic
acid and mix it well.
▪ Filter before use.

33. ▪ 0.1 % acid alcohol:
▪ 70% absolute alcohol 99 ml and conc.HCL 1.0
ml.
▪ Eosin:
▪ Eosin 0.5 g, distilled water 500ml, glacial
acetic acid 1 drop.

34. PROCEDURE
1. DEWAXNATION:
xylene I - 5 min
xylene II – 5 min
2. DEHYDRATION:
acetone I – 5 min
acetone II – 5 min
3. WASHWITHWATER.
4. STAINWITH HAEMATOXYLINEFOR 5 min.

35. 5.Wash with water.
6.ONE DIP inACIDALCOHOL and
immediatelywash in water.
7. BLUING – running tap water 10 min.
8. SATINWITH EOSIN FOR 30 sec.
9.Wash with tap water and allow for air dry.
10. MOUNTWITH DPX.

37. RESULTS
▪ Collagen: pale pink.
▪ Muscle: deep pink.
▪ Acidophilic cytoplasm: red.
▪ Basophilic cytoplasm: purple.
▪ Nuclei: blue.
▪ Erythrocytes: cherry red.

38. AUTOMATIC STAINING

43. QUALITY CONTROL
▪ Daily one QC is done in our dept.
▪ Efficacyof hematoxylin is tested by adding
few drops of it to tap water and the colour
change is noted.
▪ If it turns blue – purple immediately,it is in
working condition.
▪ If it turns red to brown, it should be
discarded.

44. ADVANTAGE DISADVANTAGE
SIMPLE PROCEDURE
COST EFFECTIVE
PLEASING COLOURS
MOST HISTOLOGIC
STRUCTURES ARE
STUDIED
DEPARAFFINIZATION IS
IMPORTANT.
AND SHOULD BE
ADEQAUTE.
AIR BUBBLES CAUSE NON
STAINING.
DEHYDRATION AFTER
STAINING IS IMPORTANT.

45. REFERENCES
▪ MANUALOF HISTOLOGICTECHNIQUESBY
SANTHOSHKUMAR MONDAL.
▪ BANCROFT’STHEORYAND PRACTICEOF
HISTOPATHOLOGICALTECHNIQUES.
▪ PRINCIPLESAND INTERPRETATIONOF
LABORATORYPRACTICESINSURGICAL
PATHOLOGY BY SHAMEEMSHARIFF.
▪ BASICANDADVANCEDLABORATORY
TECHNIQUESIN HISTOPATHOLOGYAND
CYTOLOGY BY PRANABDEY.