Theories of staining

1. PRESENTED BY: ASHWINI AJAY
GUIDED BY: DR. NANDHINI
THEORIES OF STAINING
HEMATOXYLIN AND EOSIN STAINING

2. SYNOPSIS
• INTRODUCTION
• STRUCTURE OF DYES
• COLOR INDEX/CLASSIFICATION
• PRODUCTION OF COLOUR
• STAINING MECHANISM
• PHYSICAL AND CHEMICAL THEORIES
• METACHROMASIA
• LEUCO COMPOUNDS

3. • FLUORESCENT DYES
• EFFECTS OF FIXATION
• MORDANT
• ACCENTUATORS
• IMPREGNATION
• PREPARATION OF STAINS- SOLVENTS AND BUFFER
• STAINING AND MOUNTING PROCEDURE
• HAEMATOXYLIN AND EOSIN STAIN

4. INTRODUCTION
• STAINING
Culling et al
• Treating tissues or cells with a reagent or series
of reagents so that it acquires a colour , usually
no particles of dye are seen and the stained
element is transparent.
Bancroft
• Visual labelling of some entity by attaching or
depositing in its vicinity , a marker of definite
colour or shape.

5. • Biological staining :
Is the union between a coloured dye and a tissue substrate which resists simple
washing.
• Stain is any substance which when added to living cells or to fixed structures
or structural components makes them clearly visible or detecable.

6. • Biological stains have been used to visualise and identify tissue and
cell component
• Colouring or dying or staining of tissue:
• Makes it possible to see and study the physical features and
• To study the relationship between the tissue and their constituent cells.

7. IDEAL FEATURES OF A STAIN
• An ideal stain should allow for:
• Evaluation of the architectural pattern of the tissue fragments
• Proper evaluation of nuclear morphology: i.e all the nuclear
parameters should be clearly visualised.
• Details of cytoplasmic characteristic
• visualisation and identification of diagnostic features in the
background.

8. FOR SUCCESFUL STAINING
• Specificity - Is ability to distinguish between the individual cell
components.
• Sensitivity - Capacity of a stain to demonstrate tissue substance at low
concentration.
• Satisfactory staining method….. High sensitivity + high specificity.

9. AFFINITY OF STAIN TO TISSUE
• Depends on 2 factors:
• Physical factors
Osmosis
Absorption
Selective absorption
• Chemical factors
Based on acidic and basic nature of tissue
Acidic tissue has affinity to basic dye.
Basic tissue has affinity to acidic dye.

10. BASIC STRUCTURE OF DYE
• Dyes are coloured organic compound that can be selectively bind to tissue.
• Natural Dyes:
Extracted from plants and animals
e.g.: carmine and haematoxylin
• Synthetic dye:
Products of modern petrochemical industry.
e.g. Benzene

11. • Benzene :
• It is not coloured.
• Has a ring structure which gives flexibility . ( Double bonds are note
static)
• Chemical grouping when introduced into this ring induces colour to the
compound.
• These groups are called chromophore.
• The resultant structure is called chromogen.
CHROMOPHORE
CHROMOGEN
Eg:
nitro,azo,ethylene

12. • CHROMOPHORE
• 3 TYPES:
• Nitro group
-NO2.
Dyes include picric acid and auramine
• Azo group
N=N
Dyes include orange G and congo red
• Quninoid group
• -C=C-
• Dyes include basic and acidic fuchsin, eosin , crystal violet , methylene blue , neutral red and natural
dye.

13. • Auxochrome:
• Chromogen - not a dye
• To turn a coloured component into a dye it requires the addition of
ionisable group that allows binding to the tissue. - Auxochrome
• Eg: Sulphates , hydroxyl groups, carboxyl’s. Picric acid.

14. CLASSIFICATION OF DYES
• BASED ON ORIGIN:
Natural
Synthetic
• BASED ON THE PHYSICOCHEMICAL PROPERTIES OD DYES
Flourescent ( Thioflavin)
Metachromatic ( Toulidine blue )
Leuco ( Leuco methylene blue )
Neutral ( azure-eosinate )

15. • BASED ON HISTOLOGICAL CLASSIFICATION:
• Basic dye
Cationic ( +ve )
Will stain anionic or acidic materials
Nuclear stain , sulphate , phosphate of nucleic acid.
• Acidic dye
Anionic ( - ve )
Will stain cationic or basic group
mostly stains protein and connective tissue.

16. • Neutral dye
Compounds of basic and acid dyes
both ions are coloured
Stain both nucleus and cytoplasm from a single dye bath
Romanowsky stain , other stains are : Giemsa , Leishman , Wright stain.
• Amphoteric dyes
Have both anionic and cationic groups but these are the same ions.
Can stain either nucleus or cytoplasm

17. • Natural dye
Extracted from natural source
Haematoxylin - orcein
Carmine - Litmus

18. PRODUCTION OF COLOUR
• Double valency of benzene are not stable
• Light waves - excite electrons
• Change in bond position
• Excitment of benzene - bands pushed to higher wavelength.
• Colour visible to eye.

19. TYPES OF STAINING
• Direct staining: When mordant is not necessary. ( acholoic or aniline stain ).
• Indirect staining: When mordant is applied. ( H and E method ).
• Progressive staining: staining that happens at a specific time period.
• Regressive staining : Over staining of tissue structures.
• Vital staining : When inclusion of cells or tissue are stained.
• Supravital staining: When living cells are stained after being removed from
the body
• Intravital staining: Staining of cells while still part of the body.

20. COMMONLY USED TERMINOLOGIES
• MORDANT: Metallic substance that acts as a link between the dye
and the tissue
• Mordant + dye = LAKE
• Lake is invariable basic in nature.
• Advantages:
• Insoluble in most fluids used in biological staining.
• Little altered by treatment of tissue section.

21. • ACCENUATORS:
• Chemical substance which increase the:
• Colouring power
• Crispness
• Selectivity of stain.

22. • IMPREGNATION:
• Deposition of salts on or around the cells and tissue constituent etc.
• It has the following charcterstics:
• Structures demonstrated are opaque and black
• Colouring matter is particulate
• The deposit is on or around but not in the element so
demonstrated.

23. Hematoxylin
 Dark red color
 The hematoxylin is extracted from log wood with hot water
and then precipitated out from the aqueous solution using urea.
 It is sold commercially as a crude mixture of hematoxylin and other, unide
 It comes as a brownish tan powder which is poorly soluble in water and so

24. 1. Alum haematoxylins
2. Iron haematoxylins
3. Tungsten haematoxylins
4. Molybdenum haematoxylin
5. Lead haematoxylins

28. Eosin
 Stains connective tissue and cytoplasm in varying
intensity and shades (red to pink)
 Eosin is derived from fluorescein and is available in
following types:
  
Eosin Y (eosin yellowish, eosin water soluble) Ethyl eosin (eosin S,

29. Eosin Y is most commonly used and is readily soluble in
water, less so in a alcohol thus it is sometimes sold as ‘water
and alcohol soluble’
 Preparation
Eosin Y, water soluble 5gm.
Distilled water 1000ml.
Crystals of THYMOL added to inhibit the growth of
fungi.

30. in alcohol.
 Eosin Y water & Alcoholsoluble 10gm
Distilled water 50ml 95%ethyl alcohol 940ml
 In use, sections should be treated with 95% alcohol
before staining with alcoholic eosin, and the excess stain
washed out in the same solvent.

31.  The addition of little ACETIC ACID (0.5 ml to 1000 ml
stain) is said to sharpen the staining.
 Ethyl eosin and eosin B are now rarely used, although
occasional old methods specify their use, e.g. the Harris stain
for Negri bodies.

32. HAEMATOXYLIN AND EOSIN
Armamentarium in staining
• Specially designated bench
• Staining bench Should be facing window
• Slide washing tray made of stainless steel
• Bunsen burner – to heat up the stain
• Thermostatically controlled hot plate to melt the wax
• Microscope to control staining reaction

33. Hematoxylin itself is not a stain.
On oxidation it produces HEMATIN - a poor dye but
metallic mordant, forms the most powerful stain.
 When aluminum salts– will stain blue  When ferric salt–
will stain blue-black

34. Ripening
 This process of oxidation is often referred to as ripening or
maturing.
 This can be carried out in two ways
1. Natural oxidation
2. Chemicaloxidation

35.  Carried out by exposure to light and air.
 Slowprocess
 Resultant solutions seem to retain its staining ability for a long time.
Advantage
 Ones oxidation has reached an acceptable level, the staining solution
may be used, and it last for longer,
Disadvantage
 In the planning and organization required ensuring that usable

36. Chemical oxidation:
 It is achieved by the addition of the oxidizing agents such
as mercuric oxide, sodium iodate and potassium
permanganate.
 The use of chemical oxidizing agents converts the
hematoxylin to haematin almost instantaneously, so these
hematoxylin solutions are ready for use after preparation.

37. 
1. Slides are stained in following ways
Using staining dishes
 Small grooved couplin jars with glass lids
 Large staining troughs
2. Using staining racks
 Two pieces of stout glass rods 2-4 cm apart

38. Hematoxylin and eosin technique 
Principle
 H and E are principle stain for demonstration of nucleus and cytoplasm.
 Alum acts as a mordant and the hematoxylin containing alum stains the
nucleus light blue which turns red in the presence of acid.
 The cell differentiation is achieved by treating the tissue with acid
solution. The counterstaining is performed using eosin which imparts pink color
to cytoplasm

39. Hematoxylin and eosin technique
 Removal of paraffin wax (Deparaffinization)
 Removedwithxylene(impermeabletostains)
 2-3min of xylene immersion sufficient for sections of 10 μ
thickness
 First facilitated by warming the slides at 60 degrees oven to melt
the wax
 Removal of xylene
 Xyleneisnotmisciblewithwaterorlowgradealcohols,hence dipped in two

40.  Hydration(High to low)
 After removal from xylene sections are transferred to
absolute alcohol for 1-2min until it becomes opaque
 Sections rinsed in second bath of alcohol, drained and taken to
water
 Any pigments or deposits should be removed at this stage

41.  Staining
 Slides immersed in hematoxylin (Mayer s, Harris, Gills)
 If regressive stain is used longer time is used to overstained
the structures
 Differentiation
 Sections are dipped in Acid alcohol, agitated and washed in
tap water
 Observed under microscope
 If underdifferentiated- returned to acid alcohol

42.  Blueing
 Slides after draining off hematoxylin is transferred to ammonia water for 2
min. Sections when removed from hematoxylin or acid alcohol are pink in color
 Washing turns them blue
 Counterstain(Eosin)
 Transfer the slides to 1% aqueous eosin for 2min. Wash in running water
 Dehydration( low to high)  Slides are taken through

43. 11. Clearing
 Sections transferred to xylene and left until clear
 Testedforclaritybybeingheldagainstadarkbackground
12. Mounting
 Surplousxylenewipedofffromslidesurface
 Thisstepcompletedquicklytoavoidsectiondrying  Wholeoperationtakes5-
10seconds

44. Hematoxylin and eosin technique
 Results
 Cell nuclei – Blue
 Muscle fibres – Red
 Collagen fibres – Pink  RBC – Bright red