The document discusses the theory of staining in histology. It explains that staining allows tissues to be distinguished by providing different colors. Staining works through interactions between dyes and tissue components based on factors like charge, size, and chemical bonds. Differential staining is achieved through differences in binding capacity, rate, and strength between dyes and tissues. The document outlines various dye properties, classification systems, and staining methods. It also addresses factors influencing staining quality and provides examples of common problems and their solutions.

1. Theory of Staining
Practical Implications

2. • Biological stains have been used for centuries in
histology.
• Ignorance of the mechanisms involved (except in
histochemistry).
• Understanding of the principles involved –
judicious approach to staining.
• Investigation of failures and problems with
wisdom.

3. Overview
• The need for staining?
• Basic Principle involved?
• Properties of Stains?
• Problems & Troubleshooting?
1. How and why tissues are stained?
2. Properties of Dyes
3. Basis of Differential staining
4. Factors affecting
5. Problems and solutions

4. Why do we Stain?
• Any object is seen only when it gives out light.
• Visible objects are distinguishable from surroundings only when they
are different in light color or intensity from surroundings.
• Metal impregnation, polarized microscopy, phase contrast
microscopy: difference in intensity.
• All other histology methods: different color.
• Staining: provides these different colors.
• Stains: give characteristic colors or hues to cell and tissue
components.
• Differentiation of various cells and tissues.

5. Properties of Dyes

6. Why are Dyes Colored?
White light falling on a dye molecule
Absorption of some WL of this light
Remaining WL passed on
Dye appears to be the color of that WL

7. Metachromasia
• Property of a dye to change color without change in chemical structure.
• One color at low conc; different color at high conc.
• Reason: polymerization. Eg: Toludine blue
• Low Conc: Blue; High Conc: Purple
• Factors favoring polymerization:
– Dye Conc: Low - blue; High - purple; Very High - red
– Temperature: Low - purple; High - blue
– Solvent: Aqueous - favors polymerization
Non-polar - no polymerization
– Tissue Factors: Nucleus - blue
Cartilage - purple (high density of sulphate ions)

8. T.Blue Staining of Muscle fibers
T.Blue Staining of Kidney

9. Dye Chemistry and Staining
• pH: Acidic dyes - Basic areas
Basic dyes - Acidic areas
Eg: H&E
• Molecular Size: Large - more staining time
Eg: Alcian blue & Crystal violet
• Solvency: relative hydrophilic/lipophilic character
Eg: Alcian blue - hydrophilic (can be dehydrated through alcohols)
Crystal violet - lipophilic (lost on dehydration through alcohols)
• Impurities: reduce staining intensity.

10. Dye Nomenclature & Classification
• Society of Dyers & Colorists (1999): Color Index
• Exclusively biological dyes, IHC stains etc: no standard names or
codes.
• Classification of Dyes
– Chemistry: Nitro, Azo, Phenol, Thiazols etc.
– Origin: Natural (Hematoxylin); Synthetic
– pH: Acid, Basic, Amphoteric, Neutral
– Physicochemical properties: Fluorescent, Metachromatic, Leuco

11. Additional factors in Dyes
• Mordants:
– A link binding the dye to the tissue
– No color of itself
– Used when dye has less affinity for tissue
– Mostly metal compounds: Fe, Al, Mo etc.
– Can be added to dye solution (Weigert’s H) or to tissue
(Heidenhain’s H)
• Accentuators:
– Increase selectivity or staining power of dye
– No chemical union with either dye or tissue
– Varied and obscure mode of action
– Chemical catalyst; KOH in Loeffler’s methylene blue (increases
alkalinity)

12. Basic Theory of Staining

13. Why and How Tissues Stain?
• Fundamental reason: Dye-Tissue Affinity.
• Affinity: preference of a tissue for a dye and strength of
their bonding.
• Transfer of stain from solution to section.
• Magnitude of Affinity:
 Stain-Tissue Interactions
 Stain-Stain Interactions
 Solvent-Solvent Interactions
 Stain-Solvent Interactions

14. Stain-Tissue Interactions
• Coulombic or Electrostatic Bonding:
– Attraction b/w differently charged molecules.
– H&E. –ve H to +ve nucleus & +ve E to –ve cytoplasm.
– pH, Dye conc, Temp etc affect magnitude of attraction.
• Van der Waal’s Forces:
– Dipole-dipole interactions
– Substrate groupings with tyrosine/tryptophan amino acid residues; dyes with large
aromatic rings
– Staining Elastic fibers with Congo red
• Hydrogen Bonding:
– Aqueous solutions – no role
– Wholly or partially non-aqueous solutions
– Best’s Carmine for Glycogen
• Covalent Bonding:
– Methods like Fuelgen nucleal, PAS etc

15. Stain-Stain Interactions
• Affinity b/w dye particles contributes to staining
pattern and efficiency.
• Metachromasia: polymerization of dye molecules
at high conc
• Silver Impregnation: Ag grains attracted to
already formed Ag grains

16. Solvent-Solvent Interactions
• Hydrophobic bonding in case of organic reagents
• In aqueous solutions, hydrophobic groups come
together to attain stability
• Tissue constituent and dye molecule
• Enzyme substrates in histochemistry; Fat staining
by Sudan dyes

17. Stain-Solvent Interactions
• Relative affinity of the dye molecule for the
solvent
• High affinity for solvent: dye may not leave
the solvent for tissue binding
• Sudan stain for fat: used in aqueous
solution.

18. Why do Stains remain on Tissues?
• Most of the stains: no or reduced affinity for dehydrating
fluids or mounting media.
• Metal complex stains, acidic dyes etc. belong to this
group.
• Basic dyes like Crystal violet, methylene blue etc dissolve
freely in lower alcohols. Dehydrate rapidly or use non-
alcoholic solvents or air-dry.
• Sudan stains: soluble in alcohols, clearing agents and resin
mountants. Use non-alcoholic solvents and mount in
aqueous media.

19. Basis of Differential Staining

20. • Question of selectivity
• 3 factors:
– Relative binding capacity
– Binding rate
– Binding strength

21. Relative Binding Capacity
• Number and affinity of binding sites
• Affinity: H&E.
 Nucleus - more affinity for H
 Cytoplasm - more affinity for E
• Number: Metachromasia with Toludine blue
 Nucleus - less number of binding sites;
orthochromatic blue
 Cartilage - more number of binding sites;
metachromatic purple

22. H&E of Glomeruli

23. Binding Rate
• Progressive staining methods: rate controlled
• Difference in rate of stain uptake for different structures
• Eg: Mucin staining with Alcian blue or Colloidal Iron
 Long Time – All basophilic structures stained
 Short Time – Only rapid staining mucins stained
• Difference in speed of reaction
• Eg: Oxidation with Periodic Acid in PAS staining
 Long Time – Oxidizes variety of molecules
 Short Time – Only fast reacting polysaccharide groups oxidized

24. PAS +ve Goblet cells in Intestinal Lining
Alcian Blue for Mucins

25. Binding Strength
• Regressive staining: strength controlled
• Differing bond strengths for different structures
• Differential loss of dye
• Eg: Iron H for muscle striations
 Non-selective staining of all structures
 Extraction in a solvent
 Permeable structures lose dye quickly
 Relatively impermeable structures like muscle bands retain stain

26. Cardiac Muscle Iron Hematoxylin Stain

27. Other Factors Influencing
Staining

28. Fixation & Staining
• Fixation: to prevent tissue autolysis and breakdown by
bacteria
• Ability of fixative to retain a tissue substance
• Eg: Lipids
 Well preserved with OsO4
 Poorly retained in formalin or alcohol fixed tissues
• Eg: Proteins in IHC
 Glutaraldehyde – retains proteins better but Ag sites are blocked;
so useless in IHC
 Acetone – poorer fixative of proteins but retains antigenicity of
tissue

29. Specimen Geometry & Staining
• Microscopy: 2-D image of 3-D structure
• Small difference in specimen thickness: alter staining
pattern
• Simple Geometrical Influences
 Thin sections stain faster
 Specimens with irregular surfaces stain faster (Cryosections)
 Dispersed specimens (smears) stain faster
• Complex Geometrical Influences
 Artefactual: Chatter – alternate dark and light stained areas
 Biological: Large secretory granules v/s Small granules

30. Some Terminologies &
Special Methods in Staining

31. • Progressive Staining:
 Tissue immersed in dye until only desired structures are stained
 Difficult process to control
 Dyes with good degree of selectivity
• Regressive Staining:
 Overstaining all tissue structures
 Removing from unwanted groups
 Depends on dye-tissue bond strength

32. • Negative Staining:
• Structures disclosed by outlining them with stain
• Nigrosine for microbes; Picro-thionine for bone
matrix
-ve Staining of Sperm
-ve Stained Phage virus

33. • Vital Staining:
Live cellular or tissue structures
Toludine blue for identifying dysplastic areas
in epithelium, in vivo
Biologic composition and activity difference

34. • Lipid Staining:
No chemical bonds
Dye dissolves in tissue lipids
OsO4 – covalent binding to unsaturated lipids
OsO4 staining of Myelin sheaths
in a peripheral nerve
Lipid Stains
Oil Red O & Sudan Black

35. • Combination Staining Methods:
Combination of staining methods providing sections
with greater detail than either method used alone
Verhoeff-Masson Trichome
Masson Trichome

36. Problem Avoidance &
Troubleshooting

37. Issues Concerning Staining
Procedures
• Choose fixative and embedding media suitable for
proposed stain
• Use a routine, standardized protocol
• Use controls proactively
• Keep samples of effective batches of stains for cross-
checking
• Difficult procedures: monitoring by experienced
personnel.

38. Issues Concerning Staining
Reagents
• Get reliable stains and reagents
• Ensure proper storage
• Do not use outdated reagents
• Get readymade reagents when possible

39. Some Common Problems in
Routine H&E
• Weak Staining
Stain factors
• < Conc from repeated use
• Long standing solutions
• Excess oxidation due to incr oxidiser content
• Impurities
Staining procedure
• Reduced time; Excess differentiation; Prolonged dehydration
or clearing
Tissue Factors
• Old specimen; prolonged fixation in non-buffered formalin
• Decalcified specimen

40. • Strong Staining
Stain Factors: incr Conc; decr pH
Staining Procedure: incr time
Tissue factors: HgO fixation; Lymphoid tissues
• Stain Deposits
Inadequate filtration of stain solution
• Dull red or Brown Nucleus
Overripe H; Insufficient blueing

41. Conclusion
• Knowledge of physicochemical principles
of staining
• Better understanding of histology
• Better understanding of problems too.

42. References
• Theory & Practice of Histological
Techniques. Bancroft
• Cellular Pathology techniques. Culling
• Histological staining methods. Disbrey