This document discusses the key steps and principles of histotechniques, which is the process of preparing tissue for microscopic examination. The main steps described are: 1. Tissue procurement and preparation, which involves obtaining tissue samples while following legal and ethical guidelines. 2. Fixation, which uses chemicals to preserve tissue structure. Common fixatives discussed include formaldehyde, mercuric chloride, and glutaraldehyde. 3. Further processing steps like dehydration, clearing, and embedding prepare the tissue for microscopic examination by changing the tissue properties. The document emphasizes best practices for fixation like using the proper fixative concentration and duration tailored to the specific tissue type and intended analysis. Artifacts from improper

1. PRINCIPLES AND PRACTICE
Capt Rishi Pokhrel
Dr Swati Patil
Dr Kirti Solanke
Dr Anil Dwivedi

4. Histo-techniques
• Preparation of tissue for microscopic examination
• Series of processes
• Ultimate aim – to make tissue ‘visible’ as it is
• Pathology Vs Anatomy
• Steps vary
– types of tissue & microscopy
– structure to be seen
– stains to be used
– time duration etc.

5. Steps
 Tissue procurement and preparation
 Fixation
 Dehydration
 Clearing
 Impregnation
 Embedding
 Section cutting
 Staining and mounting in slide

6. Tissue Procurement
Source of tissues
 Post-mortem bodies
 Cadavers
 Tissue of patients from pathology lab
 Animal sacrifice
 Slaughter house/ butcher shop

7. Legal & ethical issues
 PM bodies : written consent form NOK
 Cadavers : no additional consent required
 Patient tissue : written consent
 From slaughterhouse / butcher : no ethical
issues, preserve bills

8. Using animals – Indian laws
 CPCSEA has laid down specific rules regarding the
use of animals
 Anesthesia, trained vet. surgeon etc
 ? Dead or dying animals

9. Tissue preparation &
precautions
 Start fixation a.s.a.p.
 Prevent osmotic damage
 do not dry
 wash with and immerse in NS
 No unnecessary handling
 Remove excess blood, mucus etc.
 Cut with a sharp knife
 Marking of ‘cutting surface’
 Labeling and putting in ‘cassette’
 Instructions for mounting – wall, tube, stained surface etc.

11. Objectives of Fixation
 Preserve tissue in ‘life like’ condition
 Prevent autolysis, microbial decomposition and
petrification.
 Coagulate tissue to prevent loss of easily diffusible
substances.
 Render tissue unaffected to the harmful effects of
chemicals to be used in further processing.

12. Effects of fixation
 Tissue hardening – ease of cutting
 Mordant action – Fleming's fluid for
safranine.
 Optical differentiation – refractive index
 Precipitation or coagulation of proteins

13. How do fixatives act?
• Physical and chemical changes
• Reactions with proteins
– Aldehydes
– Oxidizing agents, OsO4
– HgCl2
• Reactions with nucleic acids
– Carnoy’s fluid
– Formaldehyde at raised temperature
– Ethanol and methanol
• Reactions with lipids
– Imidazoles
– Cholesterol - digitonin

14. Methods of fixation
• Immersion – commonly used
• Perfusion – ideal method e.g. embalming
• Hyaluronidase pretreatment

15. PH PENETRATION
TEMPERATURE CONCENTRATION
OSMOLALITY DURATION

16. H+ concentration / PH
• Anoxia in tissue -> CO2 -> pH
• General purpose : optimal pH 6-8
• Exceptions
– Gastric mucosa pH 5.5
– Granules of adrenaline and noradrenaline pH 6.5
• Buffers maintain desired pH

17. Choosing buffers
• Buffer should not react chemically with fixative
– Barbiturates with aldehyde
• Histochemistry : buffer should not react or inhibit incubation
medium or enzymes
– Phosphates react with G6PD
– Phosphates combine with lead salts in metal precipitation methods
for phopotase.
• As close to tissue pH as possible – esp for
Histochemistry

18. Temperature
• Routine : room temperature
• Special
– Electron microscope: 0-4 oC
• Low temperature –  rate of decomposition
• High temperature – better rate of fixation

19. Penetration of fixatives
• Rate of penetration varies with various factors.
– Glutaraldehyde > Formaldehyde
• Slow process; d = k √t
• Deeper tissue takes longer time, fixed tissue acts as
a barrier for diffusion of fixative
• Tissue slices should be thin 3-5 mm

20. Osmolality
• Close to tissue osmolality; higher side – (400 – 450
mOsm)
• Commonly used : NaCl
• Some exceptions e.g. pancreas ; cause shrinkage

21. Vehicles / additives
• (NH4)2S04 : stabilize proteins
• NaCl + (Na)2SO4 : binding of HgCl2 to proteins
• Alcian blue, ruthenium red, lanathum – preserve
ultrastructure of mucosubstances
• Tannic acid : enhanced microtubules and filaments
in EM

22. Detergents
• Used to dissolve lipids in cell membrane
• Immunoflorescent techniques – 150,000 Daltons
• Commonly used - saponin

23. Concentration of fixative
• Determined by
– Cost
– Effectiveness
– Solubility
– Type of tissue and method of fixation
• Formalin
– 5% for plant tissue
– 10% for animal tissue
– 50% for embalming

24. Time
• Formaldehyde
– Too short – reversal of fixation
– Too long
• shrinkage of tissue
• Inhibit enzyme activity - Histochemistry
• Depends upon
– Thickness of tissue
– Temperature
• Optimal 24-48 hours

25. Fixation artifacts
• Primary or secondary
• Intrinsic or extrinsic
• Solidification of colloid material
• Volume change
• Pigments – formalin, HgCl2
• False localization – Histochemistry and
immunohistochemistry

26. Volume changes
• Size of cells and tissue in slide do not reflect their
true size
• Routine preparation (formalin and paraffin wax) :
tissue shrink by 33% (net)
• Not same for all components of tissue/cells
– Collagen fibers swell
• Prolonged fixation - more shrinkage
• Nuclei in frozen sections are bigger than routine
preparations.

27. Fixation : Golden rules
1 • Size of tissue, 3-5 mm
2 • Volume of fixative, 10X
3 • Time – 24 hrs (formalin)

28. FIXATIVES & FIXING FLUIDS
Classification
1. Coagulant and non coagulant
2. General and specific
3. Primary and secondary
4. Microanatomical and cytological
5. Simple and compound
6. Routine and special
7. Physical and chemical
8. Conventional and newer

29. COMMON FIXATIVES

30. Formaldehyde
• Non coagulant
• Aldehyde - HCHO
• Gas – soluble in water; max con. 40% by
weight
• Sp. gravity : 1.124
• On storage converted to formic acid &
paraformaldehyde.

31. Effects of formaldehyde
• Proteins
• Carbohydrates
• Lipids
• Acidic and basic structures

32. Formalin
40% formaldehyde gas
in water = 100 %
Formalin

Hypotonic to
tissue fluids 
Mix with distilled
water 1:9
10% formalin
 Formic acid,
Paraformaldehyde
Add buffer
Add NaCl
 Buffered,

Normal 10%
Formalin

33. Buffered normal formalin
• 100 % formalin 100 ml
NaH2PO4 3.5 gms
• Buffer + Mix in 900 ml of water
Na2HPO4 6.5 gms
• Salt NaCl - 8.5 gms

34. Disadvantages
• Contact dermatitis
• Urea crystals – 5 gm
• (NH4)2SO4 - 1gm
• Water - 1l
• Irritant to eyes and resp. mucosa
• Pigment formation in Hemoglobin
• Pigments and turbidity
• Not suitable for certain tissue – testis, bone
marrow, spleen.

35. Formalin pigments in slides
Before staining :-
• Schridde’s method: leave slides for 30 mins
– 200 ml 75 % alcohol + 1ml of 25% ammonia liquor
• Verocay’s method : leave sections for 10 mins
– 100 ml of 80% ethanol + 1% KOH

36. Mercuric chloride
• White crystals
• Saturated solution : 7% water, 33% alcohol
• Extremely poisonous and corrosive to metals
• Seldom used alone – compound fixative
• Cytological fixative

37. Mercuric chloride
• Pros
– cytological fixative
– good fixative for proteins
– shrinks but doesn‘t distort tissue
– fixes both nucleus & cytoplasm
• Cons
– mercury pigments
– Corrosive and poisonous

38. Potassium dichromate
Used in compound fixatives or as mordant for lipids or
lipoproteins
• Pros
– excellent fixation of mitochondria, phospholipids & myelin
– acts as a mordant for mitochondria
– iron staining of mitochondria
– iron-containing pigments better fixed at higher pH
• Cons
– chromatin gets dissolved
– mitochondria gets thickened
– formation of insoluble precipitants
– brittleness of tissues
40

39. Chromic acid
• CrO3 mixed in water
• Fixative for carbohydrate
Osmic acid
• Used for cytoplasmic organelles
• Demonstration of myelin
• Expensive
• Use in EM
• Use and throw
• Use goggles while handling – vapors irritant to eyes.

40. Glacial acetic acid
• Part of compound fixative
• Good fixation for nuclei – precipitates nucleoproteins
• Counteracts the shrinking effect of others.
• pronounced swelling of collagen fibers
• Distorts mitochondria & Golgi body
Ethyl alcohol :
Histochemistry - alkaline phosphatase & lipase
hardening & shrinkage
Improper fixation of chromatin, mitochondria & Golgi
42

41. Glutaraldehyde
• Suitable for EM
• Better than formaldehyde
• cytological fixative
• poor penetration – use small tissue
• Expensive
Picric acid
• good fixative for proteins, carbohydrates & glycogen
• better staining
• enhances results with cytoplasmic stains
• much shrinkage & less hardening
43

42. Stock solutions
• Formalin 40%
• Mercuric chloride 7%
• Potassium dichromate 5%
• Chromic acid 2%
• Picric acid 1%
• Osmium tetra oxide 2%
• Acetic acid 100%
• Ethanol 100%

43. Fixing fluids

44. Formalin mercuric chloride (formal-sublimate)
• Mercuric chloride 30 gms
• Water 900 ml
• 100% Formalin * 100 ml
*Formalin added just before use as added solution is
unstable
Uses
• Secondary fixation following formalin
• Excellent Microanatomical fixative
• Cytoplasm preservation
• Brilliant staining with acid dyes
• Heidenhain’s susa - biopsy

45. Zenker’s fluid
Mercuric chloride 5gm
Potassium dichromate 2.5gm
Sodium Sulphate 1gm
Water 100ml
Acetic acid* 5ml
Uses
• Efficient micro anatomical fixative.
• Better staining with cytoplasmic & fiber stains

46. Bouin’s fluid (formalin-picric-acetic)
• Picric acid 75ml
• Formalin 25ml
• Acetic acid 5ml
Uses
• Micro anatomical as well as cytological fixative
• Used for demonstration of chromosomes
• Glycogen is well preserved.

47. Helly’s fluid (zenker-formal)
Acetic acid omitted & formalin 5 ml is added in
Zenker’s fluid.
Uses
• Testis, bone marrow, spleen, lymph node,
pituitary, pancreas

48. Carnoy’s fluid
• Absolute ethanol 60ml
• Chloroform 30ml
• Acetic acid 10ml
Uses
• Rapid fixation
• Not of much use for anatomy slides.

49. Sanfelice’s fluid
• Solution A Formalin 128ml
Acetic acid 16ml
• Solution B Chromic acid 100ml
• Mix 9ml A + 16ml B
Uses
• mitotic figures & chromosomes

50. • Flemming’s fluid : Cytological fixative, not used
• Lewitsky – Baker modification : tissue of
invertebrates
• Orth’s fluid : Mordant for mitochondria,
chromaffin reaction

51. Causes of poor fixation
• Less volume of fixation
• Less time
• Poorly penetrating fixative
• Specimen settling down on contained
• Wrong choice of fixative

52. Always necessary?
• Sclero proteins – nails
• Chitin, cellulose, inorganic salts,
• Frozen sections

53. Storage and preservation of tissue
• Formalin : store in fixative
• 70% alcohol
• Formal sublimate : 30% glycerin
• 1% phenoxetol
• Best : store a block rather than tissue

54. TAKE HOME MESSAGE
• Precautions on collecting and handling tissues
• Legal implications - animals
• Neutral normal 10% formalin is the best fixative in
most circumstances
• Remember the exceptions
• Pancreas
• Testis
• Bone marrow and spleen
• Choose fixative as per requirement – tissue or cell,
stains
• Storage of tissue

55. DISCUSSION

56. ONCE UPON A TIME IN
HAITI