Preparation of microscopic sections

1. Collection of specimen for microscopic examination

 Collection of samples soon after death
 Sample should be fixed immediately
 Tissue piece should be removed with clean and sharp knife or blade
 Optimum tissue thickness should be 5-6mm
 Greater size of tissue should be avoided as causes to decrease penetration of fixative
 Tissue piece should not be crushed or bent
2. Fixation/ Preservation
Fixation: A critical step in the preparation of histological sections by which biological tissues are
preserved from decay, there by preventing autolysis or putrefaction.
Fixative: A chemical substance used to preserve or stabilize biological material prior to
microscopy or other examination.
Objectives:
[Link] preserve cells and tissue components in a life-like state
[Link] prevent or arrest the degenerative processes, when blood supply is stopped
[Link] prevent Post mortem changes such as autolysis
[Link] maintain the integrity of cells
[Link] coagulate the cell proteins and intracellular bodies
[Link] prevent the cell from distortion or shrinkage
[Link] facilitate the proper staining of tissues
[Link] prevent the drying of tissues
Properties of good fixative
1. It should quickly penetrate into tissue
2. It should be less toxic
3. Easily available
4. It should coagulate the protoplasm of cells
5. It should be economical
6. It should prevent the tissue from shrinkage and distortion during dehydration and
embedding
 Types of fixation
Physical methods
 Heating (confined to smears of micro organisms)
 Micro-waving (37°C and 45°C)
 Cryo-preservation (applications in Histochemistry)
Chemical methods
Chemical fixation is usually achieved by immersing the specimen in the fixative
Fixing agents
[Link] (CH2O) : Most popular agent used for histopathology generally referred to as
formalin (37-40%)
Advantages
Relatively inexpensive
Precipitate protein
Lipids well preserved
Favors the staining of acidic structures (Nuclei)
Hazards/Disadvantages
 Irritant for eyes
Cause allergic sensitivity
Causes respiratory tract, skin problems, eye irritation
Carcinogenic
Formalin used in different forms are
a. 10 % Formalin
Formalin 10ml
Distilled water 90ml
b. Neutral buffered formalin solution
Formalin 100ml
Distilled water 900ml
Sodium phosphate monohydrate (Na2PO4H2O) 4gm
Anhydrous sodium phosphate (Na2HPO4) 6.5gm
Minimum time for fixation is 48 hours at room temperature, 24 hours at 35C and 6-8 hours at 50-
60C. Optimum time is for 10 days
c. Formalin saline
40% formaldehyde 100 ml
Sodium chloride 9g
Distilled water 900 ml
Fixation time 12 – 24 hours
2. Ethyl alcohol (Ethanol C2H5OH)
 Coagulants that denature proteins
 Used to preserve glycogen, pigments, elastic fibres
 Cause shrinkage and hardening of tissues
 Not recommended for prolong storage
 95% ethanol is used as a fixative for cytology smears
3. Acetone (CH3COCH3)
 It is used for rapid fixation of brain tissue for diagnosis of rabies
 It hardens, dehydrates and clears the tissue along with fixation
 Mostly used for soft tissues
 It is highly volatile and flammable
4. Acetic acid (CH3COOH)
 Penetrates very rapidly and causes rapid fixaton
 Causes swelling of cell constituents
 Used in fixing mixtures to counteract the shrinkage properties of formalin, mercuric
chloride
5. Picric acid (C6H2(NO2)3OH)
 Highly suitable for chitinous structure of skin
 Imparts a yellow colour to tissues during fixation
 A coagulant fixative & disrupts electrostatic and hydrogen bonds
 Recommended to preserve glycogen
 Hydrolyze nucleic acids so it should be avoided for DNA or RNA demonstration
 Considerable amount of shrinkage occurs during the processing of tissues fixed in picric
acid containing reagents.
 Special fixing fluids
Zenker’s fixative
Composition
Distilled water 950 ml
Mercuric chloride 50 g
Potassium dichromate 25 g
Glacial acetic acid 50 ml
Fixation time 4 – 24 hours
Recommended Applications
Used for bone marrow specimen
Provides greater cytological details
Tissue acquire greater affinity for stain
Gives good nuclear preservation but lyses red blood cells due to the presence of acetic
acid.
Bouin’s solution
Composition
Picric acid saturated aqueous solution (2.1%) 750 ml
40% formaldehyde 250 ml
Acetic acid glacial 50 ml
Fixation time 4 – 18 hours
Advantages
Great penetration power
Compatible with every staining technique
Shrinkage of tissue is minimum
Recommended for embryological specimen, gastro-intestinal tract biopsies, and
endocrine gland tissue
Carnoy’s solution
Formulation
Ethanol absolute 60 ml
Chloroform 30 ml
Acetic acid glacial 10 m
Fixation time 1 – 4 hours
Advantages
Used for quick fixation
Excellent nuclear fixative
Prefer fixative for glycogen
Often used for tissue mast cells
Disadvantages
It lyses erythrocytes
 Dissolves lipids
Produce excessive hardening and shrinkage
 Factors influencing chemical fixation
Temperature
Increase the rate of diffusion of the fixative into the tissue
Speed up the rate of chemical reaction between the fixative and tissue elements
 Potentially increase the rate of tissue degeneration in unfixed areas of the specimen
Microwave fixation may involve the use of higher temperatures, up to 65°C, but for
relatively short periods
Time
The optimal time for fixation will vary between fixatives. the fixative has to penetrate, by
diffusion, to the centre of the specimen and then sufficient time has to be allowed for the
reactions of fixation to occur. Both diffusion time and reaction time depend on the
particular reagent used and the optimum time will vary from fixative to fixative.
Volume ratio
It is important to have an excess volume of fixative in relation to the total volume of tissue
because with additive fixatives the effective concentration of reagent is depleted as fixation
proceeds and in a small total volume this could have an effect on fixation quality. A fixative
to tissue ratio of 20:1 is considered the lowest acceptable ratio but target ratio of 50:1 is
more efficient
Penetration rate
The penetration rate of a fixing agent depends on its diffusion characteristics and varies
from agent to agent
Specimen dimensions
A specimen should not be more than 4 mm thick
Ideally a 3 mm thick slice provide excellent fixation and processing
pH and buffers
Normal pH is required for each fixative.
Osmolality
The osmotic effects exerted by the fixative are again more important at the ultrastructural level
than at the level of the light microscope because it is the phospholipid membranes that are
easily damaged by excessively hypotonic or hypertonic solutions, but osmolality does have
some relevance in routine histopathology.