This document discusses the process of histology, which involves preparing tissue samples for microscopic examination. Specimens should be transported in fixatives like formalin or refrigerated. There are several steps involved including fixation, processing, sectioning, staining, and mounting. Tissue processors are used to dehydrate samples through a series of alcohols and xylene before infiltrating and embedding them in paraffin wax for sectioning with a microtome. Sections are then stained using techniques like H&E staining before examination under a microscope. Frozen sectioning and newer techniques using resins and microwaves can also be used to prepare samples for histological analysis.

2. It is processing and preparation of the tissue of body in
such a manner as to satisfactory study of the tissues
can be done.

3. Handling of Specimen
Specimen should be transported in glass, plastic or metal
container or in a plastic bag in 10% formalin.
If formalin is not available at hand, place the specimen in
refrigerator at 4oC to slow down autolysis.
fresh material is needed for the following purpose:
1. Frozen section
2. Immunocytochemistry
3. Cytological examination
4. Microbiological sampling before histopathology
5. Chromosome analysis
6. Research purpose
7. Museum display

4. Basic steps
Preparation of the tissues
Processing of the tissues
Preparation of the sections
Staining
Mounting

5. Specimen
Histology lab
Registration
Fixation
Grossing
Labelling
Dehydration
Clearing
Infiltration & impregnation

6. Embedding
Trimming
Section cutting
Deparaffinization
Hydration
Staining
Dehydration
Clearing
Mounting

7. Fixation - AIM
Prevent putrefaction & autolysis
Preservation of cells & tissue constituents
Hardening of soft tissues
Conversion of semifluid consistency of cell to an
irreversible semisolid consistency
Alteration of refractive indices to varying degree
which enables unstained components to be seen
easily.

8. Ideal fixatives
Cheap & easily available
Stable & easy to handle
Fix quickly
Minimal loss of tissue
Even penetration

9. Reagents employed as fixatives
Formaldehyde
Mercuric chloride
Potassium dichromate
Picric acid
Ethyl alcohol
Glutaraldehyde
Osmium tetraoxide

10. Decalcification
A process to remove calcium from bone and other mineralized hard
tissue in order to facilitate the process of cutting thin section .
Stages:-
I. Selection of tissue
II. Fixation
III. Decalcification
IV. Acid neutralization
V. Washing

11. Processing of tissue
 Embed the tissue in a solid medium.
Firm enough to support the tissue and enable thin
sections to be cut.
Soft enough not to damage the knife of tissue.

12. There are two kinds of tissue processors:
1- Moving tissue type
2- Moving fluid type.

13. Histokenette Automatic Tissue Processor

14. Tissue Cassettes

16. Tissue Processor With Vacuum And Fume Control

17. Vacuum Tissue Processor

18. Linear Tissue Processor With Touch Screen

19. PROCESSING SCHEDULE
Automated processing schedule
1-Overnight schedule
2-short processing schedule
Manual processing schedule

20. Over night schedule
1- 10% formaline. 0 hrs
2- 70% alcohol ½ hrs.
3- 95% alcohol ½ hrs
4- 100% alcohol ½ hrs
5- 100% alcohol 1 hrs
6- 100% alcohol 1 hrs
7- 100% alcohol 1 hrs
8- 100% alcohol ½ hrs
9- xylene 1 hrs
10- xylene 2 hrs
11- wax 2 x1/2 hrs
12- wax 4 hrs

21. Schedule for processing for small biopsy or for urgent
work
1- 10% formaline. 20 min vacuum heat
2- 95% alcohol 5 min y 450c
3- 95% alcohol 5 min
4- 100% alcohol 5 min
5- 100% alcohol 5 min
6- 100% alcohol 5 min
7- 100% alcohol 5 min
8- 100% 5 min
alcohol/ethylene 5 min
9- xylene
5 min
10- xylene
5 min
11- wax
5 min

22. Manual processing schedule
Rapid technique for thin slices of tissue
1- carnoys fluid- 45 min
2- 100% alcohol x 6 15 min each
3-xylene 10 min
4- xylene 15 min
5- wax 20 min
6- wax 45 min

23. Manual processing schedule for blockes
1- 70% alcohol 0900 hrs to 1000 hrs
2- 95% alcohol 1000 hrs to 1100 hrs
3- 95% alcohol 1100 hrs to 1300 hrs
4- 100% alcohol 1300 hrs to 1430 hrs
5- 100% alcohol 1430 to 1600 hrs
6- 100% alcohol 1600 to 1730 hrs
7- 100% alcohol overnight
8- xylene 0900 to 1000 hrs
9- xylene 1000 hrs to 1130 hrs
10- wax with vacuume 1130 hrs to 1230 hrs
11- wax with vacuume  1230 hrs to 1400 hrs
12- wax with vacuume 1400hrs to 1600 hrs

24. Processing of tissue
Important steps of tissue processing by paraffin wax
technique.
a)Dehydration.
b)Clearing.
c) Infiltration.
d)Embedding.

25. Factors influencing the rate of processing-
a)Heat ( increase the rate of penetration, but limited to
45 degree)
b)Agitation (tissue lies on the base of the container ,rate
of exchange of fluid is much less)
c) Viscosity ( quickness of impregnation due to lower
viscosity of paraffin in fluid state )
d)Vacuum ( little increase dehydration and clearing but
reduces the impregnation time)

26. Dehydration
I. Water is completely removed from the fixed
tissues.
II. Tissue blocks are placed in cassettes with the
identification number.
III . Passed through increasing concentration of
alcohol with changes in each concentration.

27. Dehydration
Dehydrating agents
1-Alcohols- ethenol, methanol, isopropanol,
Polyethylene glycols (PEG) , diaxone
2-Other dehydrants
Acetone , Tetrahydrofuran , 2,2
dimethoxypropane
Phenol,

28. Clearing
1-. It is the process in which water from cell & tissues is
removed & is replaced by a fluid in which wax is soluble
2- .Most commonly used agent is xylene.
3-. Xylene is miscible in both paraffin wax &
alcohol.
4-. Replaces alcohol & make room for paraffin.
5- Other clearing agent – toluene , benzene , chloroform
& cedar wood oil.

29. Xylene or toluene or benzene :
Advantages – Cheap and rapid in action, can be used
for almost all tissue, used for both paraffin and
celloidin embedding, benzene has less hardening
effect then xylene.
Disadvantages –
a)Make the tissue brittle if kept in the fluid for a longer
period.
b)Excessive shrinkage for delicate tissue.
c) May cause dermatitis
d)Benzene is more inflammable and toxic and known
to be carcinogenic.

30. Infiltration & Impregnation
Infiltration – xylene is eliminated from the tissue by
diffusion in the surrounding melting wax.
Impregnation – wax diffuses in the tissue by replacing
the xylene.
It maintain the intra cellular structure during the
section cutting on microtome.

31. Embedding
I.Casting or blocking.
II. Infiltrated & impregnated tissue is places in warm
liquid which forms a firm block after cooling.
III. Enables the tissue to be cut on a microtome.
IV. Most commonly used material is paraffin.
V. Leuckhard embedding box ( consisting of two L
shaped pieces of heavy metallic material brass)
arranged on a glass plate.

32. Ideally an infiltrating and embedding medium should be:
• soluble in processing fluids
• suitable for sectioning and ribboning
• molten between 30°C and 60°C
• translucent or transparent; colorless
• stable
• homogeneous
• capable of flattening after ribboning
• non-toxic
• odorless
• easy to handle
• inexpensive

34. Paraffin Melting point Feature
Commonly 54 c
used
Hard wax 60 c Hard fibrous
tissue
Soft wax 45 c Fetal &
areolar tissue

35. Celloidin media ( nitrocellulose )
1.Used when it is desired to avoid the use of heat in CNS
2.It is rubbery material.
3.Give better support to tissue like skin, sclera &
subcutaneous tissues.
Gelatin media – for friable tissues like lung.
Resin , Agar

36. MICROTOME
Used for cutting paraffin tissue sections of uniform
thickness. A knob on the machine is used to adjust
the thickness of section.
A knife is fixed in a clamp . The tissue block is drawn
across the knife edge, the top and bottom of the block
should be parallel and horizontal and at least 1 mm of
paraffin should be present on all side of tissue.
Then ribbon of sections is transferred to warm water.

37. MICROTOME
Rotary microtome – for paraffin embedding.
Rocking microtome – for paraffin embedding.
Sliding microtome – for celloidin embedding
Freezing microtome – for frozen section
Cold ( cryostat) – for frozen section
Ultramicrotome – for electron microscope
Laser microtome-for contact free slicing

39. Trimming of the paraffin block.
Attach the block to the microtome.
Cutting of the section.
Fix the section on the slides.
adhesives – starch paste
albumin ( glycerol + white egg
+ distilled water)

41. STAINING
Deparaffinized (2 jars of xylene – each 2 min)
2 jars of alcohol – each for 2 min.
Rehydrated (90% alcohol – 1min f/b 70% alcohol for 1 min)
Rinsed in water
Stained in harris haematoxylene for 2 – 5min
Washing in running water till sections turn blue

42. Differentiation (1% acid alcoholic solution for 10 sec)
Dip in 0.5% hydrochloric acid, nuclear aaper dark purple.
( bluing)
Rinse in water for 10-15 min
Counterstain (1% aqueous solution of eosin for 1-3 min)
Rinse in tap water
Dehydrate
Clearing in xylene
Mounting ( DPX / Canada balsam)

43. Frozen section
Method of sectioning of tissue after making the
tissue hard by rapid freezing.
Advantages – rapidly prepared,
 Minimum shrinkage of tissue,
 Every method of staining is allowed especially
suitable for immunochemistry and almost
obligatory for enzyme study
Essential technique for demonstration of certain
lipid and enzymes.

44. Disadvantages –
sections are thick and sometime difficult to interpret properly,
not always possible to maintain the structural element in their
natural position .
it is practically impossible to obtain and correlate serial section

45. Methods
Freezing microtome with carbon dioxide which is
popular method.
Freezing microtome with thermoelectric molecules.
Use of refrigerated microtome ( cryostat).
10 % formal saline is most common fixative used,
fresh unfixed tissue may be used.
Tissue thickness should not exceed 3 mm.

46. Advances
Use of different type of resins as alternative to wax.
Large block preparation and large sections for study
of whole pathological region of tissue.
Specimen radiography or thin section
ultrasonography on freshly removed specimen to be
sure that the whole pathology has been removed.
Microwaves for quick fixation, processing.

47. Microwave processing of tissue
Tissue is cut on small pieces ---- tissue block are
placed in isotonic saline and subjected to microwave
irradiation for 120 sec. (full power )- ( 50 % power)
dehydration by 70 % alchohal for 4 min, 100 %
alchohal for 5 min, chloroform for 5 min then
impregnation in wax for 5 min---- embedding-
section cutting.

51. THANK YOU
PRESENTED BY – Dr. Narmada Prasad Tiwari