1. Preparation of tissues for study

2. HISTOLOGY : HISTOPATHOLOGY : Tissue for study can be obtained from:
It is the branch of science which deals with the microscopic study of normal tissue
HISTOPATHOLOGY :
It is the branch of science which deals with the microscopic study of tissue affected by disease
Tissue for study can be obtained from:
Biopsies
Autopsies

3. Microtechnique
Microtechnique: is tissue preparation for microscopic examination
There are different methods used, however the basic principles are similar
It usually involves hardening of the tissue followed by sectioning (cutting)
Paraffin technique
Freezing technique

4. Histological Techniques:
1. Paraffin
Tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 1 to 30 µm thick.
From there the tissue can be mounted on a microscope slide, stained and examined using a light microscope.

5. 2. Freezing technique:
Water-rich tissues are hardened by freezing and cut frozen; sections are stained and examined with a light microscope
This technique is much faster than traditional histology (20 minutes vs. 16 hours) and are used in operations to achieve a quick diagnosis
Cryosections can also be used in immunohistochemistry as freezing tissue does not alter or mask its chemical composition

6. Protocols followed in Histotechniques
Receipt & Identification
Labeling of the specimen with numbering
Fixation
Dehydration
Clearing
Impregnation (infiltration)
Embedding
Section cutting
Staining
Mounting

7. Specimen Dissection - the stage

8. Fixation

9. Fixation This is the process by which the constituents of
cells and tissue are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture
This is achieved by exposing the tissue to chemical compounds: fixatives
Fixatives prevent autolysis and bacterial decomposition and preserves tissue in their natural state and fix all components

10. Tissue fixatives Buffered formalin (light microscope preparation)
Buffered gluteraldehyde (electron microscope preparation)
Osmium tetraoxide (electron microscope preparation, preserve and stain)
Zenker’s formal saline
Bowen’s fluid

11. No fixative will penetrate a piece of tissue
thicker than 1 cm.

12. Cassette

14. Specimen is placed in porous cassettes

15. Labeling

16. Cassettes are collected in fixatives 10% formalin
1mm/hour fixation

17. Processing

18. Tissue Processing In order to cut thin sections of the tissues, it
should have suitable hardness and consistency
when presented to the knife edge.
These properties can be imparted by infiltrating and
surrounding the tissue with paraffin wax, various
types of resins or by freezing.
This process is called tissue processing.

19. Tissue Processing It can be subdivided into: a) Dehydration
b) Clearing
c) Impregnation (infiltration)

20. Types of tissue processing
There are two types :
1. Manual Tissue Processing
2. Mechanical Tissue Processing

21. Mechanical Tissue Processing
In this the tissue is moved from one jar to another by mechanical device
Timings are controlled by a timer which can be adjusted in respect to hours and minutes
Temperature is maintained around 60 ºC
Automatic tissue processor:
Overnight
12 Baths
16 hours

22. Tissue basket

23. Tissues processor

24. Manual Tissue Processing
In this process the tissue is changed from one container of reagent to another by hand
Note:
The processing, whether manually or mechanically, involves the same steps and reagents in same sequence

25. Dehydration (removal of water)
It is the process in which the water content in the tissue to be processed is completely removed by passing the tissue through increasing concentrations of dehydrating agents
Tissues are dehydrated by using increasing strength of alcohol; e.g.
70%, 90% and 100%
Water is replaced by diffusion

26. During dehydration water in tissue has been
replaced by alcohol
The next step alcohol should be replaced by
paraffin wax
As paraffin wax is not alcohol soluble, we
replace alcohol with a substance in which wax is
soluble. This step is called clearing.

27. Clearing Some clearing agents:
Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium
Some clearing agents:
- Xylene
- Toluene
- Chloroform
- Benzene

28. Impregnation (infiltration):
In this the tissue is kept in a wax bath containing molten paraffin wax

29. The duration of the procedure can be noted down as:
Fixation
10 % Formalin saline (I) for hours
10 % Formalin saline (II) for hours
Dehydration
80 % alcohol for hour
95 % alcohol (I) for hour
95 % alcohol (II) for hour
Absolute alcohol (100%) (I) for hour
Absolute alcohol (100%) (II) for hour
Absolute alcohol (100%) (III) for hour

30. Clearing: Infiltration: Paraffin wax (I) for 1.5 hours
Xylene (I) for hours
Xylene (II) for hours
Infiltration:
Paraffin wax (I) for 1.5 hours
Paraffin wax (II) for 1.5 hours

31. Embedding

32. Embedding
Embedding: is the process by which impregnated tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning

33. Embedding:
It is done by transferring the tissue to a mould filled with molten wax & is allowed to cool & solidify
After solidification, a wax block is obtained which is then sectioned to obtain ribbons

34. Embedding tools
Moulds
Paraffin wax

35. Metal moulds

36. Disposable plastic moulds

37. Embedding tools Paraffin wax:
is a polycrystalline mixture of solid hydrocarbons. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C

38. Embedding tools
Scalpel (blade)
Forceps

39. Embedding Centre

40. Embedding Centre Molten wax (60°C) (reservoir) Used lids
Heated chambers
Wax flow adjuster
Mould
Hot surface
Cassette
Cold plates (-5°C)
Wax dispenser

41. General Embedding Procedure

42. Fill the mould with paraffin wax

43. Using warm forceps select the tissue, take care that it does not cool in the air

44. Orienting the tissue in the mould

45. Orientation Of Tissue In The Block
Correct orientation of tissue in a mould is the most important step in embedding
1. cross section
2. longitudinal section
Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy

46. Cool the block on the cold plate

47. Blocks on the cold plate

48. Remove the block from the mould

51. Blocks of embedded tissue are usually trimmed to remove the excess wax on the surface

52. Sectioning

53. Sectioning (Section Cutting)
It is the procedure in which the blocks which have been prepared are cut or sectioned and thin strips of uniform thickness are prepared
The instrument by which this is done is called as a Microtome

54. Freezing (cryostat) microtome Ultramicrotome
TYPES OF MICROTOMES:
Rotary microtome
Freezing (cryostat) microtome
Ultramicrotome

55. Microtome knives Steel Knives Non-corrosive Knives For Cryostats
Disposable Blades
Glass Knives
Diamond Knives
Microtome blades are extremely sharp, and should be handled with great care. Safety precautions should be taken in order to avoid any contact with the cutting edge of the blade

56. Steel Knives (for rotary microtome)

57. Disposable Blades

58. Glass Knives (for ultramicrotome)

59. Rotary Microtome
Block holder
Knife holder
Drive wheel

60. Micron adjustment (section thickness) 1-30µm Paraffin Block
Steel knife
Block holder
Knife clamps
Block adjustment
Thickness gauge
Angle of tilt adjustment
Operating handle.

61. Rotary Microtome
The knife is stationary (fixed) and the block holder is moved up and down in a vertical plane by the rotary action of the hand wheel
It is the most commonly used
Suitable for paraffin embedded sections
It is suitable for cutting of small tissues
Ideal for cutting serial sections

62. Ultra Microtome

63. Ultra Microtome Used for very thin section
The typical thickness of tissue cut is between nm for TEM
Knife: Diamond or Glass

64. Ultra Microtome

65. Ultra Microtome

66. Freezing (Cryostat) microtome
Frozen tissue embedded in a freezing medium is cut on a microtome in a cooled machine called a cryostat

67. Freezing (Cryostat) microtome

68. Freezing (Cryostat) microtome

69. Sectioning procedure

70. Ribbon of sections

72. Tissue floatation bath
It is a thermostatically controlled water bath
It is maintained at a temperature 5 – 6 degrees below the melting point of paraffin wax

73. Flattened paraffin sections

74. Taking the floating sections onto slide
Adhesives used for fixing the sections on the slides
Albumin solution ( Mayor’s egg albumin)

75. Taking the section onto slide
Flat, no air bubbles, no stretch or breaks

76. Staining

77. Staining
Staining is a process by which we give colour to a section. Staining of the section is done to bring out the particular details in the tissue under study
There are hundreds of stains available
The most commonly used stain in routine practice is Hematoxylin & Eosin stain

78. Staining Classification of Stains: Acid stains Basic stains
Staining steps:
a. rehydration
b. stain
c. dehydration

79. Acid Dyes In an acidic dye:
The basic component is colored and the acid component is colorless
Acid dyes stain basic components e.g. eosin stains cytoplasm
The color imparted is shade of red

80. Basic Dyes In a basic dye:
The acid component is colored and the basic component is colorless
Basic dyes stain acidic components e.g. Hematoxylin stains nucleus
The color imparted is shade of blue

81. H & E stains are universally used for routine histological examination of tissue sections

82. Result :
The nucleus stains Blue
The cytoplasm stains pink

83. Special stains
When a specific component of tissue e.g. fibrous tissue, elastic tissue, nuclear material is to be stained, certain special stains are used which specifically stain that component tissue
Examples:
PAS
Masson’s trichrome
Silver

84. Normal glomerulus of kidney is stained with H&E

85. Normal glomerulus of kidney is stained with PAS
detects glycogen, glycoproteins, glycolipids and mucins in tissues

86. Normal glomerulus of kidney is stained with Masson_trichrome
Masson Trichrome Stain
Nuclei stain black or blue-black
• Muscles stain red
• Collagen and mucus stain green or blue
• Cytoplasm of most cells stains pink
Normal glomerulus of kidney is stained with Masson_trichrome

87. Normal glomerulus of kidney is stained with methenamine silver
Silver stains reticular fibers (type III collagen)

88. Staining Tools

89. Slide rack

91. Slide rack

92. Solutions

93. Procedure of staining:
There are two types of staining:
Manual Staining
Automatic staining
Slides stained either manually or by automatic stainer, pass through same sequences of reagents.

94. Manual Staining Different reagent containers are placed in a
special sequence and the slides are removed
from one container to another manually.

95. Manual Staining

96. Manual Staining

97. Automatic stainer

98. Automatic stainer

99. Haematoxylin
Hydration
(alcohol)
Deparaffinization
(xylene)
Eosin
Dehydration
(alcohol)
Clearing
(xylene)

100. Mounting
Stained section on microscope slide is mounted using mounting medium dissolved in xylene
Examples of Mountants : DPX ( Distrene Dibutyl phthalate Xylene )
A coverslip is placed on top, to protect the sample

105. Light Microscopic Examination

106. Microscopic Examination

107. Concering Electron Microscopy
Electron beam instead of light
Gluteraldehyde fixative
Embedding is in hard epoxy plastic
Ultramicrotome
Diamond or Glass knives
Thin section ( µm).
Specimen is mounted on a metal grid

108. Ultra Microtome