Preparation of histochemical specimen

1. PREPARATION OF
HISTOLOGICAL SPECIMENS

2. Histotechniques
➢ The techniques for processing the tissues,
whether biopsies, larger specimen removed at
surgery, or tissues from autopsy or impact on
tissues due to pollutants so as to enable the
pathologist to study them under the
microscope.

3. Section cutting planes of tissue
Cross section/ Transverse section: They are obtained
by taking imaginary slices perpendicular to the main axis
of organs, vessels, nerves, bones, soft tissue, or even the
entire human body.
Sagittal Plane (Lateral Plane) - A vertical plane running
from front to back; divides the body or any of its parts
into right and left sides.
Median plane - Sagittal plane through the midline of the
body; divides the body or any of its parts into right and
left halves
Longitudinal section done by a plane along the long axis
of a structure in contrast to the other term, cross section,
which is a section that is cut transversely.

5. Steps
Protocols followed in Histotechniques are:
1. Obtaining a fresh specimen
2. Fixation
3. Dehydration
4. Clearing
5. Embedding
6. Section cutting
7. Staining
8. Mounting

6. 1.Specimen

8. Types of fixation
Fixation is a complex series of chemical events that differ
for the different groups of substance found in tissues.
There are generally two types of fixation.
1. Physical fixation
2. Chemical fixation
1.Physical fixation
 Heat fixation
 Perfusion
 Immersion
 Cryo-fixation
2.Chemical fixation
It can be achieved by using different chemicals.

9. Properties of an ideal fixative
✓ It should prevent autolysis and bacterial
decomposition.
✓ It should penetrate evenly and rapidly.
✓ It should avoid excessive hardness of tissue.
✓ It should not cause shrinkage or swelling of the
cells.
✓ It should enhance staining of tissue.
✓ It should be non toxic and non allergic for user.
✓ It should not be very expensive.

10. 2.Fixation
❖ It is a process in which specimen is treated by
exposing it to a fixative for a particular period of time
in order to facilitate the succeeding steps.
❖ The purpose of fixative is to preserve tissues
permanently in life like condition.
❖ The fixative should be 15_20 times more in volume
than specimen.

11. Good fixatives
❑ Formaldehyde
❑ Alcohol
❑ Potassium dichromate
❑ Picric acid

12. Factors affect fixation:
❑ pH.
❑ Temperature.
❑ Penetration of fixative.
❑ Volume of tissue.
❑ Concentration of fixative
❑ Fixation time.

13. Formaldehye
❑ The most commonly
used fixative is formalin.
❑ It is prepared by mixing
40% formaldehyde gas
in 100w/v distilled
water.
❑ Routinely,10% formalin
is used.
Mechanism of action:
✓ It forms cross links
between amino acids of
protein thereby making
them insoluble.

14. Bouin’s Fixative or Picric acid
 Saturated picric acid 3000.0 ml
 Formaldehyde 1000.0 ml
 Glacial acetic acid 200.0 ml
➢ Bouin’s solution is an excellent general fixative
for connective tissue stains.
➢ The yellow color can be removed with 70%
ethanol, lithium carbonate,or another acid
dye, separately or during the staining
sequence.
➢ Bouin’s solution destroys membranes,
therefore intact nuclei cannot be recovered
from Bouin’s fixed tissue and there may be
extensive shrinkage of larger specimens.
➢ Bouin’s fixative is carcinogenic, irritant and
toxic.
➢ To prevent from any difficulty, one should
wear gloves, work in ventilated area.

15. TISSUE PROCESSING
The aim of tissue processing is to embed the tissue in a
solid medium firm enough to support the tissue and give it
sufficient rigidity to enable thin sections to be cut , and yet
soft enough not to damage the knife or tissue.
Stages of processing:
1- Dehydration.
2- Clearing.
3- Embedding.

16. Dehydration
To remove fixative and water from the tissue and replace them
with dehydrating fluid.
There are a variety of compounds many of which are alcohols.
several are hydrophilic so attract water from tissue.
 To minimize tissue distortion from diffusion currents, delicate
specimens are dehydrated in a graded ethanol series from
water through 10%-20%-50%-95%-100% ethanol.
 In the paraffin wax method, following any necessary post
fixation treatment, dehydration from aqueous fixatives is
usually initiated in 60%-70% ethanol, progressing through
90%-95% ethanol, then two or three changes of absolute
ethanol before proceeding to the clearing stage.

17. Types of dehydrating agents:
Ethanol, Methanol, Acetone.
 Duration of dehydration should be kept to the minimum
consistent with the tissues being processed. Tissue blocks 1
mm thick should receive up to 30 minutes in each alcohol,
blocks 5 mm thick require up to 90 minutes or longer in each
change. Tissues may be held and stored indefinitely in 70%
ethanol without harm

18. Clearing
 Replacing the dehydrating fluid with a fluid that is totally
miscible with both the dehydrating fluid and the
embedding medium.
 Choice of a clearing agent depends upon the
following:
- The type of tissues to be processed,
and the type of processing to be undertaken.
- The processor system to be used.
- Intended processing conditions such as temperature, vacuum
and pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .

19. Some clearing agents:
- Xylene
- Toluene
- Chloroform
- Benzene
- Petrol

20. Embedding
Is the process by which tissues are surrounded by a medium such
as agar, gelatin, or wax which when solidified will provide
sufficient external support during sectioning.
 Paraffin wax
Properties :
 Paraffin wax is a polycrystalline mixture of solid
hydrocarbons produced during the refining of coal and
mineral oils. It is about two thirds the density and
slightly more elastic than dried protein. Paraffin wax is
traditionally marketed by its melting points which range
from 39°C to 68°C.
 The properties of paraffin wax are improved for
histological purposes by the inclusion of substances
added alone or in combination to the wax:
- improve ribboning.
- increase hardness.
- decrease melting point
- improve adhesion between specimen and wax

22. ORIENTATION OF TISSUE IN THE BLOCK
 Correct orientation of tissue in a mould is the most important
step in embedding. Incorrect placement of tissues may result
in diagnostically important tissue elements being missed or
damaged during microtomy.
 elongate tissues are placed diagonally across the block.
 tubular and walled specimens such as vas deferens, cysts
and gastrointestinal tissues are embedded so as to provide
transverse sections showing all tissue layers
 tissues with an epithelial surface such as skin, are embedded
to provide sections in a plane at right angles to the surface
 multiple tissue pieces are aligned across the long axis of the
mould, and not placed at random

23. The process by which tissue is trimmed
and cut into uniformly thin slices.
A microtome is a mechanical
instrument used to cut biological
specimens into very thin segments
for microscopic examination. Most
microtomes use a steel blade and are
used to prepare sections of animal or
plant tissues for histology.
Microtome

24. Principle of microtome
➢ Through the motion of the
sample holder, the sample
is cut by the knife position 1
to 2;at which point the fresh
section remains on the
knife. At the highest point of
the rotary motion, the
sample holder is advanced
by the same thickness as
the section that is to be
made, allowing for the next
section to be made.

25. Parts of microtome
All microtomes have three
main parts:
1.Base(microtome body).
2.Block holder:
❖ Holds the tissue block
in place.
3.Knife carrier and
knife:
❖ Cuts the tissue block in
sections.
❖ Knife is immovable
whereas the block
holder moves when we
move screws that are
present at the sides of
the microtome.

26. Types of microtomes
✓ Rocking microtome
✓ Rotary microtome
✓ Sledge microtome
✓ Freezing microtome
✓ Vibrating microtome
✓ Cryostat
✓ Sliding microtome
✓ Saw microtome

27. Rocking microtome
The use of rocking
microtome is common
in developing
countries.
It is relatively
inexpensive, simple to
operate and it has the
ability to produce
tissue sections of high
quality.

28. Rotary microtome
In a rotary microtome, the
knife is fixed in horizontal
position. The knife is
stationary and the block
move up and downward
direction.

29. Sledge microtome
❑ It is used to cut very
hard tissues, such as
wood, bone etc.
❑ We can get quite thin
sections by using this
microtome.

30. Freezing microtome
❑ The optimum cutting
temperature is -20
degree celsius.
❑ The freezing of tissue is
done by carbon dioxide
gas.

31. Vibrating microtome
Vibrating microtome have a
vibrating blade to cut
sections without freezing
and embedding.
This microtome is used for
cutting difficult, soft and
fres biological samples.

32. Cryostat microtome
❑ Sectioning is done on
unfixed tissue.
❑ Cryostat is a
refrigerated chamber
containing usually a
rotary microtome.
❑ Temperature may be
maintained between -15
to -30 degree celsius.

33. Saw microtome
This microtome is
specially for hard
materials such as
bones and teeth.
Their cut thickness is less
than 3 micrometer.

34. Sliding microtome
➢ A microtome in which the
object to be cut is fixed
and the knife is carried
obliquely across it.
➢ The block remains
stationary while the knife
moves during the
process of sectioning.
s

35. Microtome knives
 STEEL KNIVES
 NON-CORROSIVE KNIVES FOR CRYOSTATS
 DISPOSABLE BLADES
 GLASS KNIVES
 DIAMOND KNIVES

36. Applications of microtomy
1.Traditional histological technique:
➢ The tissue is cut in the microtome at thicknesses
varying from 2 to 25 micrometers thick. From
there the tissue can be mounted on a microscope
slide, stained and examined using light
microscope. Can be achieved by rotary
microtome.
2.Botanical microtomy technique:
➢ Hard materials like wood, bone and leather
require a sledge microtome. The microtomes
have heavier blades and can not cut as thin as a
regular microtome.

37. Care for microtomes
❑ Cover properly.
❑ Avoid rust.
❑ Application of light oil.
❑ Remove wax.
❑ Clean metallic parts.

39. Floating and picking up section
✓ Water bath must be set at temperature about 45 ºC
to 50 ºC .
✓ Small amount of alcohol or detergent should be
added into water bath to reduce surface tension and
allow the section to
✓ flatten out easily.
✓ Floating of tissue section should be done more
carefully to prevent water bubbles from being
trapped under section. Fold in section can be
removed by simply teasing with forceps.
✓ Section should be allowed to float for about 30 s.

41. Staining
Staining
It is simply a technique of dyeing a substance, quite
often of a biological system to make it or a specific part
thereof more prominent for examination.
Stain:
Stain or dyes are substance that colour a substance by
penetration.
Purpose of stains:
It reveal the inner morphology of cells or tissues. This is
possible because different stains behave differently towards
different parts of the cells or tissues.

42. Types of Staining
In vitro
Staining is done with non living cells and so can
necessarily be done out side the biological system.
Example:
Gram staining etc.
In vivo
Staining is carried out with living system.
Example:
Acridine orange (AO) /Ethidium bromide (EB)staining used as
marker for apoptosis in cell population and to locate bands of
DNA in gel electrophoresis. EB/AO combination is used for
distinguishing between live cells and apoptic cells.

43. Staining
 Stains are usually salts composed of cation and anion and
one is colored (chromophore)
 Acidic dyes stain alkaline structures; basic dyes stain
acidic structures and are used more commonly

44. Staining
 Simple stains
 Differential stains
⚫ Gram stain
⚫ Acid-fast stain
⚫ Endospore stain
 Special stains
⚫ Negative (capsule) stain
⚫ Flagellar stain
⚫ Fluorescent stains
 Staining for electron microscopy

45. Simple stains
In this staining, single dyes used to stain the
organism and it has limited clinical application. The
dye is negative and the bacterium is positively
charged and they will get stained due to the
interaction of the opposite charges. It doesn’t
provide a lot of detail on structure though.

46. Simple Stains
Figure 4.16b

47. Cheek Cell

48. Elodea

49. Epidermal Onion Cell

50. Differential staining:
They are called differential stains because they
differentiate organisms.
Example
Gram stain differential between – and +.
Acid fasting is useful because it differentiates between
an acid fast bacteria vs non acid fast bacteria.

51. Gram Stain
Figure 4.17.1

52. Gram Stain
Figure 4.17.2

53. Gram Stain

54. Gram Stain
Figure 4.17.4

55. Acid-Fast Stain
Figure 4.18

56. Endospore Stain
Figure 4.19

57. Special staining

58. Staining
❑ The embedding process must be reversed
in order to get the paraffin wax out of the
tissue and allow water soluble dyes to penetrate
the sections. Therefore, before any staining can be done, the slides
are "deparaffinized“ by running them through xylenes (or
substitutes) to alcohols to water.
❑ There are no stains that can be done on tissues containing
paraffin.
❑ Stain rehydrated sections in Hematoxylin solution for 20-40
minutes.
❑ Wash in tap water for 1-5 minutes, until sections turn blue
("bluing").
❑ Differentiate sections in 70% ethanol—containing 1% HCl—for 5
seconds. This removes excess dye, allowing nuclear details to
emerge.
❑ Wash 1-5 minutes in tap water until blue.
❑ Stain in Eosin solution for 10 minutes.
❑ Wash 1-5 minutes in tap water.
❑ Dehydrate, clear and mount.
❑ Note the use of tap water in the washing steps—tap water
provides the alkanlinity necessary for the "bluing" process.

59. Mounting
o After staining, dry it and put
on slide.
o Mount by putting albumin
and cover with glass cover
slip and slightly compress.
o Then let mounting material
be dry overnight.
o Then we will observe under
microscope.

60. Positive Staining Negative Staining

61. Negative (Capsule) Stain
Figure 4.20

62. Flagellar Stain
Figure 4.21

63. Capsule Stain
Special Stains

64. STAINING

65. Staining machine

66. Microscopic Measurements
Calibrate the ocular micrometer

68. Stage Micrometer
Ocular Micrometer
Stage Micrometer = 10 units =10*0.01mm = .1mm
Ocular Micrometer = 9 units
1 ocular division = .1mm / 9 units = 0.011mm
• Start with 4X objective
• Get stage micrometer in focus
• Line up the zero lines of the two micrometers

69. Each small division = 0.011 mm
This cell spans across 11 ocular divisions.
How wide is this cell? 11*0.011=0.121mm.

70. Dinoflagellate
4Xobjective
(40X)
10Xobjective
(100X)
40Xobjective
(400X)
Czura 2005

71. Lilium Pollen
4Xobjective
(40X)
10Xobjective
(100X)
40Xobjective
(400X)
Czura 2005

72. Frog Blood
4Xobjective
(40X)
10Xobjective
(100X)
40Xobjective
(400X)
Czura 2005

73. 100X objective
(1000X)
40X objective
(400X)
Streptococcus faecalis
Czura 2005

74. Dinoflagellate
Frog Blood
Lilium Pollen
Streptococcus faecalis