PRACTICAL 2

Histo technique, tissue processing, microscopy,

identification of cell
Microscope: An optical instrument used for magnifying
minute objects in order to study details of structure.

LIGHT MICROSCOPE - The usual type of microscope

used in clinical laboratories is called light microscope.
A light microscope can be a simple or a compound
microscope.
Magnification power depends upon-
 Length of optical tube
 Magnifying power of objective
 Magnifying power of eye piece

Other types of microscopes

 Simple microscope
 Compound microscope
 Dark ground microscope
 Polarizing microscope
 Fluorescent microscope
 Electron microscope

Simple microscope: it is a simple hand lens (convex lens).

Magnifying power is 2x to 200x.
Compound microscope:
Consist of an array of lenses which collectively give a high
magnification.
Types: Monocular & Binocular
Principle:
Objective forms an enlarged inverted image of the object,
which the ocular (eyepiece) reverses and brings closer to the
eye.
A third optical system- the condenser controls the trans-
illumination.
A compound microscope has the following parts:
„ Stand
„ Body
„ Optical system
„ Light/illumination system

Name Principal Use

Dark ground The micro-organisms are used for examination

microscope illuminated by an oblique of unstained living
ray of light which does not micro-organisms e.g.
pass through the micro- Treponema pallidum
organism. The condenser is
blackened in the centre and
light passes through its
periphery illuminating the
living micro-organism on a
 glass slide.
Polarizing The light is made plane This method is used
microscope polarised. Polariser sieves for demonstration of
out ordinary light rays birefringence e.g.
vibrating in all directions amyloid, foreign
allowing light waves of one body, hair etc
orientation to pass through.
Birefringent objects rotate
the light rays and therefore
appear bright in a dark
background
Fluorescent Fluorescent microscopy Micro-organisms
microscope depends upon illumination stained with a
of a substance with a fluorescent dye and
specific wavelength (UV then exciting it with
region i.e. invisible region) light of Ultraviolet
which then emits light at a range. Used for :
lower wavelength (visible [Link],
region) Malarial Parasite,
Amyloid etc.

Electron By using an electron beam Used to study

Microscope of light, the resolving ultrastructural details
power of the microscope is of the tissues and
increased to 50,000 to cells.
100,000 times and very eg: Storage disorders-
small structures can be glycogen storage,
visualised. lipid storage
disorders, Study of
viruses,
Tissue Processing

Steps of Tissue Processing

Methods :
1. Paraffin section method
2. Frozen section method

1. Paraffin section method

• Fixation
• Trimming
• Dehydration
• Clearing
• Impregnation
• Embedding and Block Making
• Section cutting
• Staining

FIXATION
Fixation - Is the process of preserving the cells and tissues in
a life-like manner as possible

Fixative produces the following effects:

 i. Prevents putrefaction and autolysis.
ii. Hardens the tissue which helps in section cutting.
iii. Makes cell insensitive to hypertonic or hypotonic
solutions
An ideal fixative has the following properties:
i. It should be cheap and easily available.
ii. It should be stable and safe to handle.
iii. It should cause fixation quickly.
iv. It should cause minimal loss of tissue.
v. It should not bind to the reactive groups in tissue which
are meant for dyes.
vi. It should give even penetration.
vii. It should retain the normal colour of the tissue

Fixatives can be classified for practical purposes as-

• Simple Fixatives(routine) : contain only one chemical
substance . e.g. – Formalin
• Compound fixative(special) : contains two or more
chemical fixatives. e.g. Bouin’s fixative, Zenker’s
fixative
Commonly used Fixatives are :
• Formalin
• Glutaraldehyde
• Picric Acid (Bouin’s)
• Alcohol (Carnoy’s)
• Osmium Tetraoxide
Formalin
• Most commonly used fixative
• Formalin is commercially available as saturated solution
of formaldehyde gas in water, 40% by weight/volume
(w/ v). For all practical purposes, this 40% solution is
considered as 100% formalin
• Normally 10 % solution is used. It is prepared by mixing
10 ml of commercial formalin in 90 ml of water.
• Time required for fixation is 6 – 8 hrs (4 mm thin tissue)
• Amount of fixative- 15 to 20 times the volume of the
tissue to be fixed.
• Mode of action: Polymerisation of cellular proteins

Advantages
• Cheap and easily available
• Stable and safe to handle
• Quick acting
• Give even & high penetration into tissues
• It gives good results on staining the tissues.
• causes minimal alteration in the tissue.

Disadvantages
• Hardens the tissue
 • Shrinkage of the tissue.
• Formalin pigments are formed
• Irritate the skin & conjunctiva.

• Bouin’s fluid
• Contents:
1. Picric acid(aquous) – 75 ml
2. Formalin – 25 ml
3. Acetic acid – 5 ml
• Advantages: Rapid penetration & Preserves glycogen
Provides excellent cytoplasmic and nuclear staining
• Disadvantages: Over hardens the tissue, Causes lysis of
RBCs
• Uses: Ideal fixative for testicular and bone marrow
biopsy.
Zenker’s fluid
• Contents: Mercuric chloride, Potassium dichromate &
Distilled water
• Advantages: Better staining of nuclei and connective
tissues
• Disadvantages: Causes red blood cell lysis, Removes
iron from haemosiderin, Causes tissue shrinkage

Glutaraldehyde
USE- This is used as a fixative in electron microscopy.
Disadvantages of glutaraldehyde-
i. It is expensive.
ii. It penetrates the tissues slowly.

Dehydration
It is the process of removal of water from the tissue.
The fixed tissue is dehydrated by passing through ascending
grades of alcohol

Clearing
• The alcohol is replaced by clearing agent – xylene,
toluene, chloroform & Benzene.
• These agents raise the refractive index of the tissue.
• They render it translucent & allow subsequent infiltration
by paraffin wax. (Impregnation)
Embedding & block making

Embedding is done in molten wax.(56 to 580C)

Manual Technique :
Wax blocks are prepared using L or Leuckhart’s metal
moulds.
Now, The L moulds are removed and plastic moulds are used
PLASTIC CASSETTES STEEL CASSETTES
Not reusable reusable
Specimen no. can be written Specimen no. can be written
on cassette on paper and fixed on block
Not for storage Can be used for storage

SECTION CUTTING (MICROTOMY)

Microtome is an equipment for cutting sections.
We use Rotary microtome.
Types of microtomes:
1. Rotary
2. Sliding
3. Freezing
4. Rocking

Common cells types

NAME FEATURE
Lymphocytes • Smallest WBC
• Size: approximately 7
µm in size
• Shape: Small round
cells
• Cytoplasm: scanty-
pale blue- narrow rim
around nucleus
• Nucleus: round, large
nuclei/small amount
of cytoplasm, dense
blue chromatin
• Account for 25% of
WBC count

Monocytes • Largest of WBCs

• Dark kidney bean shaped
nuclei
• No Granules but Vacoules
• Size: 15-20 µm
 • Highly phagocytic

Neutrophil • Size: 10 – 12 µm
• Shape: round to oval
• Cytoplasm: contains fine ,
pale, pink – violet
granules on H&E as well
as Leishman stain.
• Nucleus: lobulated,
number of lobes - 2 to 5
• No nucleolus
• Granules are small and
numerous—coarse
appearance
• 65% of WBC count

Eosinophil • Size: 10 to 12 µm
• Shape: Round to oval
• Cytoplasm: coarse
granules with intensely
orange to pink colour.
• Nucleus: bilobed
• 2-5% of WBC count

Basophils • Least numerous- 0-1%

• Weak phagocytic cells
• Contain histamine,
serotonin, heparin —
inflammatory chemical
reactions
• Size : Approx. 10 microns
• Cytoplasm: contains
granules which are large
 deep purple and obscure
the nucleus.
• Their counterpart in tissues
is known as mast cells.

Plasma cell: • Size: 2-3 times the size of

lymphocyte
• Shape: Round to oval
• Cytoplasm: abundant,
blue with perinuclear halo
• Nucleus: round to oval,
eccentrically placed
• Chromatin: arranged like
the spokes of a cartwheel,
also called ‘Cartwheel
appearance’

Endothelial cells • Lining cells of blood

vessels and lymphatics
• Flattened cells with
flattened nuclei
• No nucleolus

Histiocytes(macrophage): • Member of
reticuloendothelial system
• Size: 2-4 times the size of
neutrophils
• Shape: is variable, cell
margin is irregular
• Cytoplasm: pale pink, may
show phagocytosed
material i.e. fat, pigment
etc.
 • Nucleus: oval/ kidney
shaped
• No nucleolus
Foreign Body Giant Cell • Nuclei are haphazardly
arranged
• Nuclei are regular in size
and shape
• Cytoplasm may show
phagocytosed material

Langhan’s Giant Cell • This is an example of non-

neoplastic type of giant
cell.
• Nuclei may be arranged
peripherally or horseshoe
shaped manner
• Nuclei are regular in size
and shape
• They are characteristic of
tuberculosis

Tumour Giant Cells • Tumor giant cells may be

mononuclear or
multinucleate, nuclei are
large and with bizarre
shape
• The nuclei are
hyperchromatic
• Size and shape of nuclei
vary – anisonucleosis
• Nucleoli present

Reed – Sternberg Cells: • Giant cells with two

 “mirror image” nuclei
with prominent acidophilic
nucleoli, seen in
Hodgkin’s Lymphoma
(typically described as
Owl-Eye appearance)

Epithelioid cells • They are modified

histiocytes with abundant
cytoplasm
• The nucleus is vesicular
and shows elongated to
slipper shaped appearance.
• They are typical of
Tuberculosis.
•
Smooth muscle cell Elongated cells with blunt
ended nuclei, generally
arranged in interlacing bundles.

Fibroblast • Shape: Elongated

plump spindle shaped
cell, they may appear
round or oval if cut
transversely.
• Cytoplasm: Pink ,
may show laying of
collagen fibers.
• Nucleus: elongated
and with tapering
ends
• Nucleolus: one or
more, indistinct
• It may be noted that a
fibroblast is an
activated fibrocyte.