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MICRO SLIDE

PREPARATION PROCEDURE
FOR TISSUE MATERIALS

ABHILASH M R
Research Scholar
DOS in Environmental Science
MGM - 06
 History
 In the 19th century, histology was an academic discipline in its
own right. The 1906 Nobel Prize in Physiology or Medicine was
awarded to histologists Camillo Golgi and S R Cajal. They had
dueling interpretations of the neural structure of the brain based in
differing interpretations of the same images.
 What is Histology ?
 Histology is the scientific study of the fine detail
of biological cells and tissues by using
microscopes.

 To take specimens of tissues that have been


carefully prepared using special processes called
"histological techniques". It is a discipline that is
essential for the understanding and advancement
of biology, medicine, toxicology and many sub-
disciplines within those scientific subjects.
HISTOLOGICAL CLASSIFICATION OF ANIMAL TISSUES
There are four basic types of tissues:
1. Muscle Tissue
2. Nervous Tissue
3. Connective Tissue
4. Epithelium Tissue
.

Epithelium: The lining of glands, bowel, skin, and some organs like
the liver, lung, and kidney.
Endothelium: The lining of blood and lymphatic vessels.
Mesothlium: The lining of pleural and pericardial spaces.
Mesenchyme: The cells filling the spaces between the organs,
including fat, muscle, bone, cartilage, and tendon cells.
Neurons: Any of the conducting cells of the nervous system.
Germ cells: Reproductive cells (spermatozoa in men, oocytes in women).
Stem cells: Cells with the ability to develop into different cell types.
 How to prepare histology slides
 It is not usually knowledge required for first-level courses
in Anatomy & Physiology and Human Biology (e.g. A-Level in UK).
It is, however, useful to have a general awareness of the steps
involved in preparing histology slides.

The five main stages in the preparation of histology slides are:

1. Fixing
2. Processing
3. Embedding
4. Sectioning
5. Staining
 Fixing
 Samples of biological tissue are "fixed" to preserve the
cells / tissue in as natural a state. Also, doing avoid the
further contamination.

 Chemical Fixation

 In this case biological structures are preserved


(both Chemically and Structurally) in a state as close to that
of the living tissue as possible. This requires a chemical
fixative that can stabilize the proteins, nucleic acids and
muco-substances.
 Frozen Sections
 Small pieces of tissue (typically 5mm x 5mm x 3 mm) are placed in
a cryo-protective embedding medium then snap frozen in isopentane
cooled by liquid nitrogen. Sections are then fixed by immersion in a
specific fixative or series of fixatives for carefully controlled period of
time.

Advantages Disadvantages
of fixation by frozen sections of fixation by frozen sections
Give better preservation of Lack morphological detail
antigenicity
Minimal exposure to fixative Possibility of biohazard

Not exposed to the organic


solvents
Processing

 Tissue processing is done to remove water from the biological


tissues, This is important because biological tissue must be supported
in an extremely hard solid matrix to enable sufficiently thin sections
to be cut. Some typical values are:

5 μm thick for light microscopy


5 μm (i.e. 5 micrometers) = 0.005 mm = 0.000005 meter

80-100 nm thick for electron microscopy


80-100 nm (i.e. 80-100 nanometers) = 0.00008 mm to
0.0001 mm = 0.00000008 to 0.0000001 meter

Removal of water is also referred to as "dehydration".


 Embedding
Paraffin Embedding
Since water and paraffin do not mix, the first step in embedding with
paraffin is to replace the water in the tissues with a solvent that is
miscible with paraffin. Following are the steps in paraffin embedding:

1. Dehydration - It is the first part of the process. It is usually


accomplished by transferring the block of tissue through a series of
alcohol-water solutions beginning with 50 percent and running up to
water-free or absolute alcohol

2. Clearing - The alcohol is replaced by Histoclear (a non-toxic


substitute for xylol) or cedar oil, which is readily soluble in alcohol,
and in turn, is replaced by melted paraffin.
 Alternative technique

 Plastic embedding is commonly used in the


preparation of material for electron microscopy.

 Tissues are embedded in epoxy resin. Very thin


sections (less than 0.1 micrometer) are cut using
diamond or glass knives. The sections are stained
with electron dense stains (uranium and lead) so
that they can be seen with the electron
microscope.
 Sectioning
 Sectioning an embedded tissue sample is the step necessary to
produce sufficiently thin slices of sample that the detail of the
microstructure of the cells/tissue can be clearly observed using
microscopy techniques (either light microscopy or electron microscopy).

 Possible orientations at which tissue samples may be sectioned


include:
Vertical sectioning perpendicular to the surface of the tissue.
This is the most common method.

Horizontal sectioning is often done for the study of hair follicles and
structures that include hairs, hair follicles, arrector pili muscles, and
sebaceous glands in general. Such structures are sometimes called
"pilosebaceous units".
 Staining
 There are many different histology stains. Histology stains are
normally selected according to the type of tissue to be observed.
Some stains are more widely used than others while some are only
used to study very specific types of biological tissue.

 Stains react in two general ways:

1) They combined directly with the tissue,


or
2) They require that the tissues be treated first with an anchoring
substance or mordant.
Common laboratory stains
Stain Common use Specifically stains
General staining when paired Nucleic acids—blue
Haematoxylin
with eosin (i.e. H&E) ER (endoplasmic reticulum)—blue

Elastic fibers—pink
General staining when paired
Eosin Collagen fibers—pink
with haematoxylin (i.e. H&E)
Reticular fibers—pink

Toluidine blue General staining Mast cells granules—purple

Masson's Cartilage—blue/green
Connective tissue
trichrome stain Muscle fibers—red
Weigert's elastic
Elastic fibers Elastic fibers—blue/black
stain
Reticular fibers—brown/black
Reticular fibers, nerve fibers,
Silver stain Nerve fibers—brown/black
fungi
Fungi—black
Elastic fibres—dark brown
Orcein stain Elastic fibres Mast cells granules—purple
Smooth muscle—light blue
Periodic acid-
Basement membrane, Glycogen and other carbohydrates—magenta
Schiff stain(PAS)

Table sourced from: Michael H. Ross; Wojciech Pawlina (2006). Histology: A Text and
Atlas. Hagerstown, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3.
Remove the water & replace with wax-solvent
Embed the oriented specimen in molten wax

50 %
ethanol
70 %
ethanol
95 %
ethanol
label
100 %
Fresh ethanol
tissue benzene/
10% Formalin xylene
fixative Miscible with ethanol; paraffin
dissolves wax wax
After it is solid, hold the wax block & cut slices
Knife

Section

Block
Glass slide
MICROTOME - a fancy meat-
slicer - holds the wax block, &
cuts off thin slices, as the
block is slowly advanced Water-bath
mechanically
Mount the thin slices (sections) on slides

Lift out floating section on the slide


For fast biopsy, embedding is omitted - frozen sections
Knife

Section

Block is
the tissue Glass slide
FREEZING MICROTOME
holds the frozen tissue, & cuts
off thin slices, as the block is
slowly advanced mechanically Water-bath

Mount the thin slices (sections) on slides

Lift out section on the slide


When dry, remove the wax, & stain the section
Dissolve paraffin wax

Stain with Hematoxylin - blue


Wash
-
Potassium+ eosinate stain
+ charged amine, etc, groups on Stain with eosin - red
proteins bind -eosin
“Acidophilic staining”
Wash

Nuclei - blue
“Basophilic”
Cytoplasm- red
Tissue Processor
Tissue processing can be performed manually (hand processing),
but multiple specimens we are used convenient and much more
efficient to use an automated tissue processing machine ( a “tissue
processor”). These devices have been available since the 1940’s
and have slowly evolved to be safer in use, handle larger specimen
numbers, process more quickly and to produce better quality
outcomes. There are two main types of processors.

1) Tissue-transfer (or “dip and dunk”)


2) The fluidtransfer (or “enclosed”)
b) "Dip and dunk“
a) Modern enclosed tissue processor processors are still a good option
for smaller labs.
Figure 1: A fresh, unfixed specimen after surgical removal. To
prevent degeneration or drying-out the specimen should be fixed
as soon as possible.
Figure 2: A surgical specimen fixing with responsible fixative.
Figure 3: Slices about 4mm thick will now be taken from appropriate
areas and placed in the labelled cassettes for processing.
Figure 4: A tissue processor being loaded with a basket of
cassettes containing tissue specimens for processing.
Figure 5: An embedding center helps the operator by integrating
a cold plate, a hot plate and a controlled flow of molten paraffin
wax.
Figure 6: Stomach specimen being placed and oriented in an
embedding mold on the hot plate of an embedding center.
Figure 7: A block ready for microtomy.
Figure 8: A ribbon of sections being cut from a paraffin block
using a rotary microtome. Note that the sections which are
4µm thick (4/1000 of a millimetre), show little distortion or
disruption.
Figure 9: A rack of paraffin sections being loaded
onto a automated stainer for H&E staining.
Figure 10: A paraffin section being mounted on a
microscope slide after being floated out on warm
water to flatten it.
Figure 11: The H&E stain. This is a microscopic
image (micrograph) of a paraffin section of the wall of
a human appendix taken using brightfield
microscopy.
SOME EXAMPLES OF HEMATOXILIN-EOSIN STAINING

Seromucous gland

Serous gland
SOME OTHER OFTEN USED STAINING METHODS

Alcian blue Azan Resorcin-fuchsin

Schmorl Giemsa Silver-impregnation


REFERENCES

1. Weiss AT, Delcour NM, Meyer A, Klopfleisch R (2010). "Efficient and


Cost-Effective Extraction of Genomic DNA From Formalin-Fixed and
Paraffin-Embedded Tissues". Veterinary Pathology.227 (4): 834–
8. doi:10.1177/0300985810380399. PMID 20817894.
2. Adelmann, Howard (1966) Marcello Malpighi and the Evolution of
Embryology 5 vol., Cornell University Press, Ithaca, N.Y. OCLC 306783
3. Coyne J. (2012). "A squamous cell carcinoma with a Saint Valentine's
day message". Int J Surg Pathol. 20 (1):
62. doi:10.1177/1066896911434768. PMID 22287650.
4. Merck Source (2002). Dorland's Medical Dictionary. Retrieved 2005-
01-26. Stedman's Medical Dictionaries (2005). Stedman's Online
Medical Dictionary. Retrieved 2005-01-26.
5. 4,000 online histology images (2007). <http://histology-online.com>
THANK YOU

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