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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.
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.
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
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
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.
Elastic fibers—pink
General staining when paired
Eosin Collagen fibers—pink
with haematoxylin (i.e. H&E)
Reticular fibers—pink
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
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
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.
Seromucous gland
Serous gland
SOME OTHER OFTEN USED STAINING METHODS