Preparation of Histological Specimens
Preparation of Histological Specimens
Preparation of Histological Specimens
HISTOLOGICAL SPECIMENS
TISSUE FIXATION
Stages of processing:
1- Dehydration.
2- Clearing.
3- Embedding.
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.
Types of dehydrating agents:
Ethanol, Methanol, Acetone.
2- Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2 mm surrounding margin of wax.
4 Using warm forceps select the tissue, taking care that it does not cool in
the air; at the same time.
5- Chill the mould on the cold plate, orienting the tissue and firming it into
the wax with warmed forceps. This ensures that the correct orientation is
maintained and the tissue surface to be sectioned is kept flat.
7- Cool the block on the cold plate, or carefully submerge it under water
when a thin skin has formed over the wax surface.
• multiple tissue pieces are aligned across the long axis of the mould,
and not placed at random
Processing methods and routine
schedules
• Machine processing
• manual processing
CUTTING
• 4- Botanical microtomy:
• hard materials like wood, bone and leather require a sledge
microtome. These microtomes have heavier blades and cannot cut as
thin a regular microtomy.
• STEEL KNIVES
• NON-CORROSIVE KNIVES FOR
CRYOSTATS
• DISPOSABLE BLADES
• GLASS KNIVES
• DIAMOND KNIVES
STAINING
Hematoxylin and Eosin (H & E)