Introduction to Histology

By
Martin Inyimili.
 Objectives
• By the end of the lecture, the student should be able to:

Define Histology

Outline the various stages of preparing slides.

Scientific basis of the various stages of preparing slides.

 What is Histology.
• Is the scientific study of the fine details of cells and tissues using a microscope to
look at specimens of tissue that have been carefully prepared using special
techniques called Histological techniques.
• Is Greek word –”histos= tissue” and “logos=study”.
• Histopathology: microscopic examination of biological tissues to observe the
appearance of diseased cells and tissues in very fine details.
• Histo-chemistry: study of the identification and distribution of chemical
compounds within and between biological cells using histological techniques
such as staining, indicators and light.
Uses/values of histology
• Education- way to understand the function (physiology) of
cells/tissues/organs/systems. If one understands the structure then one
can deduce the function and vice versa. It also helps students
understand the microstructures of biological tissues.
• Diagnosis – to inform treatment options
• Research – Histology is indispensable in biological research
• Forensic investigations (Autopsy)- to determine possible causes of
sudden death.
• Archeology – for tissues recovered from archeological sites ie bone and
teeth, provide insights into history and past events
Steps in slide preparation
• Tissue collection and preparation
• Fixation
• Processing
• Embedding
• Sectioning
• Staining
• Mounting
• Microscopy.
Sample preparation
• Tissues are obtained from closest relatives of man= olive baboon
• 3 months Quarantine = observation for zoonoses
• Darting/anaesthesia
• Perfusion- washing with normal saline and introducing a fixative(mainly
formaldehyde) through the carotid artery.
• Immersing the animal in 10% formal saline.
• Purpose of fixation:
Preserve the tissue in a life-like manner as possible
Kills the tissue to avoid postmortem changes= autolysis and putrefaction
Cross-linking thus adding strength to the tissue
Cont’d
• Tissues are obtained in cubes of 1cm, some times less
• Larger tissues do not fix well as the fixative and embedding media do
not penetrate to all the sections.
• Trim wit a sharp blade to obtain regular and fine edges
• Put tissues in specimen bottles and label them ready for processing
Tissue harvesting and trimming.
 Decalcification
• The removal of calcium deposits in bone and other tissues that contain calcium for good
embedding procedure, carried out between the fixation and processing steps.
• Done via the following techniques:
Technique Examples Advantage Disadvantage

1 Strong mineral Nitric acid For dense cortical bone Damage cellular morphology
acids Hydrochloric acid Removes large quanties of Not recommended for delicate
cacium at a rapid rate tissues like bone marrow

2 Organic acids Acetic acid For delicate tissues like bone Act more slowly hence rquire
Formic acid marrow more time to decalcfy cortical
They are not aggressive hence bone
will not damage cellular
morphoogy

3 Chelating agents EDTA Not harsh to tissues Penetrates tissue poorly and
works slowly
Very expensive

4 Electrolysis Slow and not suitable for routine

work
Tissue Processing
• the purpose is to remove water and replace it with a medium capable
of solidifying to allow the sections to be cut. This medium is wax
• Has three stages = Dehydration, Clearing and Infiltration.
• Dehydration is done using ascending grades of alcohol from 70% to
absolute, since water is immiscible with wax.
• Clearing is replacing alcohol with a clearing agent like xylene, picric
acid, cedar wood oil, etc.
• Infiltration is filling the tissue air spaces with molten wax
Done in the oven at 60oC for 8-14 hours
 

Typical processing schedule

Process Solution Time
Dehydration 70% alcohol 60 mins
Dehydration 90% alcohol 45 mins
Dehydration Absolute alcohol 45 mins
Dehydration Absolute alcohol 45 mins
Dehydration Absolute alcohol 60 mins
Clearing Xylene 60 mins
Clearing Xylene 60 mins
Clearing Xylene 60 mins
Infiltration Paraffin Wax 30 mins
Infiltration Paraffin Wax 60 mins
Infiltration Paraffin Wax 90 mins
Blocking Out Paraffin Wax n/a
Cont’d
• Processing can be done in two way: manually or by an automatic
tissue processor. Below is an automatic tissue processor.
Embedding
• Tissues are placed into molds along with liquid embedding material
such as wax, agar or gelatin which is then hardened.
• Hardening is done by cooling for wax and agar while curing(heating) is
done for gelatin and resins e.g. epoxy.
• Orientation of tissue is done during this process
• Cooled blocks now have a tissue and wax and can be stored for longer
periods of time- can provide reference in medicine
Embedding station. Notice the tissue orientation on the left
Tissue cassettes: used both for processing and providing tissue support
during sectioning.
sectioning
• Involves ‘cutting’ and floating
• Sectioning is done using a microtome and a ribbon is obtained.
• For light microscopy, a steel knife mounted in a microtome is used to cut
4-micrometre-thick tissue sections which are mounted on a glass
• For transmission electron microscopy, a diamond knife mounted in an
ultra-microtome is used to cut 50-nanometer-thick tissue sections which
are mounted on a 3-millimeter-diameter copper grid.
• Individual sections are floated on a warm water bath and sections ‘fished
out’ on a slide containing an adhesive, probably egg albumin.
• Slides are arranged onto racks and allowed to dry in the oven at 50oC.
Rotary Microtome.
Left: Microtome showing a ribbon. Right: floating and fishing out
technique
 staining
• Biological tissues have little inherent contrast in both light and electron
microscopy.
• Staining is required for:
Tissue contrast
Highlight particular features of interest.
• There are several kinds of stains
• Three basic staining methods
 Hematoxylin and Eosin (H/E)
 Masson’s trichrome
 Weigert’s elastic stain.
 Special stains: silver impregnation for reticular fibres in the liver
 Mucin carmine for mucus secreting tissues like nasal cavities and the GIT
 Nissil & Myelin, Golgi stain and Luxol fast blue for nervous tissues
H/E
• Hematoxylin and Eosin(H/E) stain is commonly used.
• Cellular architecture
• Hematoxylin is basic and stains the nuclei blue while Eosin is acidic
and stains the cytoplasm red or pink
H/E staining. Nuclei appears dark stained while cytoplasm appears pink.
Masons Trichrome stain
• Has three stains
Iron Hematoxylin
Ponceau de xylidine (ponceau)
Either light green or aniline blue.
• Highlights colagenous fibres and muscle
• Iron Heamatoxylin = nuclei black/dark blue
• Ponceau = muscle, RBCs, fibrin and some cytoplasmic granules
• Light green/aniline blue= collagen, mucin and reticulin fibres
Masson’s Trichrome staining. Notice the collagen fibres staining green
and muscle red
Weigert’s Elastic stain
• Is alcohol based stain.
• Mainly for elastic fibers in heart and blood vessels
• Nuclei appear brownish black and black
• Elastic fibres appear black
Weigert’s elasic staning of blood vessel.
Used primarily to stain for elastic fibers. Notice the fibres and nuclei staining dark
blue
Mounting
• Involves putting a coverslip on the stained tissue.
• Requires an adhesive resin-either water based or resin (water
insoluble)
• Mainly DPX used
• Has to have the following characteristics:
Same refractive index as that of glass.
Not easily oxidized by air.
Durable
Note: avoid introducing air bubbles between the slide and the coverslip.
Mounting of a slide. Notice the pressing to remove air bubbles
Slide Storage
• Slide have to be labelled before storage.
• Mainly the tissue name or number.
• Storage on cupboards (wooden or metallic)
Caution
• The whole process of producing a slide is very costly, thus uttermost
care of slides is important to avoid breakages
• Respect those who have taken time to prepare slides, take care of the
slides.
Thank you !!!!
Questions