CHAPTER 2.

0
TECHNIQUES IN HISTOLOGY
Syllabus Content
2.1 Introduction to histology
2.2 Safety rules
2.3 Histological preparation for tissues and cells
2.3.1 Fixation
2.3.2 Dehydration
2.3.3 Embedding
2.3.4 Sectioning
2.3.5 Staining
2.3.6 Mounting
2.4 Gram staining
Histology is the study of tissue sectioned as a thin slice, using a microtome

Preparation of small thin pieces of specimen

Many biological materials are too thick to be examined properly with the microscope

The thin sectioned materials must be preserved and slide made permanent
 Source of Tissues

∗ Histological examination of tissues starts with

surgical biopsy or autopsy

A thin section of lung

tissue stained with
hematoxylin and eosin
(H&E).
Standard Histology Procedures

PROCESSING
 1) Fixation of Specimen

∗ The objectives of fixation are:

- Prevent the cell changes e.g. by bacteria or autolysis
- To improve the staining potential of tissue parts
∗ Solution used for fixation is called fixative

∗ Fixative can be made up of several chemicals such as:

- alcohols - formaldehyde
- acetic acid - mercuric chloride
- chloroform - picric acid
Any fixative should:
- Penetrate rapidly to prevent postmortem changes
- Coagulate cell contents into insoluble substances
- Protect tissue against shrinkage and distortion during dehydration,
embedding, and sectioning
- Allow cell parts to be made selectively and clearly visible

Each tissue must be preserved in specific fixatives

Tissues should be placed in fixatives as soon as possible after death

Fixation ensures that the tissue is preserved in its natural state until
processing

cassette
 2) Dehydration

Important for successful embedding in paraffin

A series of steps to remove fixative and water from specimen

Usually achieved by immersing the tissue (in cassette) in a series of

solution of ethyl alcohol in water with gradually increasing percentages
of alcohol
Changing through 30, 50, 70, 80, 95% and absolute alcohol is said to reduce some of the
shrinkage occurring in the tissue

The time required for each step depends on the size of the object, ½ to 2
hours.

Most zoologist used ethyl alcohol for dehydration whereas the botanist
prefer tertiary butyl alcohol
Automatic Tissue processor
 3) Clearing

∗ Remove or clear opacity from dehydrated tissues (in cassette),

making them transparent

∗ It is a transition step between dehydration and infiltration of

paraffin into the tissue

∗ The alcohol will not mix with paraffin and therefore fluid that
miscible with both substances must be used

∗ The hydrocarbons benzene, toluene, and xylene are commonly

used for this purpose.
Dehydration and clearing combination
∗ The use of dioxane which dehydrates and clears tissues in a
minimum steps.

Advantages:
- eliminate some shrinkage and hardening
- inexpensive

Disadvantages:
- dioxane is cumulatively toxic
- frequently contains water
 4) Infiltration with Paraffin

∗ Tissues (in cassette) are transferred directly from the clearer (etc.
benzene, toluene, and xylene) to paraffin that is in melted state

∗ By using melted paraffin, the tissue’s sponge-like holes are filled with
paraffin, enabling it to cut without compressing
∗ The melting point of paraffin is varies, 50-52°C for the softest and 60-68°C for the
hardest
∗ The choice of melting point depends on the thickness of the tissue to be sectioned,
thick sections use soft paraffin and thinner sections use hard paraffin
∗ Temperature also can influence the choice of paraffin. Hot temperature→harder
paraffin

∗ Paraffin standing in a warm oven in a melted condition for several days or weeks is
better for infiltrating and embedding purposes than freshly melted paraffin

∗ Two changes of paraffin are sufficient for most requirement.

∗ Some tissue such as horny skin, bone ~ a third change may be necessary

∗ The use of vacuum oven for infiltrating will remove air for some tissues (lung)
5) Embedding - Blocking with Paraffin

Tissues are ready to be embedded when they are thoroughly infiltrated with
paraffin
The paraffin in a container such as paper box, aluminum foil or a mold is
allowed to solidify around and within the tissue
The paraffin is poured in a mold.
Tissue is removed from the cassette and transferred in the mold using warm
forceps to prevent congealing of paraffin on forcep metal surfaces
Handle the tissues rapidly as possible to prevent the paraffin from solidifying
before the tissues are oriented in it
When small amounts of paraffin are ready to be solidified, they can be
cooled immediately in water, preferably at a temperature of 10-15°C

The perfect block is one in which the paraffin crystals are contiguous
and the paraffin appears clear and homogenous

Avoid crystallization (pockets of air produce milky spot). This can cause
difficulties in sectioning.

Remedy: return the block to melted paraffin, allow it to re-melt, and

repeat the embedding process
 6) Thin Section

The tissue-paraffin block is sectioned using cutting machine, the

microtome

Usually they are cut between 7 to 10 µm (microns) thick

Each thin sections will form a “ribbon” that stick to each other and hold
away from the knife with a camel’s hair brush

These slices, usually thinner than the average cell, are then placed on a
glass slide for mounting and staining.
Cryosection:
Water-rich tissues are hardened by freezing (not using paraffin) and cut
frozen using cryostat
This technique is much faster than traditional histology (5 minutes vs 16
hours) and are used in operations to achieve a quick diagnosis

Frozen tissue embedded in a freezing medium is cut on a

microtome in a cooled machine called a cryostat
 7) Mounting

∗ The ribbons can be mounted directly on slides, floated on water

bath or laid in order in a box

∗ Usually a water bath is used for spreading the sections

∗ Dip an albumenized slide under the sections and with a needle

hold them against the slide while removing it from the bath

∗ Drain off excess water and dry the slide on a warming table or in
an oven at 45°C for 48 hours
 8) Staining
STEP 1: PREPARATION
Remove wax with a citrus oil based solvent and
rehydrate sections through DESCENDING alcohols.
95%, 80%, 70%,50% and 30%

STEP 2: STAINING
Used HAEMATOXYLIN to stain nuclei blue (10 minutes)

Rinse with tap water

Placed in acid-alcohol

Rinse with tap water

Placed in EOSIN (10 seconds) to stain cytoplasm,collagen

and muscles fibres as red
STEP 3: DEHYDRATE
Dehydrate sections with ASCENDING grades of alcohol
(10 minutes)
30%, 50%, 70%, 80% and 95%.

Used xylene as clearing agent (15 minutes) to remove all

traces alcohol and raises refractive index to make tissue
more transparent
∗ A stain, or dye, is a molecule that can bind to a cellular structure
and give it colour.

∗ Staining techniques make the:

microorganisms stand out against their backgrounds.
They also help investigators group major categories of
microorganisms,
examine the structural and chemical differences in cellular
structures, and look at the parts of the cell.
 9) Cover Glass Mounting

∗ Apply a permanent mounting medium (e.g. Canada balsam)

along one edge of sections on slide.

∗ Rest one edge of cover slip adjacent to mounting medium and

lower gradually to ease out air without bubble formation, press
gently in place

∗ After the mounting medium dries, the slide become permanent

and can stand for years.
∗ The lab should be well-ventilated.
∗ There are regulations governing formalin and
hydrocarbonds such as xylene and toluene
∗ Meet the limits set by the Occupational Safety and Health
Administration (OSHA) that should not be exceeded

∗ Every chemical compound used in the laboratory should

have a materials safety data sheet on file that specifies the
nature, toxicity, and safety precautions to be taken when
handling the compound
∗ The laboratory must have a method for disposal of hazardous
wastes. Health care facilities processing tissues often contract
this to a waste management company

∗ Tissues that are collected should be stored in formalin and may

be disposed by incineration or by putting them through a
"tissue grinder" attached to a large sink (similar to a large
garbage disposal unit)

∗ Flammable materials may only be stored in approved rooms and

only in storage cabinets that are designed for this purpose
∗ Fire safety procedures are to be posted.
Safety equipment including fire extinguishers, fire blankets, and
fire alarms should be within easy access. A shower and eyewash
should be readily available.

∗ Laboratory accidents must be documented and investigated

with incident reports and industrial accident reports.

∗ Specific hazards that you should know about include:

Bouin's solution is made with picric acid. This acid is only sold in
the aqueous state. When it dries out, it becomes explosive
∗ Many reagent kits have sodium azide as a preservative
Flush solutions containing sodium azide down the drain with lots
of water, or there is a tendency for the azide to form metal
azides in the plumbing. These are also explosive

∗ Benzidine, benzene, anthracene, and napthol containing

compounds are carcinogens and should not be used

∗ Mercury-containing solutions (Zenker's or B-5) should always be

discarded into proper containers
Mercury, if poured down a drain, will form amalgams with the
metal that build up and cannot be removed
The Gram stain is one of the oldest, most cost-efficient, yet most under-
utilized, staining method used to identify bacteria

Bacteria can be distinguished and classified into two (2) large groups
gram-positive
gram-negative

Both Gram-positive (Gm+) and Gram-negative (Gm-) organisms form a

complex of crystal violet and iodine within the bacterial cell during the
Gram-staining procedure.

GRAM POSITIVE:
resist decolorization by alcohol or acetone because cell wall permeability is
markedly decreased when it is dehydrated by these solvents

Thus, the dye complex is entrapped within the cell, resist being washed out by
the solvents, and Gm+ bacteria remain purple following this differential stain
GRAM NEGATIVE:
cell wall permeability of Gm- organisms is increased by ethyl alcohol washing
because it removes the outer membrane from the cell wall.

This allows the removal of the crystal violet-iodine complex from within the
cell.

The decolorized Gm- cell can then be rendered visible with a suitable
counterstain, in this case Safranin, which stains them red (pink).
∗ Gram-negative bacteria are bacteria that DO NOT
retain crystal violet dye in the Gram staining protocol

∗ Gram-positive bacteria are those that are stained dark

blue or violet by Gram staining

∗ Gram-positive organisms are able to retain the crystal

violet stain because of the thick peptidoglycan layer