HISTOLOGY

LEARNING OUTCOME
State, write and explain the concepts
and theories in histological techniques
and skills.
Adapt
the
appropriate
scientific
methods and interpret the respective
data.

 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.

 Histological examination of tissues starts with

surgery, biopsy or autopsy.

A thin section
of lung tissue
stained with
hematoxylin
and eosin.

PROCESSING

 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.

 Important for successful embedding in paraffin.

A series of steps to remove fixative and water from
specimen.
Usually achieved by immersing the tissue 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, to 2 hours.
Most zoologist used ethyl alcohol for dehydration
whereas the botanist prefer tertiary butyl alcohol.

 Remove or clear opacity from dehydrated tissues,

making them transparent.
It is a transition step between dehydration
infiltration of paraffin into the tissue.

and

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

 The melting point of paraffin is varies, 50-52C for

the softest and 60-68C 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
paraffin. Hot temperature-harder paraffin.

of

Tissues are transferred directly from the clearer to

paraffin that is in melted state.

 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).

By using melted paraffin, the tissues sponge-like holes

are filled with paraffin, enabling it to cut without
compressing

 Tissues are ready to be embedded when they are

thoroughly infiltrated with paraffin.
The paraffin in a container such as paper box or
aluminum foil is allowed to solidify around and within
the tissue.
The paraffin is poured in a paper box. Tissue is
transferred in the paper box using warm forceps to
prevent congealing of paraffin on metal surfaces.
Handle the tissues rapidly as possible to
prevent the paraffin from solidifying before
the tissues are oriented in it.

 Orientation the tissue before the paraffin solidify

and marked with a slip of papers. This is important
to determine the proper surface for sectioning.
Paper of foil boxes may be fashioned as shown in
figure 3-1

 When small amounts of paraffin are ready to be

solidified, they can be cooled immediately in water,
preferably at a temperature of 10-15C.
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 remelt, and repeat the embedding process

 The embedded blocks (Fig. 5-1a) are trimmed into

squares or rectangles, depending on the shape of
the tissues

 Then, a heated instruments

(spatula or slide) is held
between the paraffin on a
wooden block or metal disc.
(Fig. 5-2a)
The
paraffin
blocks
are
mounted on a wooden block
or metal object disc for easy
sectioning handling. (Fig. 52b)

 The tissue-paraffin block

machine, the microtome.

is sectioned

using

cutting

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

Each thin sections will form a ribbon that stick to each

other and hold away from the knife with a camels hair
brush.
These slices, usually thinner than the average cell, are
then placed on a glass slide for mounting and staining.
Frozen tissue embedded in a freezing medium is cut
on a microtome in a cooled machine called a cryostat

The most common applications of microtomes are:

Traditional histological technique:
Tissues are hardened by replacing water with paraffin.
The tissue is then cut in the microtome at thicknesses
varying from 2 to 25 micrometers thick.
From there the tissue can be mounted on a
microscope slide, stained and examined using a light
microscope.
Cryosection:
Water-rich tissues are hardened by freezing and
cut frozen; sections are stained and examined with a
light microscope.
This technique is much faster than traditional histology
(5 minutes vs 16 hours) and are used in operations to
achieve a quick diagnosis.

Electron microscopy:
After embedding tissues in epoxy resin, a
microtome equipped with a glass or diamond
knife is used to cut very thin sections
(typically 60 to 100 nanometers).
This
instrument
ultramicrotome.

is

often

called

an

Sections are stained and examined with a

transmission electron microscope.

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

 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 45C for 48 hours

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 HAEMOTOXYLIN 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.

 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.

GENERAL GRAM STAINING

TECHNIQUE
o The Gram stain is one of the oldest, most cost-efficient, yet most underutilized, staining method used to identify bacteria

 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 violetiodine 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.