The typical thickness of a histological specimin is only 3 micrometers.
At this thickness virtually all tissue structures are near-transparent.
At the basic level a stain makes the specimin easier to see and because
a stain affects different areas (e.g. nucleus vs cytoplasm)
differently, a stain makes selected elements
easier to distinguish.
Choice of stain will depend on the tissue and
the aim of the examination as well as the way the specimen has been
Preparing a tissue for staining
- If a large sample is provided e.g. from a surgical procedure then
a pathologist looks at the tissue sample and selects the part most
likley to yield a useful sample - this part is removed for examination
in a process commonly known as cut up.
This is then placed into a plastic cassette for most of the rest of the
- Water is removed from the sample in successive stages by the use
of increasing concentrations of alcohol.
- Xylene is used in the last dehydration phase instead of alcohol -
this is because the wax used in the next stage is soluble in xylene
where it is not in alcohol allowing wax to permeate the specimen.
- Molten wax is introduced into the specimen. Further wax is added
to the casette surrounding the sample, producing a wax block with the
sampled embedded within.
This process is generally automated and done overnight. It is needed to
provide a sample sturdy enough for obtaining a thin section for the
slide - it can however produce changes to the tissue called artifacts -
things that are only there due to what we have done to the tissue and
which don't mean anything diagnostically. The skilled pathologist can
recognise these artifacts for what they are.
Once the wax block is finished, sections will be cut from it. This is
usually done by hand and is a skilled job with the lab personnel making
choices about which parts of the spcimen to place on slides.A number of
slides will usually be prepared from different levels throughout the
block. After this the thin section is stained and placed on s slide
with a protective cover slip. For common stains and automatic process
is normally used but rarely used stains are often done by hand.
In the UK, tissue and sections taken for histology are retained for att
least 25 years.
Commonly used stains (alphabetically)
a mixture of
methyline blue and eosin - the appearance is unsurprisingly similar to,
but not identical to the appearance under H&E and Wrights stain. It
also binds to some pathogenic organisms including plasmodium (causative
of malaria), spirochaetes (syphillis), and trypanosomes (Chagas disease
and sleeping sickness).
Haematoxylin and Eosin. A quick to apply and cheap stain that is the
most-used preparation in most pathology labs.
has a deep
color and stains nucleic acids by a complex, incompletely understood
reaction. It stains the cell nucleus well enough to distinguish varying
cell-type- and cancer-type-specific patterns of condensation of
heterochromatin (hematoxylin staining) that are diagnostically
is pink and stains
proteins nonspecifically. It stains,
cytoplasm, extracellular matrix and nuceoli.
typical tissue, nuclei are stained blue, whereas the cytoplasm and
extracellular matrix have varying degrees of pink staining. A clear
area of cytoplasm near the nucleus represents the Golgi zone.
Oil red O: This is used as a stain for neutral
lipids. It is more soluble in fat than in either alcohol or water. It
is useful in e.g. multiple sclerosis to detect the lipid-rich debris in
macrophages from myelin breakdown in the CNS.
Papanicolaou Stain: This
histology stain is used mainly on cytological specimens. Cells in smear
preparations can be stained with Pap staining. Gynecological smears
(Pap smears), sputum, urine, cerebrospinal fluid, abdominal fluid,
pleural fluid, synovial fluid, semminal fluid and fine needle
aspiration samples can all be stained with a Pap stain. This staining
technique involving five dyes in three solutions.
Periodic Acid-Schiff (PAS): This
histology stain is particularly useful for staining glycogen and other
carbohydrates, but is useful for many things. It is often used to show
glomeruli, basement membranes, and glycogen in the liver. PAS stains
glycogen, mucin, mucoprotein, and glycoproteins magenta. The nuclei
will stain blue. Collagen will stain pink.
- Nuclear stain: haematoxylin for cell nuclei.
- Counterstain 1: Orange G, for
keratin. Its original role was to stain the small cells of keratinizing
squamous cell carcinoma present in sputum.
- Counterstain 2: contains three (or two)
- Eosin Y
stains the superficial epithelial squamous cells, nucleoli, cilia, and
red blood cells.
Green SF yellowish stains the cytoplasm of all other cells.
brown Y stains nothing and in contemporary formulations it is
(blue dye) has a positive charge and stains acidic granules as well as
RNA and DNA all of which have negative charges; Eosin (a red dye) has a
negative charge and so stains some granules and hemoglobin which are
basic with positive charges, a red colour.
stain is used to
identify acid-fast organisms such as the mycobacteria responsible for
tuberculosis and leprosy.
Antibodies can now be made to attach to specific molecules - they can
hence be used to stain these molecules and make them visible allowing,
for example a helper T cell and a ctytotoxic T cell to be
differentiated, or discovering if a breast cancer has receptors for
oestrogen (which affects it's vulnerability to treatment with
Stains applied to living tissue
Stains applied to living tissue are called Vital stains
These are further divided into supravital
(introduced to living
tissue that has been removed from the body, but before cessation of the
chemical life of the cells) and intravital
(absorbed by living cells after injection into the body) stains.
if you wish to reproduce any
information from this page.
Updated June 2016