Transcript Slide 1

BASIC STAINING TECHNIQUES
LECTURE 1
1
OBJECTIVES: At the completion of this
section the student will be able to:
Know the steps involved in tissue
processing and discuss the different
types of microscopy.
lnterprete the cytological significance
of differential staining produced by
hematoxylin and eosin (H&E).
Recognize and identify cells in
microscopic tissue preparations.
Understand that morphology reflects
the function of cells.
2
Methods
•
•
•
•
•
•
3
Histochemistry
Cytochemistry
Immunocytochemistry
Organ & tissue culture
Differential centrifugation
Specialized microscopic techniques
Histology
• is the study of tissue sectioned
as a thin slice, using a
microtome.
• It can be described as
microscopic anatomy.
• Histology is an essential tool of
biology.
• Histopathology, the
microscopic study of
diseased tissue, is an
important tool of anatomical
pathology since accurate
diagnosis of cancer and other
diseases usually requires
histopathological examination
of samples.
• Histochemistry refers to the
science of using chemical
reactions between laboratory
chemicals and components within
tissue.
• antibodies are used to specifically
visualise proteins, carbohydrates
and lipids: this is called
immunohistochemistry.
Antibodies are proteins of the globulin
group(immunoglobulins) that appear in
plasma and tissue fluids after antigen
injection,their production enables the
organism to eliminate certain proteins
and other foreign matter not
recognized by self.
Histochemistry and Cytochemistry
• Tissue Preparation
Methods for the observation of
- living cells
- dead cells
8
Living Cells
Cells from tissue cultures and
unicellular organisms observed with a
phase-contrast microscope
9
Dead cells
Sample sources
Autopsy
Biopsy
Blood samples
10
Stages of the Histology Technique
Light microscopy
 Sampling
 Fixation
 Dehydration
 Clearing
 Infiltration (impregnation)
 Sectioning
 Staining
 Dehydration
 Clearing
 Mounting
11
Embedding
Sampling
Aim – is to provide a representative specimen
12
Fixation
Aim – to preserve the cells with the
“least alteration” possible from the
living state
Use of substances that coagulate
the protoplasm
Simple – formaldehyde, alcohol,
acetic acid, osmic acid, picric acid
Complex – Bouin’s fluid, Zenker’s
fluid, special mixtures
13
Some organs
needs fixation
even before the
macroscopic
preparation takes
place
10 %
formaldehyde is
the cheapest and
easiest to keep
fixative solution
Dehydration
Aim – to remove water from the sample
to allow paraffin impregnation
Done by passing tissue sample through
gradually increasing concentrations of alcohol
14
Clearing
Aim – to take the alcohol out of the
sample to allow paraffin to
impregnate the tissue
The tissue is immersed in xylene
(xylol) that is miscible both in the
dehydrating and the embedding
agent
15
The name of the
process come
from the clear
appearance the
sample gets
Benzene,
cedarwood oil,
chloroform and
others can also be
used
Infiltration
Aim – to incrust the sample inside a
material that allow easy cutting
The sample goes inside a bath of
warm paraffin
The tissue embedded in paraffin is
put in a plastic frame that can be
used later in the microtome
16
The embedded
specimen in the
plastic frame can
be (and should be)
safely conserved
for many years
It is very important
to properly label
each one of the
plastic frames
• During this 12 to 16 hour process, paraffin
wax will replace the water: soft, moist
tissues are turned into a hard paraffin
block, which is then placed in a mould
containing more molten wax (embedded)
and allowed to cool and harden.
• Embedding can also be accomplished
using frozen, non-fixed tissue in a freezing
medium.
• This freezing medium is liquid at room
temperature but when cooled will solidify.
Microtome
• A microtome is a mechanical
instrument used to cut biological
specimens into very thin
segments for microscopic
examination.
Sectioning
Aim – to cut the sample in slices thin enough to be
useful
Sections of the tissue are cut with the aid of the
microtome
The thickness, between 3 and 10 µm, is selected
depending the stain that is going to be used
The section is transferred to a glass slide
20
Sectioning
Tissue block is cut into
1~20μm thick sections by
microtome, and laid out on
the surface of warm
water and are then adhered
to gel-coated slides.
21
Staining
Aim – to add color to the
structures of the sample in order
to differentiate them
There are several phases of
staining
Every stain has a different
procedure (phases, time, extra
procedures, etc.)
Special stains color different
structures
22
Most common,
easiest to use &
cheapest stain is
hematoxylin and
eosin (H and E)
Dehydration and clearing
23
Before
After
Mounting
Aim – to put the final sample in a
medium where it is protected and can
also be observed under the
microscope
The sample is dehydrated again
A drop of mounting agent with a
similar refractive index to that of
glass is placed on the glass slide,
and covered with a glass coverslip is
added
24
Mounting medium
- DPX
- Canada Balsam
diluted in xylene
(xylol)
Freeze Drying Method
(Important during surgical biopsy diagnosis)
• Aim – to quickly prepare a glass slide
for rapid study of specimens during a
surgical procedure
• Steps involved in specimen
preparation:
– Freeze the tissue
– Dehydrate the sample under a vacuum
– Embed the dehydrated sample
The quality of the section by this method is poor, and a regular
histological technique should followed at the end of surgery.
25
***immersion of tissues in xylene dissolves the
tissue lipids, which is undesirable effect when
these compounds are to be studied.
To avoid loss of lipids, a freezing microtome has
been devised in which the tissues are hardened at low
temperatures to provide rigidity necessary to permit
sectioning.
The freezing microtome is a more elaborate and
efficient successor of the cryostat.***
Cell Smears
• Cell smears are a form of histological
preparation that does not require
sectioning. Smears can be made for
example of the blood or bone marrow.
Smears are also common for swabs or
scrapings of epithelial cells (e.g. from the
oral cavity, cervix uteri).
MATERIALS
•
- sterilized lancet or needle
- 20 clean microscope slides and coverslips
- Canada balsam or other medium for
permanent preparations
- 95% ethyl or methyl alcohol
- distilled water
- Giemsa stain
- low containers (you can make them with
aluminum sheet also)
- microscope which magnifies 200 times at least
•
•
•
•
•
•
•
TAKING THE BLOOD
MAKING THE SMEAR
FIXING
STAINING
CHECKING
COVER-SLIPPING
OBSERVATION
MAKING THE SMEAR
FIXING
• fixing technique consists of dipping the
smear in a vessel containing 95% ethyl or
methyl alcohol for 3-5 minutes.
STAINING
• To be able to observe and recognize the
different kinds of leukocyte, you must stain
them. For this purpose, normally Giemsa
stain is used.
• It is a mixture of stains, based on
methylene blue and eosin
Electron Microscopy
33
Stages of the Histology Technique
Electron microscopy
• Small samples (1 mm3 or less)
• Tissue must be fresh
• Double fixation (glutaraldehyde for proteins plus osmium
tetroxide for lipids [osmium also stains])
• Quick dehydration and clearing
• Embedding in plastic resins (Epon or Araldite)
• Cut with ultramicrotome (glass or diamond knifes) produces 30
– 50 nm thick specimens
• Mounting in copper grids
34
• Stain with heavy metals
Tissue Fixation in Electron Microscopy
Same principles as in Light Microscopy with
the following differences:
Double fixation in glutaraldehyde and osmium tetroxide
Ultra-thin sections (< 90nm thick) from plastic embedded blocks.
Thus, all the original EM pictures are black-white images.
35
TEST YOURSELF
The agents in which procedure are intended to
stabilize tissue structure by coagulating proteins and
promoting cross-linking in?
(a) Clearing
(b) Dehydration
(c) Embedding
(d) Fixation
Stains
• Acid dyes stain basic structures: (proteins,
membranes, cytoplasm). The commonest acid dye is
eosin; it is pink
• Basic dyes stain acid structures: Nucleic acids (RNA,
DNA, rER, nuclei). The commonest basic dye is
hematoxylin; it is blue
• NOTE: tissues that stain with basic dyes are termed
basophilic; tissues that stain with acid dyes are termed
acidophilic
Nuclei are
basophilic, since
they are mainly
made of DNA
37
Cytoplasm is
acidophil, since
most proteins are
basic in nature
Useful stains in histology
Regular Stains:
•Hematoxylin & Eosin (H&E)
•Trichromes (differentiate intercellular structures)
•Iron hematoxylin (for iron-containing cells, like muscle & red blood cells)
Specific stains:
Proteoglycans (PAS, cresyl violet, toluidine blue, methylene blue)
Neurons (Nissl, Ag, Osmium, cresyl violet)
Elastic fibers (orcein or resorcein)
Reticular fibers (silver stain)
Blood cells (Romanovsky stains)
Polysaccharides (PAS)
38
Haematoxylin-eosin staining
• H&E is the most
common dye used in
the study of histology
• Hematoxylin stains
acidic structures
(nucleic acids, nuclei,
rER) blue
• Eosin stains basic
structures (proteins,
membranes) pink
39
Structures that are not readily stained by hematoxylin or
eosin, are called neutrophilic structures.
40
Which of the following statements is false
about the rough endoplasmic reticulum(RER)?
(A) Prominent in cells specialized for protein
secretion
(B) Presence of polyribosomes confers
basophilia
(C) Presence of polyribosomes confers
acidophilia in the cell
(D) This organelle can be viewed with a
light microscope
Trichrome stain
• Uses three dyes to
differentiate intercellular
structures
• Particularly helpful in
highlighting red blood cells
within blood vessels
42
Trichrome stain
• Gomori trichrome
• Mallory’s trichrome
• Masson’s trichrome
•
•
•
•
43
Red keratin and muscle fibers,
Blue or green collagen and bone,
Light red or pink cytoplasm, and
Dark brown to black cell nuclei
Trichrome stain
Note the blue nuclei, the pink/purple cytoplasm, the red
blood cells and the tuquoise material that is non-cellular.
44
Trichrome stain
Note:
• Blue nuclei
• Pink color in
cytoplasm
• Deep red of blood
cells
45
Diff btw H&E and Trichrome stain
46
Iron-haematoxylin
Highlights tissue and cells
that contain iron, such as
muscle & red blood cells
47
Specific stains for proteoglycans
• Periodic acid-Schiff (PAS) reaction ALSO
USEFUL FOR THE IDENTIFICATION OF
POLYSACCHARIDES (Glycogen)
• Cresyl violet
• Toluidine blue
• Methylene blue
48
Periodic Acid Schiff (PAS) staining
PAS reaction is a method to demonstrate glycogen-like substances.
Periodic acid oxidizes the glycol groups in the glucose residues into
aldehyde groups, which then react with Schiff’s reagent, producing
an insoluble compound with a reddish purple color.
PAS reaction in a hepatocyte
49
PAS reaction in mucosa of intestine
Specific stains used for
NEURONS
• Nissl (a specialized stain for rER of
neurons)
• Silver & Gold (for fibers &
cytoskeletal elements)
• Osmic acid (for myelin, a lipid)
• Cresyl violet (proteoglycans)
50
Nissl staining
A specialized stain for rough endoplasmic reticulum in neurons
51
Silver Staining
Reticular fibres
Neurofilaments stained
with silver salts
Reticular fibers cannot be
well seen with H&E staining
52
Silver ions are deposited on the
structures and reduced to silver
particles, showing brown to black
color, (argyrophilic).
Reticular stain
53
Cresyl violet
• Highlights proteoglycans
• Most commonly used for staining nervous tissue
• This is a violet/purple stain
54
Elastin
Elastin stains elastic fibers black
55
Verhoff stain for elastin
56
Romanovsky Stain for Blood Cells
Note the following:
• Reddish-brown cytoplasm of red
blood cells
• Large (white) cell with deep blue
granules
• White blood cells are most
readily identified with the use of
Romanovsky – type stains
• E g GIEMSA
Romanovsky stains (Giemsa, Wrights, etc.) are useful for
highlighting various types of granules present in
developing and mature white blood cells.
57
Metachromasia
Phenomenon where a certain dye shows
structures in a different color from that of the dye.
The granules in cytoplasm of mast cells are shown in purple
color when stained by toluidine blue, a blue dye.
58
Toludine blue
59
Osmic acid staining
60
****The effect of osmium
tetraoxide is to preserve and
stain lipids and proteins.*****
Oil Red O
• Oil Red O is used to stain lipids a redorange color in unfixed frozen sections.
Q1. which technique is most commonly used to locate
glycogen in cells?
(a) Methylene blue staining
(b) Periodic acid-schiff(PAS) reaction
(c) Enzyme histochemistry
.
Q 2. frozen sectioning may be required to avoid the
removal of which target substance when preparing tissues
for paraffin sectioning?
(a)Basic proteins
(b)Lipids
(c)Enzymes
(d)carbohydrates
ARTIFACTS
64
• Pre-histology
• These are features and structures that have
being introduced prior to the collection of the
tissues.
• A common example of these include: ink
from tattoos and freckles (melanin) in skin
samples.
• Post-histology
• Artifacts can result from tissue processing.
Processing commonly lead to changes like
shrinkage, color changes in different tissues
types and alterations of the structures in the
tissue.
• Because these are caused in a laboratory the
majority of post histology artifacts can be
avoided or removed after being discovered.
Artifacts (not natural occurences)
Caused by a bad histological
technique
 Autolysis
 Poor sampling
 Shrinkage
 Folds
 Stain precipitation and dust
 Defects in the knife
66
The main cause
of artifacts is
poor fixation
Some tissues
usually present
artifacts, & wrong
knowledge has
been derived from
them
Suprarenal gland showing with autolysis
67
Shrinkage and rupture
68
Shrinkage
69
Folds
70
Breaking and notches
71
Knife marks
72
Edge damage
73
Poor fixation
74
Dust
75