Introduction to

Special Stains
Summary
 Why are special stains still important and
relevant today?
 What are some of the chemical principles
behind these stains?
 Some common examples that can be
prepared in student laboratory teaching.
What are “special stains”?
  H&E was first introduced in the 1870’s and the
term special stain came to refer to any technique
other than H&E used in the clinical environment.
 Whilst the H&E stain is the most common staining
method in hospital and research laboratories, it
isn’t without its limitations.
 H&E cannot visualize micro-organisms.
 H&E is not good for distinguishing connective tissue
and nerve tissue.
 H&E cannot distinguish molecular basis of disease
and immunohistochemistry might be preferred.
Six Special Stains!
1) Toluidine Blue
 Tolonium Chloride
 Useful blue cationic (basic) dye
 Cheap and simple application
 Often used to identify mast cells , stain
proteoglycans and glycosaminoglycans in
tissue such as cartilage
 
Neutrophils separated from blood sample
Stain for 3 minutes in 0.5% toluidine blue
Buccal cells
Distinguishing nerve tissue
2) Luxol Fast Blue
 Immunohistochemical methods have advanced
but are costly and reagents deteriorate quickly.
 Special stains include silver methods (such as
Gordon and Sweet’s), gold, or Luxol fast blues
to stain myelin.
 Other special stains identify nerve cells. The
techniques are important for looking and
neurodegeneration.
Distinguishing connective tissue
Reticulin
Collagen
Fibrin
3) Gordon and Sweet’s
 One of several silver methods for staining
reticulin.
 Tissue treated with potassium
permanganate to enable the silver to bind.
 Uses an ammoniacal silver solution.
 What is reticulin?
 Why is it important?
Gordon and Sweet’s (Low Power)
Liver tissue with no counterstain
Reticulin = black
Gordon and Sweet’s (High power)
Liver counterstained with what?
Reticulin = black
Cytoplasm = pink
4) Van Gieson
 Trichrome stain – producing 3 colours.
 Anionic dye and a cationic counterstain.
 Nuclear stain applied first such as Weigert’s
haematoxlin.
 Collagen stains red with acid fuchsine.
 Cytoplasm including muscle stains yellow.
 Washing in acidified water differentiates
tissue producing two colours.
 Van Giesen (High power)
Bladder
Collagen = red
All other tissue including transitional epithelium = yellow
 Van Giesen (Low power)
Collagen = red
Smooth muscle = yellow
Epithelium = yellow
Application? Might be used to localise tumours in the bladder to either the smooth muscle or connective tissue layers.
5) Martius scarlet blue stain`
 Trichrome stain.
 MSB is a trichrome method for selective
demonstration of fibrin.  Fibrin is stained
using crystal scarlet solution, while methyl
blue is used to stain the collagen.  Red
blood cells are stained yellow by the picric
acid.
Tongue (High power)
Epithelium = red
Collagen = blue
Cytoplasm = red
No visible yellow
Bladder (low power)
Collagen = blue
Erythrocytes and early fibrin = yellow
Cytoplasm = pink
Adrenal gland (100x oil immersion)
Erythrocytes clearly yellow
Collagen = blue
Cytoplasm = red
Adrenal gland (x100 oil immersion)
Early fibrin deposits = diffuse yellow staining
Collagen = blue
Glandular tissue = red
6) Masson’s Trichrome
 Trichrome stain.
 Iron-haematoxylin plus two anionic dyes.
 MSB is a variation of this.
 Weigert’s Iron-hematoxylin + 3 different solution
 Solution A = plasma stain, contains acid fuchsin,
Xylidine Ponceau, glacial acetic acid and distilled
water
 Solution B = phosphomolybdic acid and distilled water
 Solution C = fiber stain, contains Light Green SF
yellowish or Fast Green FCF, used to stain collagen
 Bladder MSB (Left), Masson’s (Right)
Nuclei = black
Cytoplasm including muscle and epithelium = red
Erythrocytes = red
Collagen = bluey green or turquoise
Tongue (low power)
MSB
Collagen = bluey green
Cytoplasm of epithelium and skeletal muscle = red
S
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1. In preparing tissue for routine light microscopic
study, which procedure immediately precedes
clearing the specimen with an organic solvent?
A. Clearing
B. Dehydration
C. Embedding
D. Staining
Which of the following staining procedures relies
on the cationic and anionic properties of the
material to be stained?
 
A. Enzyme histochemistry
B. Hematoxylin & eosin staining
C. Immunohistochemistry
D. Periodic acid-Schiff reaction
In a light microscope used for histology,
resolution and magnification of cells are largely
dependent on which component?
A. Condenser
B. Eyepiece
C. Objective lens
D. Course adjustment knob
Resolution in electron microscopy greatly exceeds that of light
microscopy due to which of the following?

a. The wavelength of the electrons in the microscope beam is shorter

than that of a beam of light.
b. The lenses of an electron microscope are of greatly improved quality.
c. For electron microscopy the tissue specimen does not require staining.
d. The electron microscope allows much greater magnification of a
projected image than a light microscope provides.
e. An electron microscope can be much more finely controlled than a
light microscope.
The CLS in the microbiology section ran out of Gram stain
supply. There were bottles of H & E stain in the storage room
and decided to use it in replacement. Will the H & E be efficient
and effective in the same use as Gram stain?

A. No, because H & E does not distinguish between a gram-

negative to a gram- positive bacteria.
B. No, because H & E does not visualize microorganisms.
C. Yes, because H & E stains bacteria and yeast cells.
D. Yes, because H & E stains bacteria, yeast cells and parasites.