Beruflich Dokumente
Kultur Dokumente
AND STAINS
INTRODUCTION
E.M.M.MANGADA, RMT 3
COLLAGENIC FIBERS
E.M.M.MANGADA, RMT 4
Collagen Type I
E.M.M.MANGADA, RMT 5
Collagen Type II
E.M.M.MANGADA, RMT 6
Collagen Type III
E.M.M.MANGADA, RMT 7
Staining reaction of Collagen
E.M.M.MANGADA, RMT 8
RETICULAR FIBERS
E.M.M.MANGADA, RMT 9
ELASTIC FIBERS
E.M.M.MANGADA, RMT 10
Basement Membrane
E.M.M.MANGADA, RMT 12
TRICHROME STAINS
E.M.M.MANGADA, RMT 13
Factors Affecting Trichome Staining
E.M.M.MANGADA, RMT 14
Nuclear Stains for Trichrome
E.M.M.MANGADA, RMT 15
Effects of Fixation
E.M.M.MANGADA, RMT 16
VAN GIESON’S STAIN
Simplest method of differential staining of collagen using
a mixture of picric acid and acid fuschin
Fixation: Mercuric chloride fixative
SOLUTION
Sat. aq. Picric acid solution 50 ml
1% aq. Acid fuschin solution 9.0 ml
Distilled water 50 ml
E.M.M.MANGADA, RMT 17
VAN GIESON’S STAIN procedure
o Nuclei- black
o Collagen-red
o Muscle, cytoplasm, RBC and fibrin- yellow
E.M.M.MANGADA, RMT 19
VAN GIESON’S STAIN notes
E.M.M.MANGADA, RMT 20
MASSON’S TRICHROME STAIN
E.M.M.MANGADA, RMT 21
MASSON’S TRICHROME STAIN results
o Nuclei- blue/black
o Cytoplasm, muscle and erythrocytes- red
o Collagen- blue
E.M.M.MANGADA, RMT 22
MASSON’S TRICHROME STAIN procedure
Deparaffinize and bring to water
Remove mercuric pigment by iodine, sodium thiosulfate sequence
Wash in tap water
Stain nuclei by celestine blue or Wiegert’s
Differentiate with 1% acid alcohol (when using celestine blue)
Wash well in tap water
Stain in acid fuschin for 5 minutes
Rinse in distilled water
Treat with PMA solution for 5 minutes
Drain
Stain with methylene blue (or light green)
Treat with 1% acetic acid, 2 minutes
Dehydrate, clear and mount
E.M.M.MANGADA, RMT 23
MALLORY’s ANILINE BLUE
E.M.M.MANGADA, RMT 24
MALLORY’s ANILINE BLUE results
E.M.M.MANGADA, RMT 25
HEIDENHAIN’S AZAN TECHNIQUE
E.M.M.MANGADA, RMT 26
HEIDENHAIN’S AZAN TECHNIQUE
results
Amyloid connective tissue and mucuos colloid-
deep blue
Nuclei- red
E.M.M.MANGADA, RMT 27
STAINS FOR DEMONSTRATION OF
FIBRIN
Gram- Weigert
Phosphotungstic acid-hematoxylin
Trichrome methods
(Martius, Scarlet ,Blue technique or MSB technique)
E.M.M.MANGADA, RMT 28
STAINS FOR DEMONSTRATION OF
FIBRIN
•Gram- Weigert
•Phosphotungstic acid-hematoxylin
•Trichrome methods
•(Martius, Scarlet ,Blue technique or MSB technique)
E.M.M.MANGADA, RMT 29
MSB technique for fibrin
E.M.M.MANGADA, RMT 32
VERHOEFF’S STAIN results
E.M.M.MANGADA, RMT 33
Orcein method (Taenzer-Unna
Orcein)
Excellent stain for elastic fibers- stains even the most
delicate and finest fibers of skin
Main advantage: simplicity of preparation
Primary stain: orcein
Differentiator: acid alcohol
Counterstain: methylene blue or alum hematoxylin
E.M.M.MANGADA, RMT 34
Orcein method (Taenzer-Unna
Orcein) results
Elastic fibers- dark-brown
Nuclei- blue
E.M.M.MANGADA, RMT 35
WEIGERT’S STAIN
E.M.M.MANGADA, RMT 36
WEIGERT’S STAIN results
E.M.M.MANGADA, RMT 37
KRAJIANTECHNIQUE
Rapid method of elastic tissue staining, for fibrin and
amyloid employing Congo red
Elastic fibers- bright red
Fibrin and connective tissues- dark blue
RBC- orange yellow
E.M.M.MANGADA, RMT 38
OTHER STAINS FOR ELASTIC
FIBERS
Weigert’s resorcin-fuschin method
Elastic fibers- brown to purple
E.M.M.MANGADA, RMT 40
SILVER IMPREGNATION TECHNIQUES
E.M.M.MANGADA, RMT 42
GORDON AND SWEET’S METHOD for
reticular fibers
Uses 5 ml of 10% aq.silver nitrate solution with
concentrated ammonia and 3% sodium hydroxide
Notes:
short treatment with iron alum solution, of <5 minutes, give
less staining of nuclei
Use of Coplin jar for the silver solution reduces the
possibility of precipitation
Sections can be counterstained with eosin, nuclear fast red,
tartrazine, or Van Gieson
E.M.M.MANGADA, RMT 43
GORDON AND SWEET’S METHOD results
E.M.M.MANGADA, RMT 44
GOMORI’S METHOD for reticular
fibers
Uses 10% potassium hydoxide solution with 40 ml of 10%
silver nitrate solution
Results:
Reticular fibers- black
Nuclei- gray
Other tissues- according to counterstain
E.M.M.MANGADA, RMT 45
RUSSELL MODIFICATION OF THE
MOVAT PENTACHRME STAIN
Use for tissue fixed in 10% NBF or acetic formalin sublimate with tissue of 5
microns thick
Employs:
Solutions containing 1% alcian blue solution, alkaline alcohol, iodine solution, 10%
absolute alcohol hematoxylin, 10% ferric chloride
Hematoxylin solution
Crocein scarlet-acid fuschin
E.M.M.MANGADA, RMT 46
RUSSELL MODIFICATION OF THE
MOVAT PENTACHRME STAIN results
Nuclei and elastic fibers- black
Collagen and reticular fibers- yellow
Ground substance, mucin- blue
Fibrinoid, fibrin- intense red
Muscle- red
E.M.M.MANGADA, RMT 47
RUSSELL MODIFICATION OF THE
MOVAT PENTACHRME STAIN notes
Differentiation of elastic fibers is usually complete in 2-3
minutes
Complete removal of alkaline alchol using runing water is
important, otherwise, subsequent staining will be
inhibited
The stain may be used to demonstrate C. neoformans
staining brilliant blue
E.M.M.MANGADA, RMT 48
OTHER STAINS FOR RETICULAR
FIBERS
Bielchowsky’s Method
Periodic Acid Schiff- Leucofuschin technique
Gold method
Laidlaw’s Silver impregnation
Reticulum- violet to blue-black
Snook’s reticulum method
Reticulum- black
E.M.M.MANGADA, RMT 49