before light green counter stain. Discard solution. 13. Aniline blue solution for 5 to 10 minutes or light green solution for 1 minute. Save solution. 14. Rinse in distilled water.
Other cellular elements—blue-black.
a. Osmium Tetroxide Stain for Fat
c. Sudan Black B Stain for Fat
Fat—black, blue, or blue-black.
Stains for Carbohydrates and Mucoproteins
a. Best’s Carmine Stain for Glycogen
b. Periodic Acid-Schiff (PAS) Reaction
Exclusively acid substances (e.g. various connective
Neutral polysaccharides (e.g. glycogen and Brunnergland mucin)—magenta.
Certain substances (e.g. most epithelial mucins and
cartilage ground substance) are colored by both Alcian
blue and PAS, yielding varying shades of purple to very
deep color. The cell bodies of fungi are red to purple,
while mucoid capsules (e.g. Cryptococcus neoformans)
Other features appear about the same as with the
f. Crystal Violet Amyloid Stain
g. Bennhold’s Congo Red Amyloid Stain
h. Toluidine Blue Metachromasia Stain
a. Modification of Mallory’s Reaction for Iron
c. von Kossa’s Method for Demonstrating Calcium
(Localizations cannot be considered reliable because of
the diffusibility of the reactants and the final color).
e. De Galantha’s Method for Demonstration of Urate
Stains for Bacteria, Fungi and Inclusion Bodies
b. Ziehl-Neelsen Stain for Acid-Fast Bacteria
c. Fite-Faraco Stain for Acid-Fast Bacilli
d. Brown and Brenn Stain for Bacteria in Tissue
e. Levaditi’s Method for Staining Spirochetes in Blocks
Background—yellow to light brown.
f. Warthin-Starry Method for Staining Spirochetes
Background—pale yellow to light brown.
g. Silver Method for Spirochetes and Donovan Bodies
Spirochetes, Donovan bodies, also fungi and bacteria—
Elastic tissue and mucin also stain deep blue.
i. Gomori’s Methenamine-Silver Nitrate Technique
(Grocott’s application to Fungi)
Fungi—sharply delineated in black
Inner part of mycelia and hyphe—old rose
j. Phloxine Toluidine Blue Stain for Malarial Parasites
Malarial parasites—pale blue cytoplasmic structures
Cytoplasm—pale rose with deep red granules
k. Parson’s Stain for Negri Bodies
Negri bodies—bright orange-red
l. Hematoxylin-Shorr S3 Stain for Inclusion bodies
Inclusion bodies—brilliant red
m. Giemsa’s Stain for Rickettsiae
Cytoplasm and connective tissue—pink
SOME STAINING TECHNIQUES IN DETAIL
Mallory’s Phosphotungstic Acid
Fixation: Zenker-fixed best. If formalin-fixed, tissue should
be mordanted from 1 to 12 hours in a saturated solution of
mercuric chloride or in Zenker’s fluid.
Technique: Paraffin, sections cut at 6 microns.
Phosphotungstic Acid Hematoxylin
Dissolve the solid ingradients in separate portions of the
water, the hematoxylin with the aid of gentle heat. When
cool, combine. No preservative is necessary. Spontaneous
ripening requires several weeks but the addition of 0.177 g
of potassium permanganate will cause the stain to ripen at
1. Deparaffinize sections through 2 changes of xylene,
absolute and 95% alcohol to distilled water as usual.
2. Remove mercury precipitate by placing in alcoholic
iodine solution for 5 to 10 minutes.
4. Clear off iodine in 5% sodium thiosulfate (hypo)
5. Wash in running water for 10–20 minutes.
6. Stain for 12 to 24 hours in phosphotungstic acid
7. Differentiate in 95% alcohol—check differentiation
8. Absolute alcohol, 2 changes.
Collagen—yellowish to brownish red
Coarse elastic fibrils—purplish tint.
van Gieson’s Stain for Collagen Fibers
Technique: Paraffin, cut sections at 6 microns.
Hydrochloric acid, concentrated 1 cc
Equal parts of Solutions A and B.
Acid fuchsin, 1% aqueous solution 2.5 cc
Picric acid, saturated aqueous solution 97.5 cc.
5. Stain in Weigert’s hematoxylin solution for 10
7. Counterstain in van Gieson’s solution for 1 to 3 minutes.
9. Absolute alcohol—2 changes.
11. Mount in DPX/Add 3 drops of saturated alcoholic
picric acid to each 50 cc of xylene used in clearing.
Mount from acidified xylene. This intensifies the
background and prevents sections from fading.
Muscle, cornified epithelium—yellow
Running water will remove van Gieson’s solution
Solution B will remove Weigert’s hematoxylin.
Fixation: Bouin’s or formalin. Mordant sections of
formalin-fixed material in Bouin’s fluid for one hour at
56oC, or overnight at room temperature.
Technique: Paraffin, cut sections at 6 microns.
Solution A and B and working solution as in Van Gieson’s
Biebrich Scarlet-Acid Fuchsin Solution
Biebrich Scarlet, aqueous 1% 90 cc
Acid fuchsin, aqueous, 1% 10 cc
Phosphomolybdic-Phosphotungstic Acid Solution
Heat water, dissolve light green,
5. Mordant in Bouin’s fixative for 1 hour at 56oC, or
overnight at room temperature.
6. Cool and wash in running water until yellow color
8. Weigert’s iron hematoxylin solution for 10 minutes.
Wash in running water 10 minutes.
10. Biebrich scarlet-acid fuchsin solution for 15 minutes.
Aqueous phosphotungstic acid 5% for 15 minutes
before light green counter stain. Discard solution.
13. Aniline blue solution for 5 to 10 minutes or light
green solution for 1 minute. Save solution.
15. Acetic water 1% for 3 to 5 minutes. Discard solution.
17. Absolute alcohol—3 changes.
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