2.

3 STAINS FOR MICRO-ORGANISMS

• Tissue sections are sometimes examined

for the presence of certain micro­organisms
with special reference to bacteria and fungi.
• Many of these micro-organisms can be seen
in routine haematoxylin eosin stained
sections, special stains are used to make
them easier to identify.
• Some of these micro-organisms may not
pick up the routine stain or they may be
very few in number and are almost
impossible to find without special stains.
• Gram stain for bacteria, acid fast stain for
mycobacterium (tuberculosis and leprosy),
silver methenamine stain for fungi, and
Giemsa stain for bacteria and blood
parasites.
2.3.1 Bacteria
2.3.1.1 GRAM STAIN

Reagent
1. Crystal violet stain:
2. Gram's iodine solution:
3. Ether

4. Acetone

5. 0.1% basic fuchsin

6. 0.1% picric acid in acetone
Staining Procedure
1. Deparaffinize and take the section to water.
2. Flood with crystal violet solution for 1 minute.
3. Rinse in water.
4. Flood with Gram's iodine for 1 minute.
5. Rinse with distilled water.
6. Blot completely dry with filter paper.
7. Decolorize with a mixture of equal parts of ether and
acetone until no more blue colour runs off.
8. Rinse in tap water.
9. Stain with 0.1% basic fuchsin for 1 minute.
10. Wash in water.
11. Blot gently but not completely dry.
12. Dip in acetone.
13. Differentiate in 0.1% picric acid in acetone until the
sections
are yellowish pink.
14. Dehydrate, clear and mount.
Result

Gram-positive bacteria - blue

Gram-negative bacteria – red
Nuclei – red
Other tissues – yellow
2.3.2 Fungi
Most fungi can be seen with H & E
stain, but usually weak.
Any fixation is suitable.
This method depends upon the
reduction of silver by the aldehyde
group produced after oxidation of
fungal wall components with
chromic acid.
It is best method for the detection of
fungi in tissue sections and the
most valuable search stain for
Pnemocystis carinii ( lung infection,
alveoli become packed with
organism)
2.3.2.1 GROCOTT-GOMORI METHENAMINE SILVER
METHOD
Reagent
1. 3% sodium thiosulphite
2. 5% cromic acid:
3. Incubating solution:
4. Light green counterstain:
5. 1% sodium bisulphite
6. 1% gold chloride
Staining Procedure
1. Deparrafinize and take section to water.
2. Oxidise in 5% chromic acid for 1 hour.
3. Wash in running tap water for 3 minutes.
4. Rinse in 1% sodium bisulphite.
5. Wash in tap water for 3 minutes.
6. Rinse well in distilled water.
7. Place in incubating solution at 60 o C in the dark for 1 hour.
8. Wash well in distilled water.
9. Tone in 1% gold chloride for 45 minutes.
10. Place sections in 3% sodium thiosulphate for 5 minutes.
11. Wash well in tap water.
12. Counterstain in light green solution for 20 seconds.
13. Wash in tap water.
14. Dehydrate, clear and mount.
Result
Fungal hyphae and yeast
bodies - black

Background - pale green

STAINING TO REMEMBER

1. Frozen section - H & E Stain

- Polychrome Methylene
Blue Stain
Stains For Particular Parts Of Tissues

1. Collagen - Masson’s Trichrome Stain

- Weigert van Gieson Stain
2. Elastic fibres - Verhoeff’s Stain
3. Reticulin fibres - Silver Impregnation
Method
Stains For Particular Substances

1. Carbohydrate - Periodic Acid Schiff Stain

2. Amyloid - Congo Red Stain
3. Haemosiderin & iron - Prussian Blue Stain
4. Calcium - Von Kossa Silver Nitrate Stain
5. Neutral lipids - Oil Red O stain
6. Cellular components of nervous system
- Haematoxylin van Gieson stain
- Cresyl fast violet stain
7. Tissue antigens
- Immunoinfluorescence Technique
Stains For Micro-organisms

1. Bacteria - Gram Stain

2. Fungi - Grocott-Gomori Methenamine
Silver Method

Overall = 16 stains to memorize

MLT Boleh…….