Special Veterinary Pathology

Staining teqniques -Fungi and spirochaetes

DEMONSTRATION OF FUNGI IN TISSUE SECTIONS

Solutions required

• Periodic acid
• Periodic acid 1.0 g
• Distilled water 100.0 mL
• Schiff reagent
• Differentiator
• Potassium metabisulfite, 10% aqueous 5 mL N/10 Hcl 5 mL
• Distilled water 100 mL
• Light Green
• Light green SF, Yellowish 0.2 g
• Distilled water 100.0 mL
• Glacial acetic acid 0.2 mL

Procedure

• Deparaffinize and hydrate slides to water Oxidise in periodic acid for 5 min Wash in running water for 15 min
• Treat with schiff reagent for 10-15 min
• Differentiate, 2 changes for 5 min
• Wash in running water for 10 min
• Stain with light green in running water for 3-5 min
• Dehydrate, clear and mount in DPX

Results

• Fungi - Red
• Background - Light green
• Glycogen, mucin, amyloid and colloid - Purplish red

Fungi - Red

DEMONSTRATION OF SPIROCHAETES IN TISSUE SECTIONS BY LEVADITI'S METHOD

• Thin pieces (1 mm thick) of tissue fixed in 10% neutral buffered formalin for 1-2 days, or older material stored in formalin can be used.

Solutions required

• 3% Silver nitrate solution:
• Silver nitrate - 3 g
• Distilled water - 100 mL

Reducing solution

• Pyrogallic acid 3 g
• Formaldehyde 5 mL
• Dist. Water 100 mL

Technique

• Rinse tissue block in tap water to remove excess formalin Soak in 95% alcohol for 24 hours
• Place in distilled water, and leave until the tissue block sinks
• Place in freshly prepared 3% silver nitrate solution and keep in the dark at 37 ° C for 4 days, changing the solution daily
• Rinse in distilled water
• Place in reducing solution at room temperature for 48 hours
• Wash in several changes of distilled water
• Dehydrate, clear in Cedar wood oil, and embed in paraffin wax as usual
• Cut sections at 5 m , mount on glass slides, when dry, dewax in xylene and mount in D.P.X.

Results

• Spirochaetes - Black
• Background tissue - Shades of yellow to light brown