Double Oxidation

Thiosemicarbazide, Schmorl

This method demonstrates fungi.

Solutions

Periodic acid solution

Aniline-acetic
Aniline	10	mL
Acetic acid, glacial	90	mL

Thiosemicarbazide
Thiosemicarbazide	1	g
Distilled water	100	mL

Schmorl's solution
Ferric chloride, 1% aqueous	30	mL
Potassium ferricyanide. 1% aqueous	4	mL
Distilled water	6	mL
Make fresh immediately before use.
Do not re-use.

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Method

1. Bring sections to water via xylene and ethanol.
2. Do a Mallory bleach.
3. Wash with running tap water.
4. Oxidise in periodic acid for 10-20 minutes.
5. Rinse well with water.
6. Place into aniline-acetic for 30 minutes.
7. Wash well with running tap water.
8. Return to periodic acid for a further 20 minutes.
9. Rinse well with water.
10. Place into thiosemicarbazide for 10 minutes.
11. Wash well with running tap water to remove all traces of thiosemicarbazide.
12. Place into freshly made Schmorl's solution for 10 minutes.
13. Wash with running tap water.
14. Optionally, counterstain with nuclear fast red or lightly with eosin.
15. Rinse well with water.
16. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Fungi  –  blue
• Nuclei  –  red
• Background  –  pink

Notes

1. It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS from J. T. Baker (via the SIRI database) says:

   Flash Point: N/A
   Lower Explosive Limit: N/A
   Upper Explosive Limit: N/A
   Unusal Fire and Expl.rds: NONE IDENTIFIED

2. Thiosemicarbazide has the formula H₂NNHCSNH₂. The hydrazine group (H₂NNH-) combines with any aldehydes generated by periodic acid oxidation. The thiocarbamyl group (-CSNH₂) is a more powerful reducing agent than are aldehydes, and rapidly reduces ferricyanide to ferrocyanide, which immediately forms a prussian blue deposit at the site.
3. The mallory bleach lightens background staining and improves contrast. It may also produce some aldehyde which is removed in step 6.
4. The oxidation-aldehyde block-reoxidation sequence removes most oxidisable carbohydrate other than fungi, and improves contrast.
5. The Schmorl's solution is Lillie's modification.

 

Reference
Hayashi, I., Tome, Y. and Shimosato, Y., (1989)
Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
Stain Technology, v 64, p 185.

Lillie, R.D. and Glenner, G. G. (1957)
Journal of Histochemistry and cytochemistry. v 5, p. 311

Llewellyn, B. D., unpublished.