Double Oxidation
Thiosemicarbazide, Schmorl
This method demonstrates fungi.
Materials
- Periodic acid solution
- Aniline-acetic
Material Amount Aniline 10 mL Acetic acid, glacial 90 mL - Thiosemicarbazide
Material Amount Thiosemicarbazide 1 g Distilled water 100 mL - Schmorl’s solution
Material Amount 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.
Protocol
- Bring sections to water via xylene and ethanol.
- Do a Mallory bleach.
- Wash with running tap water.
- Oxidize in periodic acid for 10-20 minutes.
- Rinse well with water.
- Place into aniline-acetic for 30 minutes.
- Wash well with running tap water.
- Return to periodic acid for a further 20 minutes.
- Rinse well with water.
- Place into thiosemicarbazide for 10 minutes.
- Wash well with running tap water to remove all traces of thiosemicarbazide.
- Place into freshly made Schmorl’s solution for 10 minutes.
- Wash with running tap water.
- Optionally, counterstain with nuclear fast red or lightly with eosin.
- Rinse well with water.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Fungi – blue
- Nuclei – red
- Background – pink
Notes
- It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide.
The MSDS from J. T. Baker says:Flash Point: N/A
Lower Explosive Limit: N/A
Upper Explosive Limit: N/A
Unusual Fire and Expl.rds: NONE IDENTIFIED - 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. - The mallory bleach lightens background staining and improves
contrast. It may also produce some aldehyde which is removed
in step 6. - The oxidation-aldehyde block-reoxidation sequence removes most
oxidizable carbohydrate other than fungi, and improves contrast. - The Schmorl’s solution is Lillie’s modification.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- 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., (2014)
Thiosemicarbazide-ferricyanide reduction for the histochemical demonstration of aldehydes in tissue sections.Biotechnic & Histochemistry, v 89, p 228-31.
