In this article we will discuss about the preparation of different stains used in microscopy.
1. Acetocarmine (Semi chon):
Reagents:
45% glacial acetic acid … 100 ml
Carmine powder … 10 g
Preparation:
Mix the two; heat up to boiling. Cool, filter and store for future use.
2. Borax Carmine:
Reagents:
Aqueous borax solution … 4%.
Carmine solution … 3%.
Alcohol … 70%
Preparation:
Prepare 2 to 3% carmine solution in 4% aqueous borax solution by boiling for 30 minutes. Allow to stand for 2 to 3 days with occasional stirring. Dilute with equal volume of 70% alcohol. Allow to stand and filter. Stained objects should be washed with alcohol.
3. Delafield’s Haematoxylin:
Reagents:
Saturated aqueous
ammonia solution … 400 ml
Haematoxylin … 4 g
90% ethyl alcohol … 25 ml
90% methyl alcohol … 100 ml
Glycerine … 10 ml
Preparation:
Dissolve 4 g haematoxylin in 25 ml 90% ethyl alcohol. Mix the solution with 400 ml ammonia solution. Leave the mixture exposed to air and light for 4 days.
Add 100 ml 90% methyl alcohol and 10 ml glycerine. Filter and leave to ripen for about 2 months in a bottle plugged with cotton wool.
4. Ehrlich’s Acid Haematoxylin:
Reagents:
Distilled water … 100 ml
Absolute methyl alcohol … 100 ml
Glycerine … 100 ml
Glacial acetic acid … 10 ml
Haematoxylin … 2 g
Alum (Aluminium sulphate) … 20 g
Preparation:
Dissolve 2 g haematoxylin in 100 ml alcohol. Add by steps glacial acetic acid 10 ml, water 100 ml and glycerine 100 ml with stirring. Add 20 g alum. Filter and leave the solution in sunlight for about 2 months to ripen in a bottle plugged with cotton wool.
5. Heidenhain’s Iron-Alum Haematoxylin:
Reagents:
Haematoxylin powder … 0-5 g
Distilled water … 100 ml
95% ethanol … 10 ml
Ferric ammonium sulphate … 4 g
Preparation:
Dissolve the haematoxylin powder in 10 ml 95% ethanol and put 100 ml distilled water. The stain is ready for use.
Separately dissolve 3 gm. Ferric ammonium sulphate in 100 ml distilled water. Thus 3% Iron alum mordant is produced.
Dissolve 1 g Iron alum in 100 ml distilled water in a separate vial for preparation of 1% iron alum solution.
Use:
This haematoxylin is used for staining protozoan parasites. The protozoans are mordented in 3% iron alum first, then stained with haematoxylin.
6. Eosin (Alcoholic):
Reagents:
90% alcohol … 100 ml
Eosin powder … 1 g
Preparation:
Dissolve 1 g eosin powder in 100 ml 90% alcohol. Stir and use the solution. It keeps for an indefinite period.
7. Eosin (Aqueous):
Reagents:
Eosin powder ….. 1 g
Distilled water ….. 100 ml
Preparation:
Dissolve the eosin powder in 100 ml distilled water.
8. Leishman’s Stain:
Reagents:
Leishman’s powder … 0.1 g
100% methyl alcohol … 100 ml
Preparation:
Dissolve 0.1 g Leishman’s powder (dye) in 100 ml absolute methyl alcohol at room temperature with occasional stirring.
9. Picro-Indigo Carmine:
Reagents:
I. Saturated solution of picric acid in 90% alcohol
II. Saturated solution of Indigo carmine in 70% alcohol
Preparation:
Mix one part of I with two parts of II.
10. Aceto-Carmine:
Reagents:
Dry carmine powder … 0-5-1 -0 g
Distilled water … 55 ml
Glacial acetic acid … 45 ml
Preparation:
Mix 45 ml glacial acetic acid and 55 ml distilled water and heat to boiling in a conical flask with cotton plugging. Add 0.5 to 1.0 g carmine powder. Shake well, cool and filter and store as stock solution.
11. Aceto-Orcein:
Reagents:
Orcein powder … 1 g
50% acetic acid … 100 ml
Preparation:
Dissolve orcein (1g) powder in 50% 100 ml boiling acetic acid with slow stirring. Cool rapidly, filter and store.
12. Methyl Violet 1% Solution:
Preparation:
Mix 1 g methyl violet powder with 100 ml of distilled water.
13. Safranin 1 % Solution:
Preparation:
Mix 1 gm Safranin powder with 100 ml distilled water.
14. Methylene Blue (Vital Stain):
Reagents:
Methylene blue powder … 1.5 g
Absolute ethyl alcohol … 100 ml
Preparation:
Dissolve the powder in 100 ml absolute alcohol and store.
15. Congo Red (Vital Stain):
Reagents
Congo red powder … 0.5 g
Distilled water … 100 ml
Preparation:
Stir the Congo red powder with water till the dye is dissolved. Filter out the larger suspended particles and keep as a stock solution.
16. Lugol’s Iodine:
Reagents:
Kl … 20 g
Distilled water … 200 ml
Iodine crystal … 10 g
Preparation:
Dissolve Kl in distilled water and add iodine crystal to it.
17. Gram’s Iodine Solution:
Reagents:
Iodine crystal … 1 g
Potassium iodide … 2 g
Distilled water … 300 ml
Preparation:
Dissolve Kl in distilled water and add iodine crystal to it.