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STM 312: STAINING


Staining is the addition of colour or colouring agent to a material or microbial culture to make them more visible.

E.g of stains include
 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.

LECTURER IN CHARGE: MRS ALBERT

 BLOGGER: HOC TEMIYEMI

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