Haidenhain's Iron-Haematoxylin - Using Spirogyra as a type
Proceed as follows:
1. Fix 24 hours in chromo-acetic acid. 1 per cent chromic acid........................100 c.c. Glacial acetic acid..............................3 c.c.
The volume of the fixing agent should be at least 50 times that of the material to be fixed.
2. Wash in running water 10 hours; if running water is not available, 24 hours, changing often. 3. 2 per cent aqueous solution of iron-alum (ammonia sulphate of iron), 2 hours. 4. Wash in running water, 20 minutes. If running water is not available, wash in a large quantity of water and change frequently. 5. Stain over night, or 24 hours, in 1 per cent aqueous solution haematoxylin. 6. Wash in water, 20 minutes. 7. 2 per cent aqueous solution of iron-alum, until the stain is satisfactory. This can be determined only by examining frequently under the microscope. 8. Wash in water, 2 hours. If this washing is not thorough, the continued action of the iron-alum will cause the preparations to fade. 9. Transfer to 10 per cent glycerin, and allow the glycerin to concentrate until it has the consistency of pure glycerin. It is not necessary to use an exsiccator. Merely put the glycerin into shallow dishes, and leave it exposed to the air, but protected from dust. If the material is in Petri dishes or other dishes with a large surface, 3 or 4 days will be sufficient. This process may be hastened by warming, if the temperature does not go above 35 C. If the reduction from 10 per cent glycerin to pure glycerin is accomplished in less than 24 hours, the change in the concentration is so rapid that material is likely to suffer. 10. Wash out the glycerin with 95 per cent alcohol. It will be necessary to change the alcohol several times. From 10 to 20 minutes will be sufficient if the alcohol is changed frequently. This alcohol cannot be used again for the same purpose, but it will be useful in cleaning one's hands and in cleaning dishes which have contained Venetian turpentine. 11. Complete the dehydration in 100 per cent alcohol: 10 minutes should be sufficient. 12. Most failures are now ready to occur.
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