The chief Iron-Hæmatoxylin Stain is that devised by Heidenhain. Unfortunately the procedure involved is a long one, and various modifications have been made to obviate this disadvantage. Hæmatein may be used instead of ripened hæmatoxylin.
One efficacious modification of Heidenhain’s stain is that of Rosenbusch. The smear or tissue, after fixation, must be graded downwards through the alcohols to water. Mordant for one and a half hours in a 3 1/2 per cent. aqueous solution of ferric ammonium sulphate. Stain for about three minutes in 1 per cent. solution of ripe hæmatoxylin or hæmatein in absolute or 96 per cent. alcohol, to which a drop of saturated aqueous solution of lithium carbonate, sufficient to produce a wine-red colour, has been added. Differentiate under the microscope with a very dilute solution of the ferric ammonium sulphate. Wash, gradually dehydrate, clear and mount in balsam. It must be remarked that iron-hæmatoxylin is a regressive stain, hence great care must be exercised in differentiating with the iron alum.
Gentian Violet.—A 1 per cent. alcoholic solution of gentian violet, or of methyl violet, or of crystal violet, will be found useful for staining spirochætes.
Methyl Green.—This substance is considered to be a chromatin stain, for either fresh or perhaps recently fixed tissues. A concentrated aqueous solution contains about 1 per cent. of the stain. This should be added to a 1 per cent. solution of acetic acid. It may be used for demonstrating the nuclei of ciliates.
In conclusion it is essential to remember that the actual magnification of figures of Protozoa should be given, and not merely the combination of objective and ocular that has been used, for unless the tube-length and distance of the drawing board from the ocular be also given, it is not possible to compute the magnification from such information. Drawings should always be made with the aid of a camera lucida, drawing prism or other form of projection apparatus.
APPENDIX ON TREMATODA AND NEMATODA.
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