4. Wash in water; dehydrate in absolute alcohol; clear in xylol; mount in xylol-balsam.
Nuclei of the amœbæ and granules of the mast-cells are brownish-red; nuclei of cells blue.
2. Trichomonas vaginalis and intestinalis; Cercomonas coli; Megastoma entericum; Balantidium coli; Pyrosoma bigeminum; Trypanosoma; Leishman-Donovan bodies, and allied forms are best stained with the modifications of Romanowsky’s stain; the chromatin is red-violet (macro-nucleus red, micro-nucleus black, flagellum red, protoplasm blue, basophilic granules black). For staining in sections mercuric chloride or Zenker’s fixation followed by staining with polychrome methylene-blue, Giemsa’s or the modifications of the Romanowsky method may be employed.
3. Plasmodium Malariae. For films make medium smears (not too thin); fix with equal parts of absolute alcohol and ether for ½-1 hour; or fix and stain in the same solution (Leishman-Romanowsky, Wright’s stain, etc.). For single staining methylene-blue, carbol-thionin, etc., may be employed; for double staining eosin and methylene-blue. Ehrlich’s tri-acid, or any of the eosin-methylene-blue combinations may be used (particularly the Leishman-Romanowsky or Wright’s). With the Romanowsky methods the body of malarial organism is stained blue, the chromatin varying shades of lilac, red, purplish-red or almost black. When the blood contains but few parasites 1 cc. may be drawn, mixed with 20 cc. of distilled water and centrifugated. Smears are then made of the sediment. For the staining of the plasmodium in imbedded tissues the following method is recommended by Bignami. The tissue should be fixed in formol or mercuric chloride, preferably a mixture of mercuric chloride 1 grm., sodium chloride 0.75 grm., acetic acid 0.75 grm., water 200 cc. Fix for 2 hours; after-harden in alcohol and iodine-alcohol, changing the alcohol each day for seven days. Dehydrate in absolute alcohol, and imbed in celloidin or paraffin. Stain in a saturated watery solution of magenta or in a mixture of equal parts of saturated alcoholic mixtures of magenta and orange G. Good results may, however, be obtained with Löffler’s methylene-blue. Clear in xylol; mount in balsam.
4. Coccidia and Sarcosporidia. The ordinary fixations give good results. Imbed in paraffin or celloidin. Weigert’s iron-hæmatoxylin and Van Gieson’s give as good pictures as any of the special methods advised.
5. Negri Bodies of Rabies. Examine in smears or make sections. Take portions of gray brain-substance from the cortex in the region of the fissure of Rolando (in the dog from around the crucial sulcus), from the hippocampus, and from the cerebellum. Smear cover or slide by taking a thin slice of the gray matter and compressing it between two slides, or cover and slide, or by drawing the cover across the cut surface in order to get some of the cells. Dry in the air; stain with watery methylene-blue; wash; stain with watery acid fuchsin; wash in water; blot dry; mount in balsam. Negri bodies fuchsin-red (about size of red blood cells); everything else blue. When dried in the air and then fixed in methyl alcohol for 5 minutes the smears may be stained by Giemsa’s method. For the demonstration of the bodies in sections fix in Zenker’s, imbed in paraffin, and stain by the eosin-methylene-blue method. The bodies take the eosin stain. Formol fixation, freezing-microtome and Romanowsky stain give quick results. Mann’s method for the staining of Negri bodies in sections is strongly recommended by many workers. Fix material in mercuric chloride or Mann’s fluid (1 grm. picric acid and 2 grms. tannin dissolved in 100 cc. concentrated water solution of mercuric chloride) for 24 hours; wash thoroughly in running water; imbed in paraffin. Stain in Mann’s mixture (1 per cent aqueous methyl-blue [not methylene-blue] 35 parts, 1 per cent aqueous eosin 35 parts) for 24 hours; wash in water; rinse in absolute alcohol; place in alkaline alcohol (absolute alcohol 50 cc., 4 drops of a 1 per cent solution of sodium hydroxide) for 15-20 seconds until sections become reddish; wash quickly in alcohol; wash about 2 minutes in water until superfluous color is removed; place in weak acidulated water (acetic acid) 1-2 minutes until sections are blue; quick dehydration in alcohol; xylol; balsam. Cells are blue, nucleoli and blood-vessels red; Negri bodies bright red. For quick diagnosis use acetone fixation and imbedding, stain in Mann’s fluid 2-4 minutes, and proceed as in the Mann’s method. While these bodies possess a great diagnostic importance for rabies, their exact nature must still be regarded as unsettled; they are most probably not parasites.
6. Vaccine Bodies. Fix in Flemming’s, mercuric chloride or Zenker’s; imbed in celloidin or paraffin. Stain with Heidenhain’s iron-hæmatoxylin (bodies black) or Biondi-Heidenhain mixture (bodies blue, nuclei of leukocytes and mitoses green, nuclei of epithelium and connective-tissue blue, protoplasm and connective-tissue red). These bodies are probably not parasites, but may be products of cell-degeneration.
7. Vermes. The heads, proglottides and ova are best examined in the fresh state, in physiologic saline or glycerin. Acetic acid may be used to bring out details. Berlin-blue or methylene-blue may be injected through the genital pore for the demonstration of the excretory and genital organs. Scolices and hooklets of echinococcus may be obtained by scraping the cyst-wall; examine in glycerin. Permanent preparations of cestodes, nematodes and trematodes may be made by fixing in mercuric chloride, formol or Flemming’s, after-hardening in alcohol, staining in orange G, borax carmine, alum hæmatoxylin, hæmatoxylin and eosin etc., mounting in glycerin gelatin; or dehydrating, clearing in xylol and mounting in balsam. For sections imbed in paraffin or celloidin. Trichinæ may be studied by teasing the fresh muscle; by digesting with pepsin and hydrochloric acid and examining the freed trichinæ on a warm stage; or by imbedding in paraffin or celloidin and staining with hæmatoxylin and eosin. Permanent mounts of the embryos of filaria may be made by fixing cover-glass preparations of blood or chylous fluid by heat or mercuric chloride, and staining for a few seconds with Löffler’s or a 2 per cent aqueous thionin.
CHAPTER XXVIII.
THE STAINING OF SPECIAL ORGANS AND TISSUES.
I. BLOOD AND BLOOD-FORMING ORGANS.