Ehrlich's Method. "Five parts of aniline oil are shaken up with 100 parts distilled water, and the emulsion filtered through moistened filter paper. A saturated alcoholic solution of fuchsine, methyl-violet, or gentian-violet is added to the filtrate in a watch-glass, drop by drop, until precipitation commences. Cover-glass preparations are floated in this mixture for fifteen to thirty minutes, then washed for a few seconds in dilute nitric acid (one part nitric acid to two of water), and then rinsed in distilled water. The stain is removed from everything except the bacilli; but the ground substance can be after-stained brown if the bacilli are violet, or blue if they have been stained red" (Crookshank, Bacteriology and Infective Diseases, p. 89).
Gram's Method. The primary stain in this method is a solution of aniline gentian-violet (saturated alcoholic solution of gentian-violet 30 cc., aniline water 100 cc.), which stains both ground substance and bacteria in purple. The preparation is next immersed in the following solution for half a minute or a little more:
| Iodine | 1 part |
| Potassium iodide | 2 parts |
| Distilled water | 300 parts |
In this short space of time the iodine solution acts as a mordant of the purple colour in the bacteria, but not in the ground substance. Hence, if the preparation be now (when it has assumed a brown colour) washed in alcohol (methylated spirit), the ground substance slowly loses its colour and becomes clear. But the bacteria retain their colour, and thus stand out in a well-defined manner. Cover-glass preparations decolourise more quickly than sections of hardened tissue, and they should only be left in the methylated spirit until no more colour comes away. The preparation may now be washed in water, dried, and mounted for microscopic examination, or it may be double-stained, that is, immersed in some contrast colour which will lightly stain the ground substance. Eosin or Bismarck brown are commonly used for this purpose. The former is applied for a minute or two, the latter for five minutes, after which the specimen is passed through methylated spirit (and preferably xylol also) and mounted. The result is that the bacteria appear in a dark purple colour on a background of faint pink or brown. Carbol-thionine blue, picro-carmine, and other stains are occasionally used in place of the aniline gentian-violet, and there are other slight modifications of the method.
Ziehl-Neelsen Method. Here the primary stain is a solution of carbol-fuchsin:
| Fuchsin | 1 part |
| Absolute alcohol | 10 parts |
| 5 per cent. aqueous solution of carbolic acid | 100 parts |
It is best to heat the dye in a sand-bath, in order to distribute the heat evenly. The various stages in the staining process are as follows: (a) The cover-glass with the dried film upon it is immersed in the hot stain for one to three minutes. (b) Remove the cover-glass from the carbol-fuchsin, and place it in a capsule containing a 25 per cent. solution of sulphuric acid to decolourise it. Here its redness is changed into a slate-grey colour. (c) Wash in water, and alternately in the acid and water, until it is of a faint pink colour. (d) Now place the cover-glass for a minute or two in a saturated aqueous solution of methylene-blue, which will counter-stain the decolourised ground substance blue. (e) Wash in water. (f) Dehydrate by rinsing in methylated spirit, dry, and mount. A pure culture of bacteria will not necessarily require the counter-stain (methylene-blue). Sections of tissue may require twenty to thirty minutes in the primary stain (carbol-fuchsin). This stain is used for tubercle and leprosy. With a little practice the staining of the bacillus of tubercle when present in pus or sputum becomes a very simple and accurate method of diagnosis. A small particle of sputum or pus is placed between two clean cover-glasses and thus pressed between the thumb and finger into a thin film. This is readily dried and stained as above, the bacillus of tubercle appearing as a delicately-beaded red rod with a background of blue.
Bacteriological Diagnosis. The following points must be ascertained in order to identify any particular micro-organism:
(1) Its morphology, bacillus, coccus, spirillum, etc.; the presence or absence of involution forms.
(2) Motility by the unstained cover-glass preparation ("hanging drop"); note presence of flagella.