After the film is fixed the stain desired is dropped on, allowed to act for the proper time, which will depend on the stain and the preparation, washed in water, dried thoroughly and examined with the oil-immersion lens, without a cover. If it is desired to preserve the preparation it may then be mounted in balsam. This is not necessary, as they keep just as well, provided the immersion oil is removed. To do this, fold a piece of filter paper so that at least three thicknesses result. Lay this on the slide and press firmly several times, when the surplus oil will be taken up by the paper. Slides not mounted in balsam are more apt to become dusty than those that are. This is the only disadvantage.
Gram’s Method of Staining.—It has been ascertained that some bacteria contain a substance, possibly a protein, which forms a compound with gentian violet and iodine, which compound is insoluble in alcohol, and other bacteria do not contain this substance. Consequently when bacteria are stained by Gram’s method (given below), those that contain this chemical remain colored, while if it is not present the dye is washed out by the alcohol and the bacteria are colorless and may be stained by a contrast stain. The bacteria which stain by this method are said to “take Gram’s” or to be “Gram-positive,” while those that decolorize are called “Gram-negative.” The method is:
- 1. Prepare the film as above given.
- 2. Stain with fresh anilin gentian violet 1 minute.
- 3. Wash in tap water.
- 4. Cover with Gram’s solution 1 minute.
- 5. Wash in tap water.
- 6. Wash with 95 per cent. alcohol three times or until no more color comes out.
- 7. Dry and examine.
Gram’s solution is:
| I | 1 part |
| KI | 2 parts |
| H2O | 300 parts |
This method is excellent for differentiating Gram-positive and Gram-negative organisms on the same slide. First stain by this method and after washing with alcohol stain with a counter-stain, carbol-fuchsin diluted ten to fifteen times with water is excellent. The Gram-positive bacteria are violet and the Gram-negative are red.
It is also of great value in staining Gram-positive bacteria in tissues, but the sections should be stained about five minutes in the anilin gentian violet and be left about two minutes in the Gram’s solution. Sections are to be counter-stained in Bismarck brown, dilute eosin or safranin solutions and cleared in oil of bergamot, lavender or origanum and not in clove oil or carbol-xylol, as these latter dissolve out the dye from the bacteria.
Staining of Spores in the Rod.—Prepare the films as usual. Cover with carbol-fuchsin, using plenty of stain so that it will not dry on the slide; heat until vapor arises, not to boiling; cool until the stain becomes cloudy and heat again until the stain clears, and repeat once more; wash in tap water and then wash in 1 per cent. H2SO4 three times, dropping on plenty of acid, tilting and running this over the slide three times and then pour off and use fresh acid and repeat this once. Wash thoroughly in distilled water, then stain with Löffler’s blue one to three minutes. Wash, dry and examine. The spores should be bright red in a blue rod.
This method will give good results if care is taken to secure cultures of the right age. If the culture is too old the spores will all be free outside the rods, while if too young they will decolorize with the acid. For Bacillus subtilis and Bacillus anthracis, cultures on agar slants forty-eight hours in the 37° incubator are just right. For the spores of Clostridium tetani, the culture should be three days old, but may be as old as a week.
Staining of “Acid-fast” Bacilli.—Mycobacterium tuberculosis, Mycobacterium of Johne’s disease, “grass” and “butter bacilli,” Mycobacterium lepræ, Mycobacterium smegmatis.