Gabbet’s method:

  1. 1. Prepare the film as usual.
  2. 2. Stain with carbol-fuchsin as given above for spores.
  3. 3. Wash with tap water.
  4. 4. Decolorize and stain at the same time with Gabbet’s blue, two or three minutes.
  5. 5. Wash, dry and examine.

The sulphuric acid in Gabbet’s blue removes the carbol-fuchsin from everything except the “acid-fast” bacteria, which remain red, and the blue stains the decolorized bacteria and nuclei of any tissue cells present.

Ziehl-Neelson method:

  1. 1, 2, 3, as in Gabbet’s method.
  2. 4. Decolorize with 10 per cent. HCl until washing with water shows only a faint pink color left on slide.
  3. 5. Wash thoroughly.
  4. 6. Stain with Löffler’s
  5. blue one or two minutes.
  6. 7. Wash, dry and examine.

The results are the same as with Gabbet’s method.

Staining of Capsules.—Räbiger’s Method.—Films of the organism to show capsules should be freshly prepared, dried but not fixed. Material is usually obtained from milk or blood. A drop of the fluid is placed on the middle of a slide about one-fourth of the distance from one end. The narrow edge of another clean slide is placed in this drop and then drawn lengthwise across the slide with firm pressure. This gives a thin layer which is necessary if good results are to be expected. The preparation is covered with a freshly prepared saturated solution of gentian violet in formalin and this allowed to stain for 30 seconds. Then wash lightly, dry and examine. The organisms appear deeply violet and much larger than with ordinary stains and capsules are well stained and show well.

Welch’s Method.—Prepare films as in the above method. Cover with glacial acetic acid for 10 to 20 seconds. Wash off the acid with carbol-fuchsin. Wash the stain off with physiological normal salt solution (0.85 per cent.) until all surplus stain is removed. Dry and examine. Capsules and bacteria are red.

Staining of Flagella.—The rendering of flagella visible is considered one of the most difficult processes in staining. Experience of a number of years during which whole classes numbering from one hundred to three hundred students accomplish this result shows that it is no more difficult than many other staining processes. The essentials are: (1) clean slides, (2) young cultures on agar slopes, (3) freshly prepared mordant and stain which are kept free from precipitate, (4) gentle heating. The author’s students are furnished only stock materials and make their own cultures, mordants and stains.

The slides are cleaned with pumice in the usual way. An agar slope culture of the organism to be stained from six to twenty-four hours old is selected. A bit of the culture is removed and placed in a watch-glass of water. The bacteria are allowed to diffuse of themselves without stirring. After several minutes a loop of this water is removed and three streaks are made across the slide, one in the middle and one on each side of this about one-quarter of an inch from it. This gives well scattered bacteria in one of the three streaks at least and very little other material on the slide to cause precipitates