The material for culture is very conveniently obtained by means of sterilized cotton swabs. In collecting this material the swab is removed from its test-tube and touched to the affected areas of the mucous membrane of either the nose or throat. It is then to be gently rubbed over the surface of a blood-serum culture-tube, or it may be replaced in the testtube and the inoculation of the culture-tube made later in the laboratory. In the latter case the inoculation should be made within an hour or two after the material has been collected, the infected swab meanwhile being prevented from drying by firmly replacing the cotton plug.

In cases with membrane-formation the greatest number of bacilli are on the surface or in the upper layer of the membrane, and the swab should therefore be touched to these portions rather than to the tissue beneath.

Special Methods of Staining the Bacillus Diphtheria.Owing to the fact that the bacillus diphtheria may be recognized by its peculiar morphology and characteristic staining in coverglass preparations from its growth upon certain culture-media, as already pointed out, various special staining methods have been devised for accentuating and rendering more striking to the eye the peculiar deeply stained points and granules in the bodies of the individual bacilli, which have been referred to as of great importance in the identification of the organism.

These special methods of staining are said to be of great advantage in cases where only a few specific bacilli may be suspected to be present among a large number of other bacteria.

Neisser's Method.-1. Stain for one to three seconds in a solution which is made as follows: 1 gram of methylene-blue (Gruebler), in powder, is dissolved in 20 c.c. of 96 per cent. alcohol. To this add 950 c.c. of distilled water and 50 c.c. of glacial acetic acid, and filter.

2. Wash in water.

3. Stain for three to five seconds in a solution of vesuvin (Bismarck brown), made by dissolving 2 grams of the dye (in powder) in 1000 c.c. of boiling distilled water.

4. Wash in water, and mount.

The diphtheria bacilli stained by this method appear as pale brown rods bearing bluish-black granules, usually of oval shape and of a diameter somewhat greater than the rod. The majority of the bacilli show a granule at each end or at only one end, but not rarely three granules are present, one being near the middle of the rod. More granules than these are exceptional (see Fig. 50). The bacilli must have been grown on Löffler's blood-s -serum

medium, coagulated at 100° C., and the culture must be at least nine hours and not more than twenty-four hours old.

Hunt's Method.-1. Stain in saturated aqueous solution of methylene-blue one minute without heating.

2. Wash in water.

3. Cover with aqueous solution of tannic acid, 10 per cent., ten seconds.

4. Wash in water.

for

5. Stain in saturated aqueous solution of methyl-orange one minute, without heating.

6. Wash in water.

7. Dry, and mount in balsam.

FIG. 50,-Diphtheria bacilli from blood-serum culture stained according to Neisser's method; X 2000 (Wright and Brown).

By this method the granules, etc., are dark blue or almost black, and stand out very sharply against the light-green coloring of the body of the bacillus (see Fig. 51). The solution of methyl-orange should be freshly prepared, for it deteriorates in a few days.

Bacillus of Typhoid Fever.'-Synonyms: Bacillus typhi abdominalis; Bacillus typhosus; Typhoid bacillus.

1 Eberth: Virchow's Arch. f. Path. Anat., Bd. 81, 1880; Bd. 83, 1881; Gaffky: Mitth. a. d. Kais. Gesundheitsamte, Bd. 2, 1884.

Blood-serum.-Round, grayish, viscid-looking colonies, which may attain a diameter of 2 mm. after forty-eight hours in the incubator.

Morphology.-Medium-sized bacilli with rounded ends, generally short (Fig. 52), but sometimes long or thread-like,

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FIG. 52.-Typhoid bacilli from a bouillon culture, showing characteristic irregularity in staining and variability in length; X 2000 (Wright and Brown).

and frequently showing faintly-stained, sharply-defined areas in their protoplasm (Figs. 52 and 53).

Gelatin Slant.-Broad translucent streak with wavy, irregular margins. The gelatin is not liquefied. Growth is

FIG. 53-Typhoid bacilli from a culture on potato, showing unstained areas in the bacilli and polar granules; X 2000 (Wright and Brown).

slower than that of the bacillus coli communis in the same medium.

An isolated colony, slightly magnified, on gelatin, is shown in Fig. 54.

FIG. 54-Bacillus of typhoid fever: superficial colony two days old, as seen upon the surface of a gelatin plate; X 20 (Heim).

Glucose-gelatin Stab.-Growth all along the line of inoculation in the form of confluent spherical grayish colonies, and on the surface about the point of entrance of the platinum

wire in the form of a circular translucent grayish layer. No production of gas-bubbles. No liquefaction.

Glucose Agar-agar.-Growth similar to that in the preceding. No gas-formation.

Litmus-milk.-No visible change.
Potato.-Growth occurs, but it is usually invisible.

FIG. 55.-Typhoid bacilli, from a culture on agar-agar, showing flagella, from a preparation stained by Dr. Hugh Williams; X 2000 (Wright and Brown).

Dunham's Peptone Solution.-No indol-production-i. e. no red color appearing in the twenty-four- to forty-eighthour cultures after the addition of 5 drops of concentrated sulphuric acid, c. p., and I cubic centimeter of a solution of sodium nitrite, I: 10,000.

Motility-Very marked.