uals may be found joined; generally the individuals are all separate from one another. The morphology of the bacillus is peculiar in its considerable irregularity, for among the well-formed individuals which abound in fresh cultures a large number of peculiar organisms are to be found, some much larger than normal, some with one end enlarged to a club-shape, some greatly elongated, with both ends expanded into club-shaped enlargements. These bizarre forms seem to represent an involution-form of the organism, for, while present in perfectly fresh cultures, they are so abundant in old cultures that scarcely a single well-formed bacillus can be found. It not infrequently happens that in unstained bacilli distinct granules can be defined at the ends-polar granules-thus giving the organism somewhat the appearance of a diplococcus.

The bacillus can be readily stained by aqueous solutions of the anilin colors, but more beautifully and characteristically with Löffler's alkaline methylene blue :

Saturated alcoholic solution of methylene blue, 30; I: 10,000 aqueous solution of caustic potash, 100; and an aqueous solution of dahlia, as recommended by Roux.

When cover-glass preparations are stained with these solutions, the bizarre forms already mentioned are much more obvious than in the unstained individuals, and the contrast between the polar granules, which color intensely, and the remainder of the bacillus, which tinges slightly, is marked. Through good lenses the organisms are always distinct bacilli, notwithstanding the fact that the ends stain more deeply than the centres, and it is only through poor lenses that the organisms can be mistaken for diplococci. The bacilli stain well by Gram's method, this being a good method to employ for their definition in sections of tissue, though Welch and Abbott assert that Weigert's fibrin method and picro-carmin give the most beautiful results.

The diphtheria bacillus does not form spores, and is delicate in its thermal range. Löffler found that it could not endure a temperature of 60° C., and Abbott has shown that a temperature of 58° C. for ten minutes is fatal to it. Notwithstanding this susceptibility, the organism can be kept alive for several weeks after being dried upon shreds of silk or when surrounded by dried diphtheria membrane.

No flagella have been demonstrated upon the bacillus. It is non-motile.

Fernbach has shown that when the organisms are grown in a medium exposed to a passing current of air, the luxuriance of their development is increased, though their life-cycle is shorter. The growth can also take place when the air is excluded, so that the bacillus must be classed among the optional anaërobic organisms.

The diphtheria bacillus grows readily upon all the ordinary media, and is a very easy organism to obtain in pure culture. Löffler has shown that the addition of a small amount of glucose to the culture-medium increases the rapidity of the growth, and suggests a special medium which bears his name-Löffler's bloodserum mixture:

Blood-serum,

3;

Ordinary bouillon + 1 per cent. of glucose, I.

This mixture is filled into tubes, coagulated, and sterilized like blood-serum, and is one of the best-known media in connection with the study of diphtheria.

The clinical impossibility of making an accurate diagnosis of diphtheria without a bacteriologic examination has made many private physicians and many medical societies and boards of health equip laboratories where accurate examinations can be made. The method requires some apparatus, though a competent bacteriologist can often make shift with a bake-oven, a wash-boiler, and other household furniture instead of the regular sterilizers and incubators, which are expensive.

When it is desired to make a bacteriologic diagnosis of a suspected case of diphtheria or to secure the bacillus in pure culture, a sterile platinum wire having a small loop at the end, or a swab made by wrapping a little cotton around the end of a piece of wire and carefully sterilizing in a test-tube, is introduced into the throat and touched to the false membrane, after which it is smeared carefully over the surface of at least three of the blood-serum-mixture tubes, without either again touching the throat or being sterilized. The tubes thus inoculated are stood away in an incubating oven at the temperature of 37° C. for twelve hours, then examined. If the diphtheria bacillus is present upon the first and second tubes, there will be a smeary yellowish-white layer, with outlying colonies on the second tube, while the third tube will show rather large isolated whitish or slightly yellowish colonies, smooth in appearance, but rather irregular in outline. Very often the colonies are chinawhite in appearance. These colonies, if found by microscopic examination to be made up of diphtheria bacilli, will confirm the diagnosis of diphtheria, and will at the same time give pure cultures when transplanted. There are very few other bacilli which grow so rapidly upon Löffler's mixture, and scarcely one other which is found in the throat.

Ohlmacher recommends the microscopic examination of the still invisible growth in five hours. A platinum loop is rubbed over the inoculated surface; the material secured is then mixed with distilled water, dried on a cover-glass, stained with methylene blue, and examined. This method, if reliable, will be very valuable in making an early diagnosis preparatory to the use of the antitoxin.

The presence of diphtheria bacilli in material taken from the throat does not necessarily prove the patient to be diseased. Virulent bacilli can often be discovered in the throats of healthy persons who have knowingly or unknowingly come in contact with the disease. The bacteriologic examination is only an adjunct to the

clinical diagnosis, and must never be taken as positive in itself.

The bacillus grows similarly upon blood-serum and Löffler's mixture. Upon glycerin agar-agar and agar-agar the colonies are much larger, more translucent, always

FIG. 64.-Diphtheria bacilli (from photographs taken by Prof. E. K. Dunham, Carnegie Laboratory, New York): a, pseudo-bacillus; b, true bacillus ; c, pseudo-bacillus.

without the yellowish-white or china-white color of the blood-serum cultures, and generally are distinctly divided into a small elevated centre and a flatter surrounding zone with indented edges, sometimes with a distinctly radiated appearance. It must be remarked that when sudden transplantations are made from blood-serum to agaragar the growth resulting is meagre, but the oftener this growth is transplanted to fresh agar-agar the more luxuriant it becomes.

The growth in gelatin puncture-cultures is characterized by small spherical colonies which develop along the entire length of the needle-track. The gelatin is not liquefied.

Upon the surface of gelatin plates the colonies that develop do not attain anything like the size of the colonies upon Löffler's mixture. They appear to the naked

FIG. 65.-Bacillus diphtheria, colony twenty-four hours old upon agar-agar; × 100 (Fränkel and Pfeiffer).

eye as whitish points with smooth contents and regular though sometimes indented borders. Under the microscope they appear as granular, yellowish-brown colonies with irregular borders (Fig. 65).

When planted in bouillon the organism causes a diffuse cloudiness at first, but, not being motile, soon settles to the bottom in the form of a rather flocculent precipitate which has a tendency to cling to the sides of the glass. Sometimes a delicate irregular mycoderma forms upon the surface, especially when the cultivation is made by the method of Fernbach with a passing current of air. This mycoderma, which may appear quite regular when the flask is undisturbed, is so brittle that it at once falls to pieces if the flask be moved.