Bacillus Diphtheriae is a non-motile, non-liquefying, aerobic organism from 0.4 to 1.0 u broad, by 1.5 to 3.5 u long, slightly curved and with clubbed ends. Is found in the pseudomembranes of those suffering from diphtheria. It is peculiar in that in a pure culture there will be found individuals differing greatly in size and shape. These probably represent involution forms, as they are found in greatest numbers in old cultures.

Stain

Ordinary methods, but particularly Loeffler's methyl-ene-blue. Stains by Gram's. The ends take the stain more deeply than the middle.

Culture. Ordinary Media

Is obtained very easily in pure culture. The best medium is Loeffler's blood-serum mixture. On it there appears a smooth, smeary, yellowish-white layer at the end of about twelve hours when grown at a temperature of 370 C. To make the diagnosis a swab of absorbent cotton is brought in contact with the suspected surface and the tube is then inoculated directly. A diagnosis can be made at the end of five hours. On gelatin the colonies appear as white points. On bouillon a distinct whitish pellicle forms on the surface. Also on agar, milk, and potato.

Fig. 85. - Bacillus of Tetanus with Spores (Fränkel and Pfeiffer).

Fig. 86. - Bacillus DiphtheriAe, Five Hours at 360 C.

This shows only solid staining forms.

Fig. 87. - Bacillus DiphtheriAe, Same Culture, Eight Hours at 360 C.

This also shows solid forms, many of them with parallel arrangement.

Fig. 88. - Bacillus DiphtheriAe, Same Culture, Twelve Hours at 360 C.

The bacilli stain faintly at their ends, and in some small granules are seen at the tip of the faintly stained portions.

Fig. 89. - Bacillus Diph-therle, Same Culture, Fifteen Hours at 360 C.

The bacilli stain more unevenly and the granules are larger.

(Photomicrographs by Mr. Louis Brown. The magnification is the same in all - X 2000. All of the preparations were made from growth on blood-serum.) (Francis P. Denny, in "Jour, of Med. Research.")

Pathogenesis

When introduced into the individual it causes on mucous membranes the formation of a pseudo-membrane, composed chiefly of fibrin but containing desquamated epithelium and Bacillus diphtheriae. Is generally associated with both staphylococci and streptococci, giving rise to a mixed infection. Besides the local lesion there is a marked and serious intoxication, resulting from the absorption of poisonous metabolic products.

Preparation Of The Diphtheria Antitoxin

Virulent diphtheria bacilli are grown in alkaline bouillon containing 2 per cent, peptone for a period of three or four weeks at a temperature of 370 C. The culture is then heated for two hours at 650 C. and passed through a Chamberland filter. In this fluid there is the toxin. It is kept in sterile bottles in the dark.

Fig. 90 - Bacillus Diphtheria, Same Culture as Figs. 87-90, Twenty-four Hours at 36° C.

This shows clubbed and barred forms as well as granular forms. At the lower part of the field is a paired form which shows the characteristic clubbing of the distal ends.

Fig. 91. - Bacillus Diphtheria, Forty-eight Hours at 36° C.

This is the same bacillus as in Figs. 86-90, but from a culture where the colonies were discrete. It shows long filamentous forms.

(Photomicrographs by Mr. Louis Brown. The magnification is the same in all - X 2000. All of the preparations were made from growth on blood-serum.) (Francis P. Denny, in "jour, of Med. Research.")

For the purpose of immunization the horse is the best animal, as it furnishes a greater amount of antitoxic serum in less time than when a smaller animal is used. The horse is injected hypodermically with 0.1 c.c. of the toxic nitrate. In the course of six days a larger dose is given; this is repeated about every six days till from 500 to 1000 c.c. can be given without ill effects. When the degree of immunity is high the blood is withdrawn from a vein and collected in sterile bottles. These are placed on ice for about four days and the clear serum is drawn off from the coagulated blood. This serum is the antitoxin. It is preserved by the addition of 1:1000 formaldehyde, phenol, trikresol, etc.

The strength of the serum is designated by the term "immunizing units." According to Ehrlich and Behring, the "normal serum" is so strong that 0.1 c.c. of it would protect against ten times the least surely fatal dose of toxin when simultaneously injected into a guinea-pig. At present the "immunizing unit" is considered as containing ten times the least amount of antitoxin that will protect a 300-gram guinea-pig against the action of ten times the minimum fatal dose of the toxin.

To determine the strength of any given serum the minimum fatal dose of a sterile toxin for a 300-gram guinea-pig must be ascertained. Then determine the least quantity of the antitoxic serum that will protect a guinea-pig against ten times the minimum fatal dose of the toxin. The necessary dose of antitoxic serum is expressed as a fraction of a cubic centimeter and multiplied by ten, the result equaling one unit.

The value of the antitoxin depends upon its use in the early stages, before the third day. At the outset 4000 units should be given. In later stages 8000 to 10,000 should be given every four or six hours until the characteristic effect is produced. Except in very young children, the age should not affect the dosage. Smaller doses may be used as a prophylactic in those who have been exposed to diphtheria.

In severe cases more than one injection may be necessary. Amelioration of the local and general symptoms indicates the favorable action of the serum.