rius, 0.3-0.5 per cent. of sodium formate, as suggested by Kitasato and Weil, 0.1 per cent. of sodium sulphate, suggested by the same authors, and various other chemicals. None of these additions has been sufficiently successful to merit continued favor, and at the present time this method is not employed.

5. Exclusion of Atmospheric Oxygen by Means of Various Physical Principles and Mechanical Devices.This has appealed to the ingenuity of many experimenters, and many means of accomplishing the atmospheric exclusion have been tried with success.

The most simple plan is that of Hesse, who made a deep puncture in recently boiled and rapidly cooled gelatin or agar-agar, then covered the surface of the medium with sterile oil (Fig. 69). The so-called "shake culture" is an

Fig. 68.-Buchner's method of Fig. 69.-Hesse's method of makmaking anaerobic cultures. ing anaerobic cultures.

other very simple method, suggested by Liborius and Hesse. The medium to be inoculated, contained in a well-filled tube or flask, is boiled to displace the contained air, cooled so as no longer to endanger the introduced bacteria, then inoculated, the inoculated bacteria being distributed by gently shaking. On cooling, the medium "sets," the organisms below the surface remaining under anaerobic conditions.

Kitasato first used paraffin as a covering for the inoculated medium, his recommendation having recently been revived and made successful use of by Park for the cultivation of the tetanus bacillus. The paraffin floats upon the surface of the medium, melts during sterilization, but does not mix with it, and "sets" when cool. The inoculation is to be made while the culture medium is warm, after boiling and before the paraffin sets.

Koch studied the colonies of anaerobic organisms by culti

Figs. 70, 71.-Wright's method of making anaerobic cultures in fluid media (Mallory and Wright).

70 and 71).

vating them upon a film of gelatin covered by a thin sheet of sterilized mica, by which the air was excluded.

Salomonsen has made use of a pipet for making anaerobic cultures. It is made of a glass tube a few millimeters in diameter, drawn out to a point at each end. The inoculated gelatin or agar-agar is drawn in while liquefied and the ends sealed. The tube, of course, contains no air, and perfect anaerobiosis results.

Theobald Smith has found the fermentation-tube and various modifications of it excellently well adapted to the growth of anaerobes, which, of course, grow only in the closed limb.

Hens' eggs have been used for anaerobic cultures, and in them. the tetanus bacillus grows remarkably well. Conditions of anaerobiosis are, however, not perfect, as can be shown by the behavior of the egg itself. If oxygen be completely shut out by oiling or varnishing the shell, a fertile egg will not develop.

A quite satisfactory and simple device for routine work with

anaërobic organisms has been invented by Wright* (Figs. The essential feature consists of a pipet, D, "Jour. Boston Soc. of Med. Sci.," Jan., 1900.

with a rubber tube, E, at the end, and one interruption connected by a rubber tube, C. The device will be made clear at once by a glance at the accompanying illustration. The method of employment is very simple. An ordinary tube of bouillon or other fluid culture media receives the pipet, the whole being sterilized, the cotton plug in place. The bouillon being inoculated with the culture or secretion to be studied is drawn up in the bulb of the pipet, A, by suction, until it passes the rubber interruption, C. By forcing the upper end of the pipet downward in the test-tube, a kink is given each rubber tube and the fluid contained in the bulbous part of the pipet becomes hermetically sealed.

In all cases where the presence of suspected microorganisms is to be demonstrated, it is necessary to make both aerobic and anaerobic cultures. For routine work of this kind, this method of Wright is probably the most convenient yet suggested.

CHAPTER X.

EXPERIMENTATION UPON ANIMALS.

THE principal objects of medical bacteriology are to discover the cause, explain the symptoms, and bring about the cure and future prevention of disease. We cannot hope to achieve these objects without experimentation upon animals, in whose bodies the effects of bacteria and their products can be studied.

No one should more heartily condemn wanton cruelty to animals than the physician. Indeed, it is hard to imagine men, so much of whose life is spent in relieving pain, and who know so much about pain, being guilty of the butchery and torture accredited to them by a few of the laity, whose eyes, but not whose brains, have looked over the pages of physiologic text-books, and whose "philanthropy has thereby been transformed to zoolatry."

It is entirely through experimentation upon animals that we have attained our knowledge of physiology, most of our important knowledge of therapeutics, and most of our knowledge of the infectious diseases. Without its aid we would still be without one of the greatest achievements of medicine, the serum therapy of diphtheria.

Experiments upon animals, therefore, must be made, and, as the lower animals differ in their susceptibility to diseases, large numbers and different kinds of animals must be employed.

The bacteriologic methods are fortunately not cruel, the principal modes of introducing bacteria into the body being by subcutaneous, intraperitoneal, and intravenous injection.

Any hypodermic syringe that can conveniently be cleaned and disinfected may be employed for the purpose. Forms expressly designed for bacteriologic work and most fre

quently employed are shown in figure 72. Those of Meyer and Roux resemble ordinary hypodermic syringes; that of

Fig. 72.

-1. Roux's bacteriologic syringe; 2, Koch's syringe; 3, Meyer's bacteriologic syringe.

Koch is supposed to possess the decided advantage of not having a piston to come into contact with the fluid to