fever by direct inoculation of killed typhoid cultures. This method, however, does not come under the head of anti-bacterial serum, the protection being produced by active immunization. The antibacterial bodies are important in the diagnosis of certain diseases, and will be referred to subsequently.

Antitoxine. Antitoxic Sera.-The various steps in the production of antitoxine are as follows, and diphtheria may be taken to serve as the type of the general method adopted :

(1) Preparation of a powerful toxine by growing the organism elaborating the toxine under the most favourable conditions for the development of the poison. Filtration of the fluid containing the toxine through a porcelain filter (see fig. 30), the filtrate being termed " toxine."

(2) Estimation of the power of the toxine by inoculation of guineapigs to determine the minimal dose which will produce death.

Behring denotes as a normal poison a toxine solution of which 0.01 cc. is sufficient to kill a guinea-pig weighing 250 gm. in four days. Of this normal toxine (D.T.N.) 1 cc. will kill one hundred guinea-pigs. This is the toxine unit and has a working value of 25,000, a toxine ten times as strong is expressed thus D.T.N.10; one ten times weaker D.T.N.

10

(3) Production of an antitoxic serum in a susceptible animal (horse) by repeated toxine injections as described above (p. 72).

(4) Estimation of the antitoxic power of the serum by determining how much is required to protect against the normal diphtheria toxine unit (D.T.N.). If, for instance, the serum is found to contain enough antitoxine in 1 cc. to prevent a fatal result with the injection of 1 cc. of the toxine (D.T.N.), it is called a normal diphtheria antitoxine (D.A.N.). The amount of antitoxine that is required to protect 25,000 gm. weight of guinea pigs from the minimal fatal dose of the poison (D.T.N.), is termed an IMMUNITY UNIT (I.E.).

To cure a person ill with diphtheria 600 to 1,800 1.E. are required contained in 2-6 cc. of serum; therefore the strength of the serum used is D.A.N. 300.

Ehrlich has propounded the idea that the antitoxines are exactly identical with the portions of the proteid molecule to which the toxines become bound in the production of disease; the poisonsusceptible parts of the molecule (toxophoric) are termed "side

That is, 25,000 grammes weight of living guinea-pig.

chains (seitenketten). When a toxine is introduced in small quantities, some of the "side chains" unite with the toxine, are thrown off from the cell and at once replaced. This repair takes place more and more rapidly as the side chains are more frequently torn off by the toxine. Finally the side chains are produced in greater quantity than there is toxine to fix and they then appear in the serum as antitoxine.

The production of antitoxine is apparently associated with an increase in the white blood corpuscles.

For a summary of the present knowledge of antitoxine and the theories of immunity the reader is referred to the article by Prof. Ritchie, Journal of Hygiene, April, 1902, and to the summary by Armit in the British Medical Journal, April 2, 1902.

Antibacterial Sera. In certain diseases the production of immunity is associated with the production of bodies in the serum which possess a destructive action upon the bacteria themselves. These bodies may be shown to be present in various ways:— (1) Agglutination (Gruber and Durham, Widal).

(2) Bacteriolysis: Pfeiffer's reaction.

(3) Inhibition of growth (Wright).

FIG. 31.-WIDAL BLOOD PIPETTE FOR COLLECTING BLOOD FOR AGGLUTINATION REACTION.

(1) Agglutination.-Two or three drops of blood of the immunised animal are collected in a tube having a bulb in the centre and capillary ends (fig. 31); the blood is allowed to clot and the serum collected in the capillary end opposite to that by which the blood has been introduced. A cultivation of the organism in broth is taken and a drop of the diluted serum added, the reaction being carried out in a small test tube. If the organism be, say, B. pyocyaneus and the animal was immunised to B. pyocyaneus, a well marked precipitate soon occurs. If instead of the test tube the reaction is carried out on a hanging drop slide and watched under the microscope the motile organisms will be seen to lose their motility and become aggregated into clumps. This reaction is used in the diagnosis of typhoid fever. The serum obtained from the suspected case is tested in various dilutions-50 per cent., 5 per cent., and 0.5 per cent.— and the effect noted in half an hour. The dilutions are made by

diluting the serum with sterile broth by means of a capillary pipette, and then mixing equal loopfuls of typhoid culture and serum. Two pipettes are required: a 90 cmm. and a 10 cmm. dilutions are made as follows:

The

(1) 50 per cent.: one loopful of the broth culture is mixed with one loopful of the serum to be tested on the coverslip.

(2) 5 per cent.: 90 cmm. of sterile broth are mixed with 10 cmm. of the serum, and a loopful of the mixture of serum and broth added to a loopful of the broth culture on the coverslip.

(3) 0.5 per cent.: 90 cmm. of broth are added to 10 cmm. of the 10 per cent. solution; and equal loopfuls of this and the broth culture mixed on the coverslip.

By the above method the manipulation of the cultivation is limited to the platinum loop.

The culture used should be a twenty-four-hours-old broth cultivation, and a time limit of half an hour observed in estimating the reaction.

(2) Lysogenic Action. Pfeiffer's Reaction.-Bacteriolysis.

The culture of the organism to be tested is mixed with the immune serum, and the mixture injected into the peritoneal cavity of a healthy animal. Small quantities of fluid are removed from time to time for observation; the organisms are observed to become swollen, contorted and finally dissolved.

If an animal A be immunized to another's (B) red blood corpuscles by the injection of increasing doses of washed red discs, the serum of the injected animal (A) will eventually be found to cause laking (hæmolysis) of the red corpuscles of B when serum A is added to the blood of B in a test tube at body temperature.

(3) Inhibition of Growth. Wright's Method.-The cultivation of the organism to be tested is mixed with various dilutions of the serum and melted gelatin. The mixture is introduced into capillary tubes and the presence or absence of colonies noted after incubation. The immune serum prevents the development of the corresponding organism. Control experiments without serum addition should always be made.

We are now in a position to review the various theories of immunity that have been advanced from time to time to explain the foregoing phenomena.

Pasteur suggested the theory of exhaustion, by which he endeavoured to explain the production of immunity as due to the

using up by the infecting organisms of some portion of the tissues especially adapted to their growth; and that when this hypothetical proteid fragment was exhausted the organisms could no longer live or find substances fitted for their activity, and therefore gradually perished.

This supposition is entirely negatived by the production of passive immunity by the injection of antitoxic sera, which can hardly exhaust the tissues of the injected animal.

The Theory of Retention supposes that products inimical to the bacteria present are retained in the animal body, and that due to their presence the organisms die out, just as they do in an old cultivation in a test tube. Such a suggestion is, however, at variance with the main facts given above, and is also opposed to the fact that death of an animal can take place by an overdose of toxine, although its serum is antitoxic for other animals.

Phagocytosis. Metchnikoff, who first advanced this theory, which is particularly related to the question of natural immunity, supposes that certain cells of the blood, termed phagocytes, especially function as destroyers of bacteria. Of these cells two main varieties are present in the human subject: (a) polymorphonuclear leucocytes or microphages, and (b) the larger varieties of connective tissue cells endowed with amoeboid movement (macrophages). These cells are endowed with the power of ingesting foreign bodies, retaining them in their protoplasm until they are either wholly or partially digested, when if the phagocyte retains its vitality the remains are extruded. Such a phenomenon can be directly observed by keeping amoeboid cells of this class upon a warm slide in the presence of bacteria. The bacteria may be watched during the whole process of engulfing, &c., and are to be seen within the plasm of the cells. The phagocytes will be seen to move in the direction of the organisms, impelled probably by the secretions of the bacteria; at other times the cells recede as if to withdraw beyond the influence of a too powerful poison; these movements are severally known as positive and negative chemiotaxis.

Metchnikoff observed that in a susceptible animal these phagocytic movements were slower, more ill-defined, or were absent, while in an animal possessed of a high degree of immunity the cells were relatively more active and ingested the bacteria with apparent ease and avidity; moreover, he also observed that the cells of a susceptible animal acquired greater power of dealing with

bacteria by this process of phagocytosis in proportion to the degree of immunity conferred by artificial means, in fact as immunity increased so did the phagocytic power of the cells. Natural immunity, therefore, would be due under this conception to a relatively robust and active state of the phagocytes, while susceptibility would result from a slow and indifferent phagocytic power. The phagocytes have, as it were, become more and more educated in the hunting of bacteria.

It is, however, difficult to entirely reconcile the facts given above with the production of immunity by the injection of toxines without the bacterial bodies themselves. That a large number of bacteria are destroyed by the phagocytes is undoubted, and, moreover, in certain diseases in which bacteria are present in the blood a marked phagocytosis is developed, but the presence of such an extra development of white blood cells appears to be more in the nature of a concomitant phenomenon than the means by which immunity is effected.

Natural Immunity.-We have so far considered the question of immunity from the standpoint of the pathological phenomena concerned in the resistance and susceptibility to various diseaseproducing bacteria. The number of pathogenic bacteria is, however, small when compared with the numerous species which exist as saprophytes, and which do not, even when introduced into animals, produce symptoms of poisoning.

But animals are not always susceptible to even pathogenic organisms, for instance, the common fowl is highly resistant to tetanus, the common mouse is immune to tubercle, while the field mouse is susceptible, the guinea-pig is resistant to the pneumococcus, while the rabbit is peculiarly susceptible, and so on.

In some cases it has been shown that the animal naturally immune has some antitoxic power in its serum, and that the blood is also antibacterial, but this is only in a few cases and is not sufficiently generalised to account for the immunity possessed by many animals.

Animals, therefore, possess a natural immunity to certain diseases which may be due (a) to the power of animal tissues generally to destroy bacteria (b) to the ease with which the toxines of the infecting organisms are neutralised in the body. Both of these processes may be in operation at the same time and by appropriate means one or other may be so lowered by artificial