also, it may be successfully demonstrated in the blood (blood from the heart, veins, rose-spots); but, to be sure, it is so often not found that the importance of the blood examination for substantiating the clinical diagnosis is not great. Kühnau found the typhoid bacillus in the blood from the veins of the arm in 10 out of 41 cases (Z. H. xxv, 492). Neufeld obtained very good results in the blood from rose-spots by making use especially of fluid nutrient media (Z. H. xxx, 498). It has also been very frequently demonstrated in the kidney, liver, and bile (Chiari), and especially in the urine (H. Neumann, C. B. VIII, 80). Regarding the occasional presence of enormous numbers of typhoid bacilli in urine, see Petruschky (C. B. XXIII, 577). Statements regarding successful cultivation from typhoid stools are comparatively rare (compare p. 237). The typhoid bacterium can itself cause the various complications in the clinical picture of typhoid. It has been demonstrated with certainty as the only cause of cases of serous and suppurative inflammations of the spinal cord, of the brain and its membranes, of the lungs and kidneys, and in the erysipelatous, phlegmonous, and suppurative processes of typhoids (in bones, skin, testicle, lymph-glands, parotid, thyroid, spleen, etc.). The pyogenic function of the B. typhi is no more contested, and has also been demonstrated by experiments upon rabbits. However, in many (the majority?) cases mixed infection with Micrococcus pyogenes, Streptococcus pyogenes or lanceolatus, etc., must be held responsible for the complications.

Results of Experiments Regarding Pathogenic Action in Animals.-According to the fairly universal view in Germany at present, the production of an infectious disease, analogous to typhoid fever in man, has never been successfully accomplished in any animal by any mode of infection. As a rule, bacteria introduced subcutaneously rapidly die, at least, do not multiply, and the injuries resulting may be produced in a similar manner by filtered cultures. Thus, the results are due to intoxication and not to infection. (Sirotinin, Z. H. 1, 465.) Also Petruschky favors the idea of an intoxication rather than an infection (Z. H. xii, 261).

Chantemesse and Vidal (A. P., 1892, 755) and Sanarelli (A. P., 1892, 721) were able, on the contrary, to so increase the virulence by all sorts of artificial means that they obtained varieties which are truly pathogenic for animals. Chantemesse (H. R., 1897, 1103) was even able to produce sickness in rabbits and monkeys by highly virulent cultures introduced into the stomach, and the animals died with typhoid symptoms (clinical and anatomical). Thus the Bact. typhi becomes acclimated to the animal body.

Special Methods for the Demonstration of the Bact. typhi.

It is usually easy to cultivate them from the spleen and lymph-glands of a fresh typhoid cadaver; still, not infrequently more colonies of the Bact. coli are obtained than typhoid. The case is different when the bacteria are to be sought for in water, feces, etc. The fact that the demonstration of the Bacterium typhi when in mixtures with other bacteria appears to be very difficult for all investigators has led to numerous suggestions to replace the simple gelatin plate method by better ones. A great distrust is aroused against all of these suggestions, since every new author criticizes the suggestions of his predecessor and usually discards them.

1

The two principal methods which have been employed

are:

1. Preliminary Culture.-The suspicious water is placed in nutrient media which contains an antiseptic, and kept twenty-four to forty-eight hours in the incubator. Water bacteria, especially a number of liquefying varieties, die, while the Bact. typhi and coli, which are more resistant to disinfectant agents, multiply in the incubator. Unfortunately, the rapidly growing forms of Bact. coli, besides Bact. vulgare, streptococci, and oïdium, multiply more intensely than the Bact. typhi, and when plates are prepared

1 An idea of the difficulty is given by the fact that many authors were not able at all to isolate typhoid bacteria from typhoid stools, and that Nicolle, Grimbert, and Chantemesse declare it to be impossible to recover typhoid bacteria from water, containing abundant Bact. coli, to which they had been added.

from the preliminary culture, almost with absolute certainty many coli forms are obtained, but also, according to most of the critical writers, much fewer typhoid bacteria than were in the original fluid (Lösener).

2. The direct preparation of plates from gelatin which contains materials interfering with growth: phenol, hydrochloric acid, methyl violet, potato juice, etc. Lösener, who has tested all these methods, recommends the following as the only useful one: Plates are prepared directly upon gelatin containing 0.03 to 0.05% phenol. The plates are best prepared, according to Kruse, by inoculation upon the surface (Tech. Appendix). Upon this carbol-gelatin the colonies of Bact. typhi and coli grow in the usual manner; many others, especially liquefying varieties, are, on the contrary, greatly retarded. From all colonies resembling typhoid inoculations are made into liquefied 2% grape-sugar agar (about a dozen tubes) and the shake cultures thus prepared are placed in the incubator. The tubes in which there is no fermentation are studied further, as indicated on page 239.

Almost simultaneously with Lösener, Elsner studied, in Koch's Institute, the methods for the ready demonstration of typhoid bacteria by means of special nutrient media, and instead of the potato-gelatin of Holz,1 which had given unsatisfactory results in the hands of many writers, he recommended a new feebly acid potato-gelatin containing 1% iodid of potassium. (See Tech. Appendix.) (Z. H. XXI, 25.)

According to Elsner, scarcely any bacteria except Bact. typhi and coli grow upon his nutrient medium, the liquefying varieties not at all. Bact. coli grows very well, and after twenty-four hours presents already perfectly developed colonies.

In contrast to this, the Bact. typhi grows very slowly; after twenty-four hours the colonies are scarcely visible with low magnification, and after forty-eight hours they appear as small, clearly shining colonies, like water drop

1 According to Holz, if carbolic acid is added to potato-gelatin, even the typhoid bacteria grow in a non-characteristic manner; if the addition is omitted, then very many liquefying germs are not at all disturbed in their growth.

lets or exceedingly finely granular, contrasting with the large, markedly granular, brown-colored colonies of the Bact. coli.

The method is said to give very good results, and usually allows of the isolation of the typhoid bacterium from stools, and the results are said to be most perfectly in harmony with Pfeiffer's typhoid reaction (see below). Compare also Jemma (Münch. med. Woch., 1897, No. 33) and Sterling (C. B. xxII, 334).

Special Differential Diagnosis of the Bact, typhi, Especially from the Bact. coli.

The following peculiarities must all be demonstrated: 1. Rods, short to thread forms; active motility; abundant, long, peritrichous flagella; not stained by Gram's method.

2. White film upon gelatin which is not liquefied.

3. No formation of gas from grape- or milk-sugar in a shake culture.

4. Uniform cloudiness of sugar bouillon in fermentation tubes without formation of gas. No formation of acid from milk-sugar, abundant from grape-sugar.

5. No coagulation of milk.

6. Indol not produced in peptone solution.

7. Finally, Lösener places value upon the demonstration by means of cultures in Petruschky's litmus whey (at 37°) that the questionable typhoid bacterium in about fortyeight hours does not produce more than 3.0 c.c. of decinormal acid from 10 c.c. of milk, while the coli bacteria form more than 8 c. c. 1

8. Marked agglutination by specific serum (see below).

9. Of less value in the diagnosis are: (1) The microscopic appearance of the gelatin plate, as it may be almost identical with the Bact.

1 Upon all these points a very satisfactory uniformity has been reached. To be sure, the uniformity depends in part upon an agreement, which is, that all those bacteria which do not present these peculiarities of the typical typhoid culture are simply declared to be different from typhoid, under the assumption that the typhoid bacterium does not vary. How little probability this assumption possesses in the face of the enormous variability of the closely related Bact. coli, requires no discussion.

coli. (2) The delicate growth upon potato, since there are typhoid bacteria which grow as luxuriantly as Bact. coli. In order that a potato culture may be of diagnostic value, two pieces from the same potato must be placed in a dish and inoculated, one with the culture in question, the other with a certain typhoid culture (Germano and Maurea). According to these authors, with whom Lösener agrees, a deviation from the growth of true typhoid bacteria upon the same potato is sufficient to exclude a diagnosis of typhoid. (3) Growth upon nutrient media to which are added antiseptic substances (phenol, formaldehyd, acids, etc.). The Bact. coli always tolerates these somewhat better than the typhoid bacterium.

The Diagnosis of Bacterium typhi is excluded:

If one of the following properties is demonstrated: 1. Absence of motility, flagella absent or located at the pole, typical spores, staining by Gram's method. 2. Absence of growth at body temperature.

3. Coagulation of milk. Formation of gas in grapesugar agar or fermentation tubes.

4. Liquefaction of gelatin.

A beautiful example of a thorough differential diagnosis between mud and typhoid bacteria is given by Houston (C. B. XXIV, 518).

Serum Diagnosis of Typhoid.1

In doubtful cases the typhoid diagnosis may very often be verified by the serum test. Since we have been acquainted with the Gruber-Durham agglutination reaction in vitro, almost always this is employed instead of R. Pfeiffer's more detailed reaction in the abdominal cavity of the guinea-pig. Cultures upon slanted agar, eighteen to twenty-four hours old at 37°, are used for the test, and

1 If one has no immune serum, still, according to Laschtschenko, he may differentiate the Bact. typhi from the Bact. coli in the following manner (H. R., 1899, No. 3): Several test-tubes, each containing 2 c.c. of fresh defibrinated rabbit's blood, obtained by venesection, are provided. To these are added two drops of a dilute suspension of the culture in question. The suspension is prepared by mixing 1 loopful of an agar culture with 10 c.c. of bouillon, and then diluting 0.5 c.c. of this with 9.5 c.c. of bouillon. In the case of Bact. coli which have not been cultivated too long, the bacteria are never dead in six to seven hours, and usually are much more numerous, while the Bact. typhi (ten cultures!) were always much less in number, no matter whether the culture had been isolated for a short or long