No. 6. DEPAKTMENT OF AGRICULTURE. 293 



PART I. 



ISOLATION AND STUDY OF PURE CULTURES FROM VARI- 

 OUS SOURCES IN MAN AND CATTLE. 



Method of Isolating Pare Cultures. — The isolation of cultures has 

 been obtained by the method devised by Dr. Theobald Smith,^ the 

 principal features of which are: (1) The test-tubes, which are fitted 

 with a cap like that of the well-knewn Miquel flask, ground on. The 

 small tubulation in the cap is plugged with glass-wool. (2) The 

 medium, which is dog's serum. The animal is bled with the strict- 

 est aseptic precautions, and the blood conducted to sterile jars 

 through sterile rubber tubes. The serum is drawn off with pipettes 

 and distributed at once into the test-tubes, put in the hardening 

 oven, and coagulated at a low temperature {7G°C.). Prepared in 

 this way the serum needs no sterilization, and is much softer than 

 when a higher temperature is employed. (3) The tubes are kept 

 always inclined, (i) Tlie tuberculous tissue selected for planting 

 in our culture tubes is not crushed oor rubbed over the surface, as 

 advised in other methods. Young nodules from the ome«tum, spleen, 

 liver, or a lymphatic gland are cut out, and a block of considerable 

 size is placed on the surface of the serum. After remaining from 

 two to three weeks in Uie i«icubator at 37.5° to 39° C, the tissue is 

 pressed against the sides of the tubes with a stout platinum wire, 

 and rubbed over the surface of the medium. Examination of the 

 fluid squeezed out of the tissue at this time will generally indicate 

 the filial result. The tubes are replaced in the incubator, and 

 within a week or ten days colonies of the tubercle bacillus usually 

 appear. The nodules for inoculation of the tubes are best obtained 

 from guinea-pigs, which are to be inoculated with the original mat- 

 ter from which we wish to obtain cultures. As soon as these show 

 marked illness they are chloroformed and the cultures made. In 

 general terms, the more recent the lesions the greater the chances 

 of successful culture. As a rule, guinea-pigs can be killed in from 

 three to four weeks after inoculation. If our material is of human 

 origin the inoculation should be intraperitoneal; if bovine, subcu- 

 taneous inoculation will generally cause a sufficient rapid involve- 

 ment of the organs. (.5) The incubator should contain a dish of 

 water, to insure an abundance of moisture in the atmosphere. With 

 the same object in view, it should be opened as seldom as possible. 



The only modification of the above method in our work has been 



. the addition of a 50 per cent, solution of glycerin in water to the 



serum in such proportion that the resulting mixture contains 5 



per cent, of glycerin. Ibis seems to make coagulation more tardy, 



