58o ELECTIVE LOCALIZATION OF BACTERIA 



these media but the disease from which the patient was suffering was successfully re- 

 produced in animals with the streptococci isolated, when cultures made according to 

 standard bacteriological methods (low columns of broth on aerobic agar or blood-agar 

 plates) remained sterile or yielded only aerobic saprophytes. Because of these ob- 

 servations and since saprophytic streptococci do not produce lesions and disappear 

 quickly from the blood, we have learned in the study of elective localiza tion to inject 

 into animals the primary (often mixed) culture in glucose brain broth in order not 

 to miss the pathogenic strain or to have it lose specific localizing power during its isola- 

 tion in pure culture. 



The glucose brain broth is prepared by dissolving 8 gm. of dehydrated Bacto- 

 broth in i,ooo cc. of distilled water, adding 8 gm. of sodium chloride, 2 gm. of chemi- 

 cally pure glucose, and, after coohng, 10 cc. of Andrade's indicator; it is also prepared 

 from beef infusion, adding 0.2 per cent glucose in the usual way and titrating to pH 

 7.4. Several bacteriological peptones on the market have been used with about equally 

 good results. The glucose brain agar is prepared from the broth by adding 4 or 5 gm. 

 of powered agar to each liter, just sufficient to set. The media are placed in tubes 

 20 by 1.5 cm. To each tube are added three pieces of calf or beef brain, each about i cc, 

 and two or three pieces of calcium carbonate in the form of crushed marble to insure 

 correct reaction. The column in each tube, after the brain and marble are added, is 

 brought to about 12 cm. The media are sterilized for twenty minutes in the autoclave 

 at 17-pound pressure. Growth in these media, kept at 35°-37° C, usually occurs with- 

 in twenty-four hours, and in most instances, especially in the case of cultures from 

 pulpless teeth, begins at the bottom of the tube and forces its way to the top as oxygen 

 is consumed. This usually occurs within twenty-four hours in the glucose brain broth, 

 while in the glucose brain agar growth to the top, or more often to within i cm. of the 

 top, frequently takes from two to three days. Negative cultures are observed daily 

 for a week or ten days before they are discarded. Colonies in the agar may be exceed- 

 ingly small or develop only around the pieces of brain at the bottom and may be 

 easily overlooked, or they may be so numerous as to give the appearance of a negative 

 culture. 



The animals, chiefly rabbits, were injected usually with eighteen-hour to twenty- 

 four-hour primary cultures in glucose brain broth, sometimes with pure cultures in this 

 medium obtained from single colonies in the soft glucose brain agar, or from inocu- 

 lated animals and after many rapidly made subcultures (four to eight each twenty- 

 four hours in previously warmed media). Subcultures from aerobic growth on blood 

 agar commonly gave negative results. The number of bacteria injected as a routine in 

 later studies was approximately from one-fifth to one-tenth as great as was used 

 earlier. Depending on the size of the animal, the source of the strain or the particular 

 purpose in mind, 5 or 7 cc. of the primary growth in glucose brain broth of material 

 from the focus or infection atria were injected intravenously as a routine into each 

 of two rabbits. The injections were usually not repeated although sometimes several 

 injections were given daily. The animals selected from healthy stock were fed in the 

 usual way and were observed daily. Those that died from the effects of the injections 

 were examined as soon after death as possible, and those that survived were chloro- 

 formed in from two to four days after the last injection. Search for lesions was made 



