EXPERIMENT STATION BULLETINS. 489 



Experiment III. > 



FILTRATION BXPERI3IENTS. 



The imsatisfactory results furnished under Exj^eriments I and II 

 seem to demand a complete change of methods. The main difficulty 

 heretofore, has been that of successfully differentiating the typhoid and 

 lactic bacteria by the use of a special medium for plating. To obviate 

 this difficulty, the plan was formulated of growing B. typhosus in the 

 products only, of the lactic organisms. The hypothesis is, the products 

 of the lactic bacteria and not the bacteria themselves exert a deleterious 

 influence upon the typhoid bacteria. 



The most feasible way of carrying out this plan appears to be that 

 of growing the lactic organisms in a suitable medium from which they 

 could be easily filtered; the filtrate would thus contain their products 

 in solution and, when properly controlled would be wholly free from 

 organisms. The typhoid bacteria could then be introduced into this 

 germ-free filtrate and plates made at any time in ordinary agar, thus 

 making the use of a differential medium unnecessary. 



By a preliminary test, lactose broth was found to be the medium which 

 would best lend itself to the abundant growth of the lactic organisms 

 and permit of filtration of the cultures. 



Medium: — This broth was made by adding li/^ per cent lactose and 

 1 per cent peptone to the meat infusion. The first lot of broth was 

 made +1^°? the second +15°, nearer perhaps the average acidity of 

 milk. The broth was then placed in 375 cc. Erlenmeyer flasks and steril- 

 ized. 



Apparatus. — The broth cultures of the lactic organisms were filtered 

 through a Pasteur-Chamberland "F" bougie used in connection with 

 Novy's filtering apparatus; Reichel's filtering apparatus was also used 

 in one or two trials. 



The lactic organisms used in this series of experiments were Strept. 

 lacficns. Bact. luctis acidi (lab.), Bact. lactis acidi No. 226, Bact. lactis 

 acidi, Harding, Bact. acidi lactici "Lactone," Bact. lactis acidi (stock), 

 and Bact. acidi lactici No. 2, and in addition, three new cultures received, 

 in the meantime, from the Geneva Experiment Station, Bact. lactis 

 acidi (from cheese), Bact. lactis acidi (from whey), and Bact. lactis 

 acidi (from starter). 



MetJiods.— Two or three hundred cubic centimeters of lactose broth 

 were inoculated with a known lactic organism and incubated at 37°C. 

 The acidity of the culture was determined from time to time by titrating 

 with N/2d NaOH. Each sample was boiled before titrating to expel 

 volatile acids. The exact time of titration and the degree of acidity were 

 recorded each time. 



When the maximum acidity had developed, the culture was allowed to 

 remain at least 24 hours longer at 37°C. then filtered into a sterile flask 

 (the bougie and flask had been autoclaved 10 minutes at 121°C.). 



When from 150 to 200 cc. of the broth culture had been filtered, two 

 flasks of sterile lactose broth (50 cc.) were inoculated with 5 cc. of the 

 filtrate and togetlier witb the filtrate, incubated at 37°C.* If no growth 

 occurred in any of the flasks, a known number of typhoid organisms was 



♦The check flasks were inoculated with the filtrate to allow the lactic organisms to de- 

 velop if they passed the filter. They would probably not have been able to grow In the 

 filtrate containing an excess of their own products. 



