THE PRESENCE, TYPE, AND POSSIBLE SIGNIFICANCE OF LACTOSE-FEEMENTING BACILLI 321 



and triple strengths. One tube is inoculated with 20 c.c. of the water ; two tubes 

 with 10 CO., 4 tubes with 5 c.c, 5 tubes with 1 cc, 5 tubes with '1 c.c, and 5 tubes with 

 •01 c.c. All are incubated at 37' C, only tubes that give both acid and gas being counted as 

 having given the reaction. If only one tube in the first seven of these gives a reaction, then 

 faecal bacilli are present in 60 c.c, and if three out of the five 1 cc tubes give a positive 

 reaction, then ftEcal bacilli are considered to be present in 1 c.c. The same applies more or 

 less to the 1 c.c. and '01 c.c. groups. The next step in the procedure is to isolate the 

 various faecal organisms present ; and in order to do this satisfactorily, the tube containing 

 10 c.c. of water or more, that has given a positive reaction in 18 hours, is selected. 



It is an important point that a tube containing at least 10 c.c. of the water sample and 

 giving the positive reaction within 18 hours, be selected. If tubes containing a smaller 

 amount than 10 cc of the water sample be selected, the danger is that only one or 

 two fiBcal bacilli may be present, and, if a tube be selected for subculturing purposes which 

 has been longer in the incubator than 18 hours, the chances are that many of the lactose 

 fermenters have had their growth inhibited by the increasing acidity of the medium. 



Having selected then a tube containing at least 10 c.c. of the water sample, which has isolation of 

 been incubated for not more than 18 hours, three to five platinum loopfuls are removed ^^'^^\ 



organisms by 



and inoculated into 10 c.c. of sterile water. The latter is then vigorously shaken, and one plating 



loopful of this mixture is then plated out on lactose bile-salt-neutral-red-agar, which 



is incubated at 37° C, for 48 hours. At the end of that time 15 to 20 colonies are found to 



be present in the plate. A segment of the plate is marked off if more than this number 



is present. Ten colonies are in any case picked off, and each single colony is inoculated 



into a tube containing a few drops of sterile water. From these tubes further inoculations 



are made into broth, glucose-peptone, and four different, special sugars and incubated 



at 37' C. The inoculated broth tube is examined at the end of 18 hours for motility and 



the production of indol. The glucose-peptone is incubated for 48 hours and, at the end 



of that time, if a strong growth is present, a few drops of a very concentrated solution 



of caustic potash are added for the purpose of finding if the Voges and Proskauer 



reaction is given. If this is present, a bright orange-red colour is produced. It may 



take 24 hours before this colour is apparent. The four sugars that are inoculated are Differential 



those recommended by MacConkey,' namely saccharose, dulcit, adonit and inulin. In ^"S-" media 



these, a positive reaction is represented at the end of 48 hours incubation at 37 C. 



by the production of acid and gas. In carrying out these tests the problem of the 



constancy of the reaction of the organisms in the various sugars was not taken into 



account, it being accepted as a fact that the same species of bacillus always fermented 



the same sugar. 



In connection with the indol test the paradimethyl benzaldehyde reaction was always 

 used, as it seems to be a very delicate and reliable one. 



With regard to the Voges-Proskauer reaction, considerable difference of opinion exists, 

 but there can be but little doubt that it appears a good test for the B. cloacie, B. lactis 

 aerogenes and Oxi/tocus perniciosus group.* 



As will be seen from the results of the following examinations, a large number of the 

 organisms will be found to be non-motile. The question of motility, as a means of 

 differentiating an organism of the coli group should not, in the opinion of the writer, be 

 regarded seriously as compared with the reactions in the sugars and the indol test. 



It may be stated here that in order to simplify the nomenclature of the faecal 



' MacConkey, A. (July, 1905), "Lactose fermenting bacteria in fsecss." Journal of Hyyieuc. 



• Vide page 326 



