LABOKATOEY AND GREENHOUSE STUDIES. 67 



gelatin at 37° C. for 48 hours, after which they are placed in an ice 

 box to permit hardening. The solidifying of the gelatin is taken as a 

 positive indication of the presence of Bacillus coli, and those tubes 

 in which the gelatin did not harden are considered as i)ositively free 

 from Bacillus coli. 



Cultures of tlie coconut organism and Bacillus coli (from animals) 

 were incubated in gelatin at 37° C. for 48 hours and then placed in 

 an ice box to harden. In six hours all of the tubes, including coconut 

 No. 1, were perfectly firm. 



The culture designated as coconut No. 1 was descended from one 

 of many cultures made from the same isolation (505 E,). In order 

 to see if they all behaved alike, transfers were made into gelatin 

 from all that were still alive nme months after isolation. There were 

 in all 11 of these cultures from as many colonies. These tubes were 

 incubated at 37° C. and tested daily for liquefaction by removing to 

 an ice box, where they might harden. Seven days after inoculation 

 all showed good growth and were perfectly firm when placed at a low 

 temperature. Ten days after inoculation the check tubes (two) were 

 perfectly firm; four of the cultures were slightly soft; and the 

 remaining seven were in the nature of a thick liquid. Coconut No. 1 

 was the thinnest of these, but even that was not entirely liquefied. 



Attempts were made to plate out coconut No. 1 to ascertain if by 

 any chance it had become contaminated. A variety of colonies was 

 obtained on the plates of plain agar, but transfers from various forms 

 of these colonies to litmus milk, nitrate bouillon, Dunham's solution, 

 neutral red in fermentation tubes, and gelatin all gave the reactions 

 typical of the coconut organism. Platings were also made from the 

 other coconut organisms and from Bacillus coli (Theobald Smith, 

 XIV) with exactly the same results. On plain agar the variety of 

 forms was great, ranging from the small, round, iridescent colonies 

 to large, thin, homogeneous, much-branched colonies. Transfers 

 from specific colonies to litmus milk gave the typical reaction for the 

 coconut organism, and platings from these tubes gave in every case 

 on plain beef agar the same variety of forms. In view of this work 

 it does not seem possible that the culture coconut No. 1 was con- 

 taminated, but rather, as has been suggested, that it was a modified 

 form of the original organism. 



According to the recommendations of the Society of American 

 Bacteriologists six weeks is the time fixed to ascertain the liquefy- 

 ing power of any organism when the cultures are grown in an ordi- 

 nary gelatin box. Bacillus coli and all six of the coconut cultures 

 answer to this test. According to the methods used by some workers 

 and mentioned in the foregoing paragraphs, 48 hours at 37° C. (before 

 placing in an ice box) is sufficient time in which to ascertain the lique- 



228 



