48 



to determine its Gram characteristics and other reactions, particularly fer- 

 mentation of lactose broth, as previously explained. 



The objections to the preliminary enrichment method are: 



(1) It is difficult to estimate the incidence of the colon forms for 

 by employing Ic. c., 0.1 c. c., etc., there is considerable error introduced 

 in calculating the number of organisms present in a unit volume of water. 



(2) There is a danger of loss of the colon forms which may be killed 

 off before isolation is attempted either by (a) products of their own meta- 

 bolism or (b) by over-growths of other organisms. 



In glucose broth inoculated with a mixture of Fact, coli and Strepto- 

 cocci, the former usually predominated for the first 24 hours, but thereafter 

 the number of Streptococci was considerably greater, in some instances 

 having completely choked out or killed the colon types in 39 hours. 



The danger of losing colon strains due to overgrowth and death may 

 be minimized considerably, if not completely eliminated, by (1) reducing 

 the concentration of fermentable substances to 0.5 percent (as recommended 

 by Burling and Levine) and (2) attempting isolation at the earliest pos- 

 sible time (after 12 to 24 hours or at the first appearance of gas). It will 

 be noticed from Table XXI. that at the first appearance of gas the colon 

 forms were far in excess of the Streptococci, in fact practically in pure 

 culture. With these precautions the preliminary enrichment method will 

 be found very reliable and convenient. 



THE PRESUMPTIVE TESTS 



The method for isolation of members of the colon group described 

 above, comprising as it does the reaction of the isolated organism in milk, 

 production of indol, reduction of nitrates, gelatin liquefaction, and mo- 

 tility was in general use between 1905 and 1910. The method, however, 

 was soon found to be tedious and inconvenient for routine work. The 

 time required for such a complete analysis was at least nine days, whereas 

 for practical purposes it is desirable to have a report available within 

 24 to 48 hours. Furthermore, there is no evidence that the determination 

 of the characters enumerated shed any light upon the sanitary significance 

 of the bacilli isolated. For routine water work, it is essential to have some 

 test which is simple and rapid and which can be relied upon as a reason- 

 able index of the probable presence of the colon group in a large pro- 

 portion of instances. Such a test is known as a presumptive test for 

 the colon group. From his wide experience with the glucose broth pre- 

 liminary enrichment method, Whipple in 1903 observed that a consider- 

 able portion of tubes showing characteristic fermentation were found, on 

 further examination, to contain colon-like organisms. He therefore sug- 

 gested that dextrose broth alone might be employed as a rapid presumptive 

 test of the probable presence of Bact. coli. The media which have been 

 and are most commonly employed for the presumptive test are (1) glucose 

 broth, (2) lactose peptone bile, and (3) lactose broth. Other media have 

 also been suggested, as for example the Eijkman test (glucose peptone 



