95 



served. This is described by Kline as follows: 



"The cream is torn or altogether dissociated by the development of 

 gas so that the surface of the medium is covered with stringy, pinkish-white 

 masses of coagulated casein enclosing a number of gas bubbles. The main 

 portion of the tube formerly occupied by the milk now contains a color- 

 less, thin, watery whey, with a few casein lumps adhering here and there 

 to the sides of the tube. When the tube is opened, the whey has a smell of 

 butyric acid and is acid in reaction. Under the microscope the whey is 

 found to contain numerous rods, some motile, others motionless." 



The method employed by Creel, which was said to be very efficient 

 for isolation in pure culture is given herewith. 



Petri dishes are selected having covers considerably larger than the 

 inner plates. Agar is poured into the inner dish, allowed to harden and 

 then inoculated by smearing over the surface with material from a broth 

 or lactose bile tube suspected of containing an anaerobe. The inoculated 

 plate is then inverted into the large cover. Three grams of pyrogallic 

 acid are placed in the cover, one c. c. saturated potassium * hydroxide is 

 inserted with a pipette, and the two dishes are immediately sealed with 

 melted paraffin. Incubation is at the body temperature. 



In the Standard Methods of Water Analysis A. P. H. A. 1912, a de- 

 tailed procedure for the detection of these anaerobes is recommended as 

 given below: 



"B. sporogenes* is indicated by a vile odor which is produced in the 

 liver broth fermentation tubes used in the regular test for general gas form- 

 ing bacteria. The specific tests are made as follows: 



1. Inoculate various dilutions (usually 0.1, 1.0 and 10.0 c. c.) of 

 water, or of sewage in higher dilutions, into fermentation tubes containing 

 liver broth and incubate for 24 hours at 37 C. If B. sporogenes is pres- 

 ent in the dilutions used, there will be vigorous gas formation, accompanied 

 by an offensive odor, and numerous large spores will be present. 



2. Transfer the entire contents of each tube showing gas plus char- 

 acteristic odor into separate sterile Erlenmeyer flasks or large test tubes 

 and heat to 80 C. for 10 minutes. 



3. One (1) c. c. (not more) of broth containing sediment is with- 

 drawn from the bottom of each of the flasks or tubes which have been 

 heated, and is planted separately into a second set of sterile liver broth 

 fermentation tubes and incubated for 24 hours at 37 C. after which time 

 gas formation and characteristic odor will again be observed. Microscopic 

 examination will reveal the presence of numerous large sluggishly motile 

 bacilli containing spores. Usually B. sporogenes is now present in pure 

 culture. 



4. A stab culture made from this 24 hour liver broth culture into 

 dextrose liver gelatine or nutrient gelatin will demonstrate the presence 

 of B. sporogenes by the following characteristic growth. After 48 hours 

 incubation at 20 C., a distinct anaerobic growth will be observed begin- 



*Term employed to designate the Welchii group and not a specific organism. 



