96 



ning about two centimeters below the surface. Liquefaction will be well 

 advanced and gas bubbles will accumulate at the top of the liqufied area. 



5. In order to obtain colonies of B. sporogenes on agar plates it is 

 necessary to transplant a few drops of broth and sediment from the second 

 set of fermentation tubes, in step 3, into a third set of tubes and incubate 

 for three to five hours at 37 C. After that period a distinct anaerobic 

 growth will be observed in the closed arm, and a few bubbles of gas will be 

 seen at the top. The B. sporogenes is now in the vegetative state and this 

 is the only condition in which it will grow on the plates. 



The contents of the closed arm are transferred to the open bulb by 

 tilting forward, and plated in dilutions of 1.0 to .00001 c. c. on dextrose 

 liver agar, and incubated for 12 to 18 hours in hydrogen at 37 C. Typical 

 colonies will then be visible consisting of one or more gas bubbles sur- 

 rounded by a delicate white fringe. The plate cultures also have a dis- 

 agreeable cheesy odor. 



6. From one of these typical colonies a deep stab culture is made 

 into dextrose* liver agar and incubated for 24 hours. A distinct anaerobic 

 growth will be observed along the line of puncture and sometimes the 

 agar is split into two or three layers by the gas evolved. 



7. A sub-culture may also be made into litmus milk and incubated 

 for 48 hours, anaerobically, after which time there will be a complete 

 separation of curd and whey and a strong odor of butyric acid. Some- 

 times the curd adheres to the sides of the tubes and has a peculiar shredded 

 appearance." 



It should be emphasized that none of the foregoing methods will 

 yield all the sporing lactose fractors. Detailed procedures for isolation 

 of specific forms together with the special media necessary are described 

 in the English Report on Anaerobic Bacteria and Infections, to which ref- 

 erence has already been made. 



The Aerobic Sporing Lactose Fermenters. Meyers, in 1918, 

 isolated a spore forming lactose fermenter capable of growing on the sur- 

 face of Endo agar, from the water supplies of Newport and Covington, 

 Kentucky and from tannery wastes. 



The organism is Gram negative, grows readily on Endo agar, pro- 

 ducing a red colony in 24 hours, which shows a distinct metallic luster 

 after 48 hours, and in Clark and Lubs medium it is alkaline to Methyl 

 Red and positive for the Voges Proskauer Reaction. The more important 

 characteristics as detailed by Meyers are: 



Agar slant. Growth quite distinctive. At 37 C., in 24 hours, thin 

 transparent veil-like growth over entire surface except the very top. Growth 

 lobate along upper edge. Microscopically in 24 hours mainly vegetative 

 forms, in 48 hours spore-bearing forms and later only free spores. 



Endo's plates at 37 C. In 24 hours, colonies pink with red center, 

 irregular contour, one to two m. m. diameter, little or no sheen. Colonies 

 48 hours, deep red, much sheen in colonies and surrounding medium. Lat- 

 ter point distinctive. 



