TECHNIC OF BACTERIOLYTIC TESTS 357 



solution or hot water. With a second sterile pipet mix the contents of 

 tube No. 1 by carefully sucking it in and forcing it out of the pipet sev- 

 eral times, and place 0.1 c.c. in Petri dish No. 1. Then, with the same 

 pipet, transfer 1 c.c. oo the second tube, mix well, and place 0.1 c.c. in 

 Petri dish No. 2; next transfer 1 c.c. to the third tube, mix, and place 

 0.1 c.c. in the third Petri dish. Continue in this way until the last tube 

 is reached, when 1 c.c. is discarded into a germicidal solution and 0.1 

 c.c. placed in the tenth Petri dish. To each Petri dish now add from 8 

 to 10 c.c. of neutral agar at 41 C., and mix thoroughly. After the 

 plates have hardened they are turned over in order that the water of 

 condensation may collect on the cover. They are then incubated for 

 twenty-four hours, and the colonies carefully counted. 



We now have the following dilutions of culture : 1 : 50, 1 : 100, 

 1 : 500, 1 : 1000, 1 : 5000, 1 : 10,000, 1 : 50,000, 1 : 100,000, 1 : 500,000, 

 and 1 : 1,000,000. Since but 0.1 c.c. of these dilutions were plated, the 

 total number of colonies in each plate must be multiplied by 10 in order 

 to obtain the approximate number per cubic centimeter of the various 

 dilutions. 



To secure fairly uniform results, the various dilutions must be 

 thoroughly mixed and the pipeting be accurately performed. We have 

 found that this method usually gives better results than are obtained 

 by plating but one or two of the higher dilutions, which are used as a 

 basis for calculating the number of bacteria in the other dilutions. 



Filling the Pipets. With a wax pencil make a mark upon the capillary 

 stem of a sterile looped pipet at a point 2 cm. from the lower end, and 

 fit a rubber teat to the barrel. The point of the capillary stem is now 

 broken off between the finger and thumb, the lower portion is sterilized 

 in the flame, and the air is expelled from the teat. 



Mannite broth is then aspirated into the pipet until the bulb is about 

 two-thirds full and the capillary portion contains an air column rising 

 to at least 5 cm. from the end (Fig. 101). 



The end of the capillary stem is now inserted into the tube contain- 

 ing the patient's serum, and the serum is allowed to flow in until it 

 reaches the pencil mark. 



The orifice of the pipet is now raised above the surface of the serum, 

 and a small bubble of air is admitted into the tube, to serve as an index 

 for the measurement. The end of the capillary stem is now carried 

 into the tube containing the highest dilution of the organism, and the 

 culture is allowed to flow in until the bubble of air has been carried just 

 past the pencil mark. 



