CULTIVATION OF BACTERIA 55 



is gently stroked over the medium in plate one, and then, without 

 turning, the same portion of the swab is smeared over a second and 

 a third plate. If the material to be examined is liquid a small 

 quantity is deposited on .the surface of the medium by means of a 

 platinum loop and the loop is then stroked lightly over the medium 

 in several plates without recharging. 



A method of plating is frequently used by means of which the 

 dilutions are carried out in definite proportions so that the num- 

 ber of bacteria present may be estimated. 



The procedure is as follows : To 9 c.c. of sterile water 1 c.c. 

 of the material to be examined is added and the resulting mixture 

 is thoroughly shaken to separate the organisms. One c.c. of this 

 1 in 10 dilution is added to 9 c.c. of sterile water, producing a 1 

 in 100 dilution. After thorough shaking the process may be 

 repeated, giving as a result a 1 in 1000 dilution, and so on until 

 the desired limit has been reached. One c.c. of each dilution is 

 placed by means of a sterile pipette in a Petri dish previously 

 numbered, and melted agar cooled to 40 C. is poured into the 

 center of it. Mixing is accomplished by gently rotating the plates 

 before the medium solidifies, care being taken that the medium 

 remains flat on the bottom and is not smeared over the sides. 

 After growth has occurred the colonies are counted and the number 

 of bacteria present in the material examined estimated. 



For example, if on the Petri dish containing the 1 in 100 dilu- 

 tion 180 colonies appear that number is multiplied by 100, and it 

 is assumed that approximately 18,000 organisms were present 

 in 1 c.c. of the material examined. The dilution showing between 

 one hundred and two hundred colonies is chosen as the most 

 representative for counting. Higher numbers are difficult to count ; 

 also crowding may have checked the development of some of the 

 organisms. Counting may be facilitated by dividing the Petri 

 dish into sections by lines made with a colored pencil or by placing 

 the dish on a Wolffhiigel counting plate (Fig. 19). Whenever 

 possible all the colonies should be counted ; if they are too numer- 

 ous and a counting plate is used, squares from representative parts 

 of the dish are counted and the total number of colonies estimated. 



