METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 207 



and cooled, is dipped to a depth of about 0.5 c.c. in a mixture of bac- 

 teria in fluid media, or touched to a growth in solid media, and then 

 rotated two or three times in a tube of the sterile melted medium. 

 Without sterilizing the needle, the process is repeated in the second 

 and third tubes. Each tube is then rotated between the palms of the 

 hands, to distribute the organisms thoroughly, and poured individually 

 into the sterile Petri dishes. The medium is flowed uniformly over 

 the bottom of the dish and set aside to harden. 



It will be seen that the first tube inoculated contains the greatest 

 number of organisms, and that the third tube would theoretically 

 contain but few. The colonies in one of the plates will be so widely 

 separated that they can be "fished" with the platinum wire without 

 the danger of touching other colonies, and transferred to fresh, sterile 

 media. The success of this procedure depends largely upon a rigorous 

 observance of details. The mouths of the tubes and the cotton plugs 

 should be flamed thoroughly before inoculation is practiced, and the 

 transfer of the contents of the tube to the Petri dish must be done 

 carefully to prevent contamination. *The cover of the Petri dish should 

 be raised with the left hand, but directly over the bottom, to prevent 

 the entrance of adventitious bacteria from the air. The mouth of the 

 tube should not touch the bottom or edge of the Petri dish and, finally, 

 the cover of the latter should be replaced at once. 



After the medium has hardened the plates are incubated gelatin 

 plates at 20 C., agar plates at 37 C. It is customary to invert agar 

 plates during incubation; when agar cools and becomes solid a con- 

 traction takes place which squeezes out some fluid. (This is well 

 defined in slanted agar as the water of condensation.). If the fluid 

 were allowed to remain on the surface of the agar plate it would con- 

 vert the surface potentially into a broth culture, in which the various 

 organisms would mix in hopeless confusion. Inversion of the plates 

 prevents the accumulation of moisture on the surface to a large degree; 

 the water of condensation collects on the cover instead. The porous 

 tops recommended by Hill may advantageously be used they absorb 

 moisture as it is formed. Gelatin plates are not inverted; fluid is 

 not expressed as the medium solidifies, and liquefied gelatin formed 

 during the growth of actively proteolytic organisms would collect on 

 the cover and probably contaminate the entire plate. 



2. Streak Method. The isolation of pure cultures of bacteria by the 

 streak method differs from the plate method in that the medium 

 (gelatin, agar, blood serum) is not inoculated in the fluid state; the 



