146 BIOLOGY AND TECHNIQUE 



sterile Petri dishes. The pouring should be done with great care. 

 The cover of the dish is raised along one margin simply far enough to 

 permit the insertion of the end of the test tube, the plug of which has 

 been removed and the lips passed, with a rotary movement, through the 

 flame. The medium is poured into the dish without the lips of the 

 tube being allowed to touch either the bottom or the cover of the dish. 

 The cover is then replaced and the medium allowed to harden. 



When agar has been used, the dishesxmay be placed in an incubator 

 at 37 C. It is well to place the plates upside down in the incubator. 

 This prevents the condensation water, squeezed out of the agar dur- 

 ing hardening, from collecting on its surface, and forming channels for 

 the diffuse spreading of bacteria. The same end may be attained by the 

 use of Petri plates provided with porous earthenware lids, as suggested 

 by Hill. Simple inversion of the plates, however, usually suffices. When 

 gelatin has been used, the plates are allowed to remain in a dark place at 

 room temperature or in a special thermostat kept at 22-25 C. 



Colonies in agar, kept at 37.5 C. , usually develop in eighteen to twenty- 

 four hours; those in gelatin or agar at room temperature in from twenty- 

 four to forty-eight hours, depending upon the species of bacteria which 

 are being studied. Often in the second dilution, more frequently in 

 the third, the colonies will be found well apart and can then be "fished." 

 The process of " colony-fishing" is one which requires practice and should 

 always be done with care, for upon its success depends the purity of the 

 sub-culture obtained. Colonies should never be fished under the naked 

 eye, no matter how far apart and discrete they may appear, since not 

 infrequently close to the edge of or just beneath a larger colony there 

 may be a minute colony of another species which may be too small to be 

 visible to the naked eye, but which, nevertheless, if touched by accident 

 will contaminate the sub-culture. 



For proper " fishing," the Petri plate with cover removed, should be 

 placed upon the stage of the microscope and examined with, a low power 

 objective, such as Leitz No. 2 or Zeiss AA. The sterilized platinum 

 needle, held in the right hand, is then carefully directed into the line 

 of focus of the lens, while the small finger of the hand is steadied upon 

 the edge of the microscope stage. WTien the point of the needle is 

 clearly visible through the microscope, it is gently depressed until it 

 is seen to touch the colony and to carry away a portion of it. The 

 needle is then withdrawn without again touching the nutrient medium 

 or the edges of the glass or the lens, and transferred to a tube of what- 

 ever medium is desired. In this way, individuals of one colony, de- 



