LABORATORY AND TEACHING METHODS 623 



in a row after previously sterilizing them wrapped in a crepe napkin in the hot-air 

 oven. 



Take three sterile test tubes numbered i, 2 and 3 and fill with the liquefied 

 nutrient to be used. Plug each tube with cotton and flame the plugs, which should 

 be removed readily from the mouths of the tubes. 



Add one loopful of inoculum to tube No. i. After replugging, rotate the tube 

 between the palms of the hands with an even circular movement to diffuse the in- 

 oculum throughout the medium; avoid jerky movements as these cause bubbles 

 of air to form in the medium. 



Sterilize the platinum loop and add two loopfuls of diluted inoculum to tube 

 No. 2 and mix as before. In a similar manner transfer three loopfuls of liquefied 

 medium from tube No. 2 to tube No. 3 and mix thoroughly. 



Flame the plug of tube No. i, remove it, then flame the lips of the tube; slightly 

 raise the cover of Petri dish No. i, introduce the mouth of the tube; then elevate 

 the bottom of the tube, pour the liquefied medium into the Petri dish to form a thin 

 layer. Remove the mouth of the tube and close the "plate." If the medium has 

 failed to flow evenly over the bottom of the plate, raise the plate and tilt it to rectify 

 the fault. 



Pour plates No. 2 and No. 3 in a similar manner from tubes Nos. 2 and 3. Label 

 the plates with the distinctive name or number of the inoculum, the number of the 

 dilution, also the date. 



In this way colonies may be obtained quite pure and separate from each other. 

 They may be described as such, and may then be transferred as pure cultures to 

 other media in other test-tubes. 



In plate No. i probably the colonies will be so numerous and crowded, and there- 

 fore so small, as to render it useless. In plate No. 2 they will be more widely sepa- 

 rated, but usually No. 3 is the plate reserved for careful examination, as in this the 

 colonies are usually widely separated, few in number and large in size. 



Agar plates are poured in a similar manner, but the agar must be melted in boil- 

 ing water and then allowed to cool to 42°C. or 45°C. in a carefully regulated water 

 bath before being inoculated and the entire process must be carried out very rapidly 

 otherwise the agar will have solidified before the operation is completed. After 

 the agar has hardened it is incubated at 37°C. and the plates are inverted as this 

 prevents flooding of the agar surface by the squeezing out of the water of condensa- 

 tion as the agar hardens. Gelatin plates are not inverted. 



Streak Method. — The isolation of pure cultures of organisms by the streak 

 method differs from the plate method in that the medium (gelatin, agar, blood 

 serum) is not inoculated in the fluid state but the necessary dflution to secure iso- 

 lated colonies is secured by drawing a glass rod with its end bent into a triangle, 

 as recommended by Bergey, several times across the surface of the sterile medium 



in Petri dishes by lifting the cover while so doing. The <^ glass rod 



has been previously infected with the material to be studied qualitatively. It is 

 preferable, according to Bergey, to place a small quantity of the mixed culture 

 in the center of the first plate of a series, and thence distribute the material 

 over three or more plates in succession with the glass spreader. Eventually a 

 degree of dilution is reached where distinct colonies are in evidence. 



