462 HILDA HEMPL HELLER 



1, expel it, draw up fresh agar and expel it into tube 2. For cul- 

 tures containing abundant organisms, give tube 2 2 inches of agar 

 measured in the capillary portion of the tube. For ordinary cultures 

 give 5 inches, for B. N.ovyi, etc., give about two capillaries full. 

 Place the inoculum throughout the length of the agar while with- 

 drawing the pipette, but do not blow air into the agar of tube 2. 

 Roll tube 2. Flame the Pasteur pipette. By means of it place 

 agar from tube 2 in tube 3 to the amount of 0.5 to 1 inch on the 

 upper or thick portion of the Pasteur pipette. Roll the tube. 

 Incubate aerobically at 37°. If actively growing species are 

 present, incubate twelve hours. Otherwise incubate eighteen to 

 twenty-four hours. For blackleg, Clostridia, and unknown shy 

 types, incubate four days. Examine the colonies with a hand lens. 

 Look for permeating growth. It is better, in fishing from a tube 

 containing more than one type of colony, to fish once more onto 

 a series of agar tubes. Final isolation should be made from colo- 

 nies of mixed cultures. Study the tubes carefully with a hand lens, 

 noting minute colonies and aerobic growth. Select the tube to be 

 fished, and, if possible, select the colonies desired. Take a well- 

 made, strong Pasteur pipette of fairly large bore, bend it at right 

 angles where the capillary begins, break the tip, flame the whole 

 capillary. Remove the plug from the tube and loose fibers of cot- 

 ton from its opening, insert the Pasteur pipette along the side to 

 the bottom, remove and empty it of agar ; re-insert it, and blow the 

 whole column of agar into a sterile Petri dish. The large Pasteur 

 pipette may be used many times. One-half Petri dish serves for 

 each tube. Take a short-stemmed Pasteur pipette, hold it in the 

 flame, draw the capillary out to a hair-like tube, and break it off 

 fairly short. Suck up the desired colony and expel it into a tube 

 of meat medium or tube 1 of another agar series. Draw out the 



constantly but slowly in the same direction, not forwards and backwards. Con- 

 tinue till the hot portion softens and contracts to about four-fifths of its former 

 diameter. Never pull the glass while it is in the flame. Remove the rod from 

 the flame and wait a second, then pull slowly. If the glass is pulled too soon or 

 too quickly the fine bore is formed from the hottest portion only, and not from 

 all the heated glass, the bore is small, and its walls are thin and weak. An hour's 

 continuous practice is necessary to begin with; the art, once learned, is extremely 

 useful and is not forgotten. 



