METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 209 



emulsion of bacteria in a fluid medium is placed on the surface of a 

 sterile thin plate of glass in such a manner that a drop of the con- 

 taminated fluid hangs in the opening in the stage ordinarily occupied 

 by the condenser. The drop is manipulated by a mechanical stage, 

 guided by direct observation with a one-sixth-inch lens until a single 

 organism appears in the field of vision. The sterile capillary pipette 

 is carefully brought upward until the tip engages the dependent drop; 

 the organism will be seen to enter the pipette, which is then lowered and 

 removed from its attachments to the microscope. The single cell is 

 transferred to a suitable medium and incubated in the usual manner. 



B. Anaerobic Bacteria. 1. Plating Methods. The cultivation of 

 anaerobic bacteria which do not grow in the presence of atmospheric 

 (free) oxygen requires special apparatus and technic. The simplest 

 method, and one which is successful if gas-forming bacteria are absent, 

 is to make dilutions in dextrose agar precisely as described under 

 Plating in the preceding paragraphs. The tubes should be filled to a 

 depth of 10 cm. with the medium, and tubes of relatively large dia- 

 meter 2 to 3 cm. are preferable. The tubes, previously heated to 

 the boiling point, and rapidly cooled to 43 to 45 C. to prevent reabsorp- 

 tion of oxygen, are inoculated by the dilution method, rotated between 

 the hands to distribute the organisms uniformly, and cooled rapidly 

 in an upright position. 



Colonies appear within the depths of the media after incubation; 

 in the thinly seeded tubes these colonies are discrete, and they may 

 be removed without contamination, either in sterile capillary pipettes 

 introduced through the surface of the medium, or after breaking the 

 tubes from the side. It is, of course, necessary to sterilize the outside 

 of the tube if it is to be broken. A greater degree of anaerobiosis may 

 be obtained within the tubes if after solidifying they are placed neck 

 downward with the cotton plugs removed, in a beaker containing 

 freshly prepared alkaline pyrogallate solution. 1 Growth of anaerobic 

 bacteria upon the surface of agar or blood serum may be obtained in 

 this manner. 2 Those bacteria which produce gas during their growth 

 cannot be isolated in pure culture in deep agar tubes; the liberation 

 of gas bubbles fragments the medium and permits the various colonies 

 to coalesce. 



1 Five grams of dry pyrogallic acid are placed in a beaker and covered with 15-25 c.c. 

 of water: when dissolved a layer of kerosene or paraffin oil about 1 cm. in depth is added, 

 and a 10 per cent, solution of sodium hydroxide is introduced below the oil layer with 

 a pipette. 



2 See Rickards, Cent. f. Bakt,, 1904, I Abt., xxxvi, 557. 



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