METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 189 



The cleaned glassware test-tubes, flasks, or fermentation tubes 

 is then stoppered with non-absorbent cotton cotton batting which 

 has a long staple or fiber. The cotton plugs must be carefully fitted 

 neither too loose, which would permit of the passage of adventitious 

 microorganisms, nor too tight, for obvious reasons. The cotton plugs 

 are conveniently prepared from a layer of cotton batting about two 

 inches square (for a test-tube of ordinary diameter, about 15 mm.), 

 which is laid squarely over the orifice. The center of the square is 

 gently pushed down into the neck of the tube for a distance of about 

 three-fourths to one inch; sufficient cotton protrudes from the tube 

 to be conveniently grasped by the fingers and removed. It is fre- 

 quently advisable to cover the cotton plugs with two or three layers 

 of filter paper, which prevents an accumulation of dust on the cotton. 

 Wide-mouthed containers are sealed with several layers of unglazed 

 paper fastened in place with a piece of twine. Flasks are frequently 

 not plugged with cotton; the neck is simply covered by an inverted 

 beaker of appropriate size. 



Glassware should always be sterilized before media is placed in 

 it; this is readily accomplished by dry heat. A hot-air sterilizer is 

 used, in which a temperature of 180 C. is maintained for one hour. 

 A higher temperature must be avoided, to prevent charring of cotton 

 plugs. The heat must be increased gradually and diminished gradually, 

 to prevent cracking of the glass. By this process not only is the 

 utensil rendered sterile, the plugs of cotton retain their shape when 

 withdrawn, as well. 



A majority of the bacteria pathogenic for man and many parasitic 

 and saprophytic forms as well require relatively complex organic 

 compounds containing carbon, hydrogen, nitrogen, and oxygen, 

 together with other elements for their nutrition. These foodstuffs 

 provide both the structural and fuel requirements of the organism, 

 as explained in the chapter on Bacterial Metabolism. Experience has 

 shown that a medium containing meat infusion, peptone, and salt is 

 a satisfactory one for many bacteria. This medium may be enriched 

 by the addition of various ingredients to meet the requirements of the 

 more fastidious organisms. 



Meat infusion is prepared from finely comminuted lean meat 1 freed 

 from fat. 500 grams of meat are intimately mixed with 1000 c.c. water 

 and allowed to infuse over night in the refrigerator. It is then strained 



1 Beef hearts make a very satisfactory meat infusion and their cost is much less than 

 the better cuts of meat. 



