CULTURE MEDIA 53 



glass dishes (Petri dishes) is referred to as plating. The advantages of this 

 method are the possibility of procuring discrete colonies when quantities are 

 planted that would give a confluent growth in tubes, greater facility of study- 

 ing the appearance of individual colonies as they occur on the medium, greater 

 possibility of removing a single colony from the medium with a platinum loop 

 without contaminating it by contact with others. Therefore, when substances 

 contain several species of organisms and it is desirable to isolate one or more of 

 them, plating is usually the most convenient method; also, when it is desirable 

 to determine the number of bacteria per cubic centimeter in a substance, plating 

 usually affords the best means of so doing. 



Most bacteria associated with disease grow best or only grow at or near the 

 temperature of the human body, hence cultures are usually placed in an incu- 

 bator at 37C. to facilitate bacterial growth. Saprophytic bacteria are usually 

 incubated at room temperature or at 2OC. or 25C. 



Aerobic bacteria, those that require oxygen to develop, grow well in bouillon 

 and on the surface of solid media in tubes or flasks stoppered with cotton plugs. 

 Sufficient oxygen percolates through the cotton when the tubes are kept in a 

 room, closet or incubator where the atmosphere is air. 



Anaerobic bacteria, those that grow only in the absence of free oxygen, are 

 usually planted in media containing i to 2 per cent, of glucose, because glucose 

 is a reducing agent and free oxygen does not exist in media containing it. 



When better means are not available anaerobes can be cultivated by taking 

 tubes half -full of solid glucose agar, making deep stab inoculations in the center 

 of the media, and covering the surface with an inch or more of sterile liquid 

 petrolatum. 



Anaerobic cultures are usually made by placing the inoculated tubes or Petri 

 dishes in air-tight jars, from which the oxygen is removed either by extraction, 

 displacement or absorption, or a combination of these methods. For extrac- 

 tion and displacement specially devised jars, fitted with taps and stop-cocks 

 are required. Exhaustion is achieved by pumping out the air and leaving a 

 vacuum. Displacement is achieved by introducing hydrogen through one tap 

 while the other is open so that air and oxygen pass out as hydrogen enters. 

 When the atmosphere is entirely devoid of oxygen the stop-cocks are closed. 

 Hydrogen is generated for this purpose in a Kipp's apparatus, from sulphuric 

 acid and zinc. It is passed, in the order mentioned, through three wash bottles; 

 the first containing 10 per cent, lead acetate solution, the second 10 per cent, 

 silver nitrate solution and the third a 10 per cent, solution of pyrogallic acid in 

 10 per cent, sodium hydrate solution. It is then delivered into the jar contain- 

 ing culture tubes or plates. 



Absorption of oxygen does not require special apparatus, any jar having a 

 ground-glass stopper or screw cap that can be made air-tight will do. The tubes 

 or plates are placed in the jar, on a support that elevates them an inch or more 

 above the bottom. About J^ inch of pyrogallic acid is placed in the bottom of 

 the jar, potassium hydrate solution is poured upon it and the jar quickly sealed. 

 Potassium pyrogallate is formed and it absorbs the oxygen. To 2 to 4 Gm. 



