56 PRINCIPLES OF BACTERIOLOGY 



quantities are used, by means of a glass funnel. The main precaution 

 to be observed is not to let the media soil the neck of the tubes and flasks, 

 as this would cause the fibres of the cotton plugs to adhere to the sides 

 of the tubes when the media dried, and make it difficult to remove 

 the plugs wholly when we wished to inoculate the contents of the 

 tubes. 



The tubes and flasks, plugged with sterile cotton and full of media, 

 are put in the steam sterilizer for one-half hour on three consecutive 

 days, or in the autoclave for fifteen minutes for two consecutive days. 

 A portion of the tubes containing nutrient agar are laid in a slanted 

 position before cooling, after the final sterilization, so that a larger sur- 

 face may be obtained. 



THE CULTIVATION OF BACTERIA. 



Bacteria can seldom be identified by their microscopic and staining 

 characteristics alone. We can only study their forms, arrangement, and 

 motility or lack of motility. To go beyond this we have to grow the 

 micro-organism in pure culture on the various culture media and per- 

 haps also in animals. It is necessary, also, to have the proper conditions 

 as to temperature, moisture, access of oxygen, etc. 



When we make cultures from any material, we are very apt to find 

 that instead of one variety of bacteria only there are a number present. 

 If such material is placed in fluid media contained in test-tubes, we find 

 that the different varieties all grow together and become hopelessly 

 mixed. When, on the other hand, the bacteria are placed on solid 

 media they develop about the spot where they were inoculated. If dif- 

 ferent varieties, however, are placed too near together, they overgrow 

 one another; it is thus advisable to have a greater surface of nutrient 

 material than is given on the slanted surface of nutrient agar or blood- 

 serum contained in test-tubes. This need is met by pouring the media 

 while warm on flat, cool, glass plates or into shallow dishes. 



Technique of Making Plate Cultures. In making plate cultures two 

 methods are carried out. In the first the material with its con- 

 tained bacteria is scattered throughout the fluid before it hardens; in 

 the second it is streaked over the surface of the medium after it has 

 solidified. Nutrient agar and nutrient gelatin, the two substances used 

 for plate cultures, differ in two essential points, which cause some differ- 

 ence in their uses. Nutrient 1 per cent, agar melts near the boiling 

 point and begins to thicken at about 36 C. It is not liquefied by bac- 

 terial ferments. Nutrient 10 per cent, gelatin melts according to the 

 variety used, at the low temperature of about 23 to 27 C., and solidifies 

 at a point slightly below that. It is liquefied by many bacterial ferments. 

 When we wish to inoculate fluid nutrient agar for plate cultures we have 

 to take great care that in cooling it to a point which will not injure the 

 bacteria, about 41 C., we do not allow it to cool too much and thus 

 solidify and prevent our pouring it into the plates. To prevent this, 

 when a number of tubes are to be inoculated, they are placed while still 



