THE ^UTiUTKXN OF BACTERIA 



2. Dissolve these ingredients by boiling them over a gas flame 

 with constant stirring. A better method, and one that obviates 

 the danger of burning the material, is to cook it in the autoclave 

 at 10 Ib. steam pressure (see Exercise 12). After cooking for 

 twenty minutes add 50 cc. distilled water to replace that lost 

 by evaporation. 



3. Filter immediately through a thin layer of wet absorbent 

 cotton in a funnel. Place the filtered medium in a funnel with 

 delivery tube and pinchcock, as shown 



in Fig. 5. 



4. Fill test tubes to the depth of 

 about 4 cm. with the liquid agar, taking 

 care that none is smeared on the inner 

 surface of the tube near the mouth, as 

 this will cause the cotton to stick to 

 the glass when the plugs are removed. 



5. Plug the mouths of the test tubes 

 with cotton. The plugs are rolled from 

 strips of cotton about 5 cm. wide, and 

 should fit firmly enough to allow the 

 tube to be lifted by that part which 

 projects; at the same time they should 

 be loose enough to permit interchange 

 of gases. The plug should extend into 

 the tube for about 3 cm., and should be 

 in contact with the wall throughout this 

 distance. The part of the plug which 

 projects from the mouth of the tube 



should overhang the lip of the tube enough to prevent dust 

 from lodging there (Fig. 6). 



6. Sterilize in the autoclave according to directions given in 

 Exercise 12. 



7. After sterilization lay the tubes in an oblique position, so 

 that the agar may solidify in the manner shown in Fig. 7. This 

 produces what is known as an agar slant, or agar slope, and is 

 much used because it gives a larger surface for the development 

 of the bacterial colony. 



FIG. 4. Funnel arranged 

 for filtering agar media 



The layer of absorbent cotton 

 is supported by a wad of ex- 

 celsior or clean straw 



