172 LABORATORY GUIDE IN BACTERIOLOGY 



3. As soon as growth has appeared transfer some of 

 the growth from the first flask to the second. 



4. As soon as growth has appeared in the second 

 flask transfer some of the growth to the third flask. 



5. Repeat the procedure until all five flasks have 

 been inoculated. This method of transferring repeat- 

 edly gradually weeds out all bacteria but those which 

 are able to grow in the solution, and these may thus 

 be obtained in pure culture. 



6. Examine these bacteria by making stains, hang- 

 ing drop, spore stain, etc. 



7. Test each flask for nitrites with the starch iodin 

 test as follows. Prepare the following solutions: 



a) Dissolve 2 grams starch in 100 c.c. of water by 

 boiling. 



b) Dissolve one gram potassium iodid in 100 c.c. of 

 distilled water. 



The potassium iodid solution must be freshly pre- 

 pared. Add a few drops of diluted H2SO4 to liberate 

 nitrous acid. Then add i c.c. of each of the solutions 

 to s c.c. of the fluid to be tested. If nitrous acid is 

 present, iodin wUl be liberated and will give a blue 

 reaction with starch. 



EXERCISE 2. ISOLATION OF NITRITE BACTERIA 



Experiment i. — Agar method — 



1. Prepare the agar medium described on p. 38. 



2. Fill 10 tubes, and sterilize in the autoclave. 



3. Pour plates froni this medium inoculated with 

 the contents of the flasks prepared in Exercise i in 

 various dilutions. 



4. Incubate at room temperature until growth has 

 appeared. 



