PREPARATION OF UREASE FROM UREA BACTERIA 37 



5. Incubate the gelatin plates at 20 C. 



6. Examine the plates every forty-eight hours for a 

 period of ten days. The urea organisms are often character- 

 ized by a distinct halo around the colonies. Under the 

 low power of the microscope the halo is composed of dumb- 

 bell-shaped crystals. 



7. Make transfers to tubes of urea gelatin and incubate 

 for two days. 



8. Now test the ammonia-producing power of the pure 

 cultures by inserting sterilized strips of Nessler's paper 

 or turmeric paper in the upper part of the tube. 



9. Prepare a stained mount of these two organisms. 



Exercise 3 

 Preparation of Urease From Urea Bacteria 



1. Inoculate two large Erlenmeyer flasks, looo-c.c. 

 capacity, containing 100 c.c. each of urea bouillon (m. 19) 

 with a pure culture of urea fermenter. A very active 

 culture should be used. 



2. Incubate at 30 C. until ammonia formation is very 

 evident. 



3. In order to secure the urease free of bacteria, filter 

 aseptically through a Berkefeld filter. 



4. Prepare a 10 per cent, solution of urea in distilled 

 water. Now mix equal volumes of the nitrate and the 

 urea- water solution. 



5. Incubate the enzyme culture at 48 to 50 C. until a 

 large part of the urea nitrogen is converted into ammonia. 



6. The action of the urease may be measured by Nessler- 

 izing aliquot portions of the treated and untreated urea- 

 water. 



