118 LABORATORY MANUAL OF MICROBIOLOGY 



Exercise 29 



An^robic Nitrogen Fixation (Clostridium pasteurianum and 

 Related Organisms) 



Prepare four small flasks (or 6-ounce Signet bottles) with 150 

 cubic centimeters of Winogradsky's solution (Medium 11). 

 Arrange to have the liquid high in the necks of the flasks (or 

 bottles). 



As soon as possible after sterilization inoculate all of the flasks 

 with a pasteurized soil extract. Heat 50 grams of soil with 200 

 cubic centimeters of water for 15 minutes at 80°C. 



Allow the coarse particles to settle and pipette 5-cubic centi- 

 meter portions into each of the four flasks. 



Arrange as follows: 



(a) 1 and 2, controls (sterilize immediately, or add 5 cubic 

 centimeters of sulphuric acid. In order to avoid too rapid evolu- 

 tion of carbon dioxide, the sulphuric acid should be added slowly). 



(6) 3 and 4, cultures of anaerobic bacteria. Incubate all flasks 

 at 28°C. 



One week after incubation examine under the microscope a 

 drop taken from the lower layers of the liquid. This may be done 

 by inserting a pipette. Make a wet mount using Meissner's 

 solution. What forms are seen? Note the color. Make a 

 permanent spore stain, Dorner method. 



After 21 days transfer the entire contents to a Kjeldahl flask 

 and analyze for total nitrogen. 



Exercise 30 



Isolation of An^robic Nitrogen-fixing Organisms (Clostridium 

 pasteurianum and Related Forms) 



From the impure cultures of Exercise 26 or 29 make isolation 

 plates on agar medium (Medium 12). 



From the bottom of an actively fermenting culture take 1 or 2 

 drops and spread on the surface of the glucose peptone agar in 

 Petri dish. Dilute by spreading over the surface of at least three 

 plates. To secure the best results use Petri dishes with porous 

 covers. 



