82 LABORATORY MANUAL OF MICROBIOLOGY 



Suction Flask. — The suction flask H is provided with a constant 

 pressure valve / which is made from small glass T-tube in which 

 a rubber band holds a small rubber disk over the lower end. By 

 the aid of this valve and the screw cock G, the flow of air can be 

 easily regulated and should not exceed about 120 bubbles per 

 minute. 



1 cubic centimeter 0.5A^ NaOH =c= 3.0 milligrams of carbon. 

 Cubic centimeters NaOH added— cubic centimeters HCl used X 



3 = milligrams carbon. 



SEED STERILIZATION 



Although a great number of methods employing various agents 

 have been recommended for removing microorganisms from seed, 

 only a few of the more promising ones will be given. Where it is 

 not necessary to render the seeds free of bacteria, but merely to 

 destroy the majority of the flora, alcohol may be used. 



Among the chemicals that have proved satisfactory for steriliz- 

 ing seed, mercuric chloride, hypochlorite of lime, and silver 

 nitrate are the most commonly used. The effectiveness of these 

 substances depends on many factors: strength of solution, time 

 of exposure, temperature, pressure, and nature of the seed coat. 



Sterilization hy Mercuric Chloride in Vacuum. — Select a large 

 heavy walled desiccator and connect to vacuum pump. 



Fill test tubes or flasks about one-half full of seed, cover with 

 0.25 per cent solution of mercuric chloride and place in the 

 desiccator. 



Exhaust for 3 to 5 minutes depending upon the kind of seed. 

 This should remove the air particles from around the seed coats 

 and allow the disinfectant to come in direct contact with the 

 seed. 



At the end of this time remove the mercuric chloride solution 

 and run in a small amount of sterile water, shake vigorously, 

 empty, and repeat this process three or four times. 



Remove some of the seed to sterile Petri dishes and pour over 

 them a layer of nutrient agar. 



After the agar hardens, invert and place in the incubator at 

 20 to 25°C. In 2 or 3 days the seed should germinate. If 

 bacteria or molds are present, they may be readily noted on 

 the agar. 



