110 LABORATORY MANUAL OF MICROBIOLOGY 



mercuric chloride in a partial vacuum. Rinse in sterile 

 water. 



In the case of peas, cowpeas, soybeans, etc., which are to be 

 grown in pots of sterilized sand, the seed may be dipped in hot 

 water (65°C.) for 4 minutes to kill the nodule bacteria. Spread 

 out and dry quickly in an atmosphere free from dust. 



Place three to five seeds each of alfalfa or red clover into a 

 large tube containing agar Medium 106. 



Plant the peas, soybeans, and related plants, four or five 

 seeds, in half-gallon pots of sand which has been sterilized 4 

 hours in an autoclave at about 110 to 115°C. Cover the seed 

 with about 1 inch of sand and add sterile distilled water. 



To inoculate, prepare a water suspension of a young culture of 

 the desired organism and from this take 1 cubic centimeter for 

 each tube or pot. 



Peas or Soybeans (Pots) Alfalfa or Clover (Tubes) 



(a) 1 and 2 uninoculated. 1, 2, and 3, uninoculated. 



(6) 3 and 4 inoculated with known 4, 5, and 6, inoculated with known 



culture. culture. 



(c) 5 and 6 inoculated with an un- 7, 8, and 9 inoculated with an un- 

 known culture. known culture. 



{d) 7 and 8 inoculated with an un- 10, 11, and 12, inoculated with an 



known culture. unknown culture. 



Under favorable conditions, nodules will begin to form on the 

 clover and peas in 10 to 15 days. With alfalfa and soybeans it 

 requires about 21 to 28 days. 



Keep cultures in a greenhouse, free from dirt and dust. 



Plants in open pots should be watered from time to time 

 with sterile distilled water. About once a week, add about 30 

 to 40 cubic centimeters of the nitrogen-free modified Crone's 

 solution. 



After 4 to 6 weeks examine for nodules; note the number, 

 size, shape, and location. 



Exercise 21 

 Artificial Cultures for the Inoculation of Legumes 

 Prepare two 6-ounce Signet bottles of mannitol agar (Medium 

 77) about 30 cubic centimeters of agar in each. After steriHza- 



