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MANUAL OF MICROBIOLOGICAL METHODS 



versely, in the ''beta procedure," the serum is diluted while antigen is 

 held constant. 



A practical application of the optimal-proportions method and one 

 which will serve to illustrate general procedure for optimal-proportions 

 determinations is the Ramon flocculation test (beta method) used for 

 toxins and toxoids. 



To a series of test tubes (12 by 75 mm) add increasing amounts of anti- 

 toxin as shown in Table 23. This is ordinarily done with a 0.2-ml pipet 

 cahbrated in 0.001 ml. Then to each tube is added 2 ml of the toxin or 

 toxoid. The tubes are shaken, placed in a water bath at 50°C, and 

 observed continuously. The tubes in which flocculation is going to 

 appear will first become cloudy. Record the time at which discrete 

 floccules are first seen in one of the tubes. This tube represents the 

 optimal ratio between the particular batches of antitoxin and toxin used. 

 In Table 23, this ratio is represented by tube 6. If a more precise end 

 point is desired, the titration may be repeated, using smaller increments 



Table 23. Beta Procedure for Optimal Proportions Using Diphtheria 

 Toxin and Antitoxin (Ramon Flocculation) 



of toxin between 0.050 and 0.060 ml. In the case of diphtheria toxin and 

 antitoxin as used here, the L/ value may be calculated. This is defined as 

 the amount of toxin or toxoid which yields optimal flocculation with one 

 standard unit of antitoxin. Let us assume that the antitoxin in this case 

 had 425 units per milliUter, or 0.00235 ml = 1 unit. Since 0.055 ml of 

 antitoxin yielded optimal flocculation with 2.0 ml of toxin, 



'S _ 



0.00235 0.055 

 Lj = 0.086 



The toxin thus has 11.6 flocculating units per milUliter. The 7/ value 

 for the toxin could then be used to standardize an unknown antitoxin. 



