ESTIMATION OF THE OPSONIC CONTENT OF THE BLOOD 217 



It has been recommended that the cultures should not be more 

 than twenty-four hours old. This, however, is not necessary for 

 all organisms. Knorr has shown in my laboratory that the same 

 degree of phagocytosis is obtained with cultures of the staphylococcus 

 more than a month old, as with young cultures. In the case of 

 the typhoid and the colon bacillus, Wright recommends the use 

 of cultures only four hours old, as with older cultures the resultant 

 spherulation of the organisms is such that approximative results 

 only can be obtained. 



In the case of the tubercle bacillus, Cole obtained the best results by 

 starting with living cultures on glycerin agar, which had been killed 

 by exposure to sunlight for twenty-four hours. Some of the mate- 

 rial is then scraped off, ground up in an agar mortar with 1.5 per cent, 

 saline and centrifugalized to remove clumps. Cole states that if 

 contamination is guarded against the supernatant fluid may be used 

 for at least a month. I have not had occasion to use emulsions pre- 

 pared in this manner, and am familiar only with emulsions made 

 from dead and ground-up bacilli. A small quantity of this material 

 is placed in an agate mortar and thoroughly triturated with 1.5 per 

 cent, saline, which is slowly added drop by drop. The resultant 

 emulsion may be freed from coarser clumps by centrifugation, but 

 the smaller ones are practically impossible to remove. I have worked 

 with heated and unheated, with extracted and non-extracted bacilli, 

 with 0.1 and 1.5 per cent, saline, but I have not yet seen an emulsion 

 of tubercle bacilli that was uniform. 



In the case of the tubercle bacillus, Wright recommends that the 

 emulsion should be of such strength that in the actual experiment 

 one or two bacilli only are found on an average in each cell. 



The Experiment Proper. Having prepared the patient's serum, 

 normal control serum, washed corpuscles, and the bacterial emulsion, 

 these "reagents" are placed in a small rack, or in a dishful of sand 

 covered with a piece of white filter paper, perforated to receive the 

 tubes, and marked accordingly. 



Mixing pipettes (Fig. d or e, Plate III) are prepared from glass 

 tubing having an outside diameter of approximately 6 mm. To 

 this end pieces of tubing are cut, measuring about 15 cm. in length, 

 heated in the middle in the flame of a Bunsen burner until soft, 

 and then drawn out after removal from the flame, so that capillary 

 stems are obtained about 10 to 15 cm. long, with a diameter of from 



