108 AGGLUTINATION 



takes place in the dilution of i :ioo; a positive result in the dilution of i : 50 can 

 only be considered as probably positive. 



As has been said, broth cultures may be used for the agglutination test if the 

 bacteria grow diffusely and regularly within the bouillon. This is not the case, how- 

 ever, with all bacteria, as for example, the cholera vibrio which produces a thin 

 pellicle upon the surface of the broth. 



b. A gar Cultures. Kolle and Pfeiffer have advised instead of broth 

 the use of agar cultures. The bacteria are washed off, and an even emul- 

 sion made in physiological salt solution, or in a dilution of the serum for 

 examination. 



The details of the procedure are as follows: 



Into a row of test-tubes is placed i c.cm. of various dilutions of the serum for exami- 

 nation, e.g., i : 10, i : 50, i : 100, i : 200, i : 500. A normal serum is similarly 

 diluted as a control. One other test-tube is to contain i c.cm. of saline only. 



A full loop of an eighteen to twenty-four hours old agar culture is evenly and finely 

 rubbed up in each of the above test-tubes as follows: 



The test-tube is held almost horizontally in the left hand between the thumb and 

 index finger; a platinum loop between the thumb and index finger of the right hand is 

 filled with the bacteria from the agar culture, and placed in the tube containing the 

 serum dilutions. The bacteria are then gently and thoroughly rubbed up on the 

 moistened wall of the tube but not within the fluid. By rolling the test-tube slightly, a 

 part of the rubbed up bacteria is washed into the fluid and the remaining bacterial mass 

 is again triturated. This process is repeated until all the bacteria are washed into the 

 fluid. Thus, a homogeneous suspension is obtained. 



The author has found this method of Peiffer and Kolle most accurate. 



It is worthy of note in this connection, that the controls show no clumps 

 or granules. (Pseudo-agglutination). There are some bacteria which 

 can be evenly emulsified only with great difficulty, while others are very 

 easily agglutinated even by normal serum. In either case the test is not 

 conclusive. 



For the hanging-drop method, blood is collected in a Wright capsule or 

 a small test- tube-, 6 to 8 drops of blood suffice. The blood is allowed to clot 

 or the serum is hastened by centrifugalization. Four loopfuls of broth or 

 saline (or equal amounts as measured by a Wright pipette) are placed on 

 each of two slides. To one of these one loopful of the serum (or one equal 

 part as measured by the Wright pipette) is added and thoroughly mixed. 

 From this mixture one loopful or equal measure is mixed with the broth or 

 normal saline upon the second slide; thus making serum dilutions of i 15 

 on the first slide and i : 25 on the second slide. A loopful of typhoid cul- 

 ture is placed on the center of each of two cover slips. To the first is 

 added one loopful of the serum dilution 1 125, and to the second is added 

 one loopful of the serum dilution i : 5 thus making a dilution of i : 50 and 



