MACROSCOPICAL AGGLUTINATION. 127 



agglutinations (as paratyphoid with typhoid serum) may occur in i 

 to 40 or possibly higher. It is very unusual for sera to agglutinate 

 any other bacteria than its specific one in dilutions as high as 1-80. 

 2. For the macroscopical or sedimentation test, take a series of 

 small test-tubes (3/8 x 3 in.) and deposit i c.c. of salt solution in each 

 of the series. Now, having taken an empty test-tube, drop 4 drops of 

 serum in it and then add 1 2 drops of salt solution. This approximately 

 gives i c.c. of a 1-4 dilution of the serum. With a rubber-bulb capillary 

 pipette, which has been graduated to hold 16 drops or i c.c. draw up the 

 contents of the tube containing the i to 4 serum and add it to the next 

 tube containing i c.c. of salt solution. This gives a dilution of i to 8. 

 Now mix thoroughly by drawing up and forcing out with the bulb 

 pipette, and then withdraw i c.c and add to the next tube containing 

 i c.c. of salt solution. This gives a dilution of i to 16. Having mixed as 

 before, again withdraw i c.c. of the mixture and add it to the i c.c. in the 

 next tube. We now have a dilution of i to 32. Again withdrawing i c.c. 

 and adding it to the fourth tube containing i c.c. of salt solution we have 

 a dilution of i to 64. In tube i there is i c.c. of a dilution of the serum 

 of i to 8; in tube 2, there is i c.c. of a dilution of i to 16; in tube 3, of 

 i to 32. Tube 4 contains 2 c.c. of i to 64. Now adding i c.c. of a cul- 

 ture of typhoid or any other organism, we have the dilution of the 

 serum in each tube doubled. Tube i now contains a serum in dilution 

 of i to 1 6, acting on the bacteria; tube 2 of a i to 32; tube 3 of i to 64. 

 Now place these tubes in the incubator and after 2-5 hours or over- 

 night, we examine for the clearing up of the supernatant fluid. If the 

 serum in a certain dilution agglutinates, the clumps gravitate to the 

 bottom and the upper part becomes clear. If so desired, these dilutions 

 may be carried on to i to several hundred in the same way. It is safer 

 to work with dead cultures instead of living ones. To prepare, in- 

 oculate a flask of bouillon containing about 150 c.c. w r ith typhoid or any 

 other culture. Allow to grow for 18 to 24 hours and then add i c.c. of 

 formalin. One percent of formalin is frequently used to kill the cul- 

 tures. At the end of 24 hours the sterile cultures may be used as 

 with the live cultures.* 



*A very convenient method in general use in Germany is the following: Make 

 dilutions of serum in ordinary test-tubes (J by 6 inches) as described for the small 



