144 PRACTICAL METHODS IN IMMUNITY 



culture or salt solution suspension of the organism to be tested gives a dilution of 

 i to 40. One loopful of the 1-20 diluted serum and 3 loopfuls of the bacterial sus- 

 pension give a dilution of 1-80. These two dilutions answer in ordinary diagnostic 

 tests. The red pipette with a i-ioo or 1-200 dilution may be used where dilutions 

 approaching i-iooo are desired. Having mixed the diluted serum and the bacterial 

 suspension on a cover-glass, we invert it over a vaselined concave slide and examine 

 with a high power, a dry objective (1/6 in.). It is simpler to make a ring of vaseline 

 to fit the cover-glass and make the mixture of diluted serum and culture in the center 

 of this ring or square. Then apply the cover-glass, press it down on the vaseline 

 ring and examine as with the ordinary hanging drop. In making dilutions it is 

 preferable to use salt solution, as the phenomenon of agglutination requires the 

 presence of salts. Ordinarily, thirty minutes is a sufficient time to wait before 

 reporting the absence of agglutination. Agglutination is more rapid at body 

 temperature than at room temperature. In reporting agglutination, always give 

 time and dilution. It is absolutely necessary that a control preparation be prepared 

 in every instance; that is, one with the bacterial culture alone or with a normal 

 serum of the same dilution as the lowest used. Some normal sera will agglutinate 

 in i to 10 dilution, and group 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 the specific one in dilutions as high as 1-80. 



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

 (3/8X3 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 12 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 dilu- 

 tion of i to 1 6. 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 culture 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 16, acting on the bacteria; tube 2 of a i to 32; 

 tube 3 of a i to 64. Now place these tubes in the incubator and, after two to five 

 hours or overnight, 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, take a twenty-four-hour agar slant culture of 

 typhoid or paratyphoid and emulsify in salt solution (about 6 c.c. to a slant). 



By adding o.i of i% of formalin to the typhoid emulsion and placing in the ice- 

 box the cultures will be found sterile in about three days. The emulsion should be 

 shrken twice daily while undergoing sterilization in the ice-box. Such cultures 



