AGGLUTINATION 169 



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 i to 80. 



Macroscopic Agglutination. 2. For the macroscopical or sedimentation test, take a 

 series of small test-tubes (% X 3 inches) 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 i to 4 dilution 

 of the serum. It is more exact to make the i to 4 dilution with a graduated pipette. 

 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 2 c.c. of 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 now 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 in- 

 stead 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 

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

 are not easily contaminated and appear to retain their agglutinable qualities for 

 several months. The macroscopic methods are preferable with such dead cultures. 



A very convenient method in general use in Germany is the follow- 

 ing: Make dilutions of serum in ordinary test-tubes (% X 6 inches) 

 as described for the small test-tubes. 



Then take a loopful (2 mg.) of culture from an eighteen to twenty-four-hour-old 

 agar culture and emulsify it thoroughly in the dilution in the first test-tuberepeat 

 the process in the second tube and so on. This procedure is much safer than when 



