SEROLOGICAL METHODS 



207 



Tube agglutination test. Serum is diluted in a series of 10- by 75-mm 

 test tubes using a twofold dilution series as illustrated in Table 21. 

 Into the first tube 0.8 ml of saline solution and 0.2 ml of antiserum are 



Table 21. The Tube Agglutination Test 



Saline, ml 



Serum, ml 



Transfer to next tube, ml . . 



Antigen, ml 



Reciprocal of final serum 



Tube No. 



0.8 

 0.2 

 0.5 

 0.5 



0.5 



0.5 

 0.5 



dilution ! 10 20 



0.5 

 0.5 



0.5 

 0.5 



0.5|0.5 



6 ! 7 



0.5 0.5 



0.5 0.5 

 0.5 0.5 



0.5 



0.5 

 0.5 



0.5 



0.5 

 0.5 



0.5 



0.5 



40 I 80 ! 160 320 I 640 \ 1280 I 2560 



I i I 



Antigen 

 control 



0.5 



0.5 

 



* Discard 0.5 ml. 



pipetted. These are mixed by taking a clean pipet and alternately 

 sucking up about 0.5 ml into the pipet and blowing it back into the tube 

 several times. Following this, 0.5 ml of the 1:5 dilution in tube i is 

 transferred into tube 2. This process of mixing and then transferring 

 0.5 ml into the next tube is followed with each tube in the series, using a 

 clean pipet for each transfer. When the last tube is reached, 0.5 ml is 

 discarded. An additional tube containing only saline solution is included 

 for a control on the antigen suspension. Next, 0.5 ml of antigen suspen- 

 sion is placed in each tube, and the tubes mixed by shaking. The test is 

 placed in a constant-temperature water bath for 2-4 hr. The tempera- 

 ture at which the water bath is set may be 37-55°C, or any constant tem- 

 perature within this range, depending on the system being studied. As 

 a rule, the arbitrary selection of 37° will yield excellent results with bac- 

 terial antigens. The clumping may be read after this preliminary incuba- 

 tion, but as a general rule, more pronounced reactions are secured follow- 

 ing storage in the refrigerator for 12-18 hr. Clumping may also be 

 accelerated by centrifuging the tubes. 



When a tube agglutination is read, each tube starting with the antigen 

 control is tapped 4-5 times with the finger tip until the cells are resus- 

 pended. The antigen control should display uniform turbidity in con- 

 trast to the clumping seen in a positive reaction. The size of the clumps 

 will depend on the potency of the antiserum and the physical nature of 

 the antigen. Cells like the ''O" antigen suspensions of Salmonella usu- 

 ally produce a granular agglutinate, while flagellated or ''H" cells yield a 

 more ''fluffy" type of clump, and capsulated organisms often produce a 

 solid button which cannot readily be broken up. As the serum becomes 



