112 MEDICAL BACTERIOLOGY 



With a sterile capillary or graduated pipette make a i : 20 dilution of the 

 patient's blood or serum, using sterile water. Mix a drop of the diluted serum 

 with an equal quantity of the typhoid culture and place on the cover glass, invert 

 it and place on the hanging-drop slide. Examine under the microscope imme- 

 diately and again at intervals for % hour. 



If the bacteria are motile and not clumped when first observed and later 

 form clumps agglutination has occurred and the reaction is positive. If clump- 

 ing does not occur within ^ hour the reaction is negative. 



This test can be made, using dead bacilli in place of the living organisms. 

 As it is more convenient for general practitioners, a preparation giving satis- 

 factory results, known as "Picker's Typhus Diagnosticum," has been placed on 

 the market. 



QUANTITATIVE AGGLUTINATION TESTS 



Under strict aseptic precautions 2 cc. to 5 cc. of blood is withdrawn from a 

 vein, allowed to coagulate and the clear serum obtained. 



A dozen perfectly clean, sterile, 5 cc. capacity test-tubes are placed in a rack 

 and i cc. of sterile salt solution is put into each tube. One-tenth cc. of patient's 

 serum is placed in the first tube and mixed with salt solution; then, i cc. of the 

 contents of this tube is transferred to tube No. 2, mixed with the salt solution 

 and i cc. is removed from this tube, and mixed with the salt solution in No. 3, 

 and so on until the last tube has been reached. Then, each tube, except the first 

 tube, contains i cc. of diluted serum, the dilutions from tube No. 2 to tube No. 

 12 being as follows: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 

 1:5120, 1:10,240, 1:20,480, 1:49,960. 



One cc. of a 24-hour-old bouillon culture of the typhoid bacillus is added to 

 each tube. 



The tubes are incubated at 37C. for 3 hours, and then examined for agglu- 

 tination. In this manner the titre limit of the serum, or the highest dilution in 

 which it will agglutinate, is determined. 



After incubation, tubes in which agglutination has not occurred have the 

 same appearance as before incubation the fluid is uniformly cloudy, there 

 is no precipitate. Tubes in which agglutination has occurred contain clear 

 fluid and show a precipitate. 



If the macroscopic appearance is doubtful the contents of the tube is 

 poured into a thin, flat dish and examined with microscope, clumps of 

 bacteria being observed if agglutination has occurred. 



AGGLUTINATION TESTS WITH LABORATORY SERA 



Guinea-pigs and rabbits may be immunized against the typhoid bacillus by 

 intraperitoneal inoculations with the typhoid bacillus given as follows : 



First. Injection ^ cc. of 24-hour-old bouillon culture, heated to 56C. for 

 J^Jiour. 



Second. Injection i cc. of same. . 



Third. Injection 2 cc. of same. 



