84 THE PRINCIPLES OF IMMUNOLOGY 



Dreyer, who has given much attention to agglutination in the diagnosis 

 of typhoid and paratyphoid fevers in individuals who have been vaccinated 

 against these diseases, maintains that heat and chemicals other than formal- 

 dehyde are inferior to the latter in killing and preserving the bacterial suspension. 

 He has given great attention to standardization of the reaction, an important 

 but not infallible precaution, where a patient, as in the army, is likely 

 to be examined in different laboratories during the course of the disease. 

 On this basis Dreyer has shown that saline emulsions from agar cultures are 

 inferior to broth cultures. Scheimann maintains in addition that the broth 

 cultures furnish a more permanent standard. Laboratories in which such 

 standards are prepared determine the optimum density of the agglutinable 

 cultures and also keep the emulsions until the early deterioration of agglu- 

 tinability produced by the formalin has reached a stationary point, after 

 which the standards remain practically unchanged for ten months and 

 probably longer. 



The primary test is carried out in small test tubes, with each dilution 

 one-half that of the preceding one. This simplifies making the dilutions, 

 especially if only one serum is to be tested. A row of twelve tubes is placed 

 in a rack and each tube receives 0.5 c.c. salt solution. To the first is added 

 0.5 c.c. immune serum, the mixture blown in and out of the pipette three times 

 and 0.5 c.c. transferred to the next tube, the processes repeated and 0.5 c.c. 

 transferred to the next tube, and so until the last tube is reached. In order 

 to preserve the constant volume in each tube, 0.5 c.c. is discarded from the 

 last tube. Thus there are dilutions 1-2, 1-4, 1-16, 1-32, 1-64, 1-128, 1-256, 1-512, 

 1-1024, 1-2048, 1-4096. To each tube is added 0.5 c.c. bacillus emulsion, thus 

 doubling each of the dilutions, so that instead of ranging from 1-2 to 1-4096, 

 they range from 1-4 to 1-8192. In the twelfth tube are placed 0.5 c.c. salt 

 solution and 0.5 c.c. bacterial emulsion to serve as a control of the emulsion 

 and prevent error due to spontaneous clumping of the organisms. The tubes 

 are placed in a water bath at 37 C. for one hour and then in the refrigerator 

 over night. The clumping is observed with the naked eye, the clumps being 

 visible and settling more rapidly than the bacterial emulsion. Should 1-512 

 of the final dilution show agglutination and 1-1024 fail to show it, the titer 

 lies between these two, and it is advisable to set up a series of tubes 1-500, 

 1-600, 1-800, 1-900, i-iooo, and repeat. The same, of course, is true of the 

 weaker dilutions, although beyond i-iooo the scale is more easily placed in 

 grades of 200 rather than 100. The preparation of such dilutions is illustrated 

 as follows: 



1 0.5 c.c. serum + 4.5 c.c. saline = i-io dilution 



2 0.5 c.c. No. i + 12.0 c.c. saline = 1-25 dilution 



3 0.5 c.c. No. i + 4.5 c.c. saline = i-ioo dilution 



4 0.5 c.c. No. 2 + 4.5 c.c. saline = 1-200 dilution 



5 0.5 c.c. No. 3 + i.o c.c. saline =1-300 dilution 



6 0.5 c.c. No. 2 + 5.5 c.c. saline =1-350 dilution 



7 0.5 c.c. No. 3 + 1.5 c.c. saline = 1-400 dilution 



8 0.5 c.c. No. 2 + 8.5 c.c. saline = 1-450 dilution 



9 0.5 c.c. No. 4 + 8.5 c.c. saline = 1-500 dilution 



Should we wish to determine a titer between 1-200 and 1-500, dilutions 

 4-9 are placed, 0.5 e.c. in each of six tubes, 0.5 c.c. emulsion added to each, 

 and in a seventh tube 0.5 c.c. saline and 0.5 c.c. emulsion as a control. The 

 tubes are placed in the water bath and incubated as before. Similar protocols 

 may be made if higher dilutions are required for the final test. Some workers 

 prefer to set up primary dilutions of i-io, 1-50, i-ioo, 1-200, 1-500, i-iooo, 

 1-2000, 1-4000, but this has no particular advantages as compared with the 

 primary titration outlined above. 



Microscopic Titration. The microscopic method may be employed with 

 the same method of dilution and mixing, simply removing a drop for obser- 

 vation in a hanging drop preparation at the end of the period of incubation 

 and examining with a 4-mm. lens. Another somewhat less accurate method is 

 to place one loopful of each dilution on a coverslip and mix with a loopful of 

 bacterial suspension, inverting the slip on a hollow ground slide, sealing with 

 vaseline, incubating and reading the result. A still less accurate method is 

 to place on coverslips or slides a row of loopfuls of salt solution, adding a 

 loopful of serum to the first drop, mixing, transferring a loopful to the second 



