60 LABORATORY COURSE IN SERUM STUDY 



Add to each tube about 0.1 c.c. of a 24 hour broth culture of B. 

 typhosus, diluted 1-500. This should be measured by making a mark 

 on a sterile capillary pipette, filling accurately to this mark and dis- 

 charging the contents against the side of the tube, close to the level of 

 the fluid, being careful not to touch the upper portion. The actual 

 amount used is unimportant, but precisely equal amounts must be added 

 to each tube. Shake. Incubate the tubes two hours at 37 C. At the 

 end of this time convey two loopsful from each tube into melted and cooled 

 agar tubes and pour plates in the usual way. Transfer of two loops from 

 tube eight should also be made into agar in this way immediately after 

 mixing without incubating. 



In careful experiments, two additional controls, the one containing 

 0.5 c.c. of 1-50 normal serum, the other containing 0.5 c.c. of 1-50 im- 

 mune serum without the addition of typhoid bacilli, should also be set 

 up and transfers should be made from them into agar to prove the sterility 

 of the reagents used. A successful experiment should show an enormous 

 number of colonies, over 10,000 to the plate, in plates Nos. 5, 6 and 7. 

 Plate 8 should also show a large number of colonies, though perhaps less 

 than the other controls. In plates 2 and 3 there should be very few col- 

 onies or none at all; in plates 1 and 4 some reduction in the number, 

 though in some cases there may be no observable difference between 

 these plates and the controls. 



Each student needs: Five f-inch test tubes, twelve petri dishes, 

 twelve tubes of agar, four one-c.c. pipettes. 



LESSON IX 

 I. TITRATION OF AGGLUTININS BY MACROSCOPIC METHOD 



THE clumping of bacteria by immune serum causes a homo- 

 geneous suspension to form flocculi easily visible to the naked eye, 

 which, on standing, settle to the bottom of the tube, making 

 the supernatant fluid clear. The macroscopic test is considered 

 more accurate than the microscopic since one is less often de- 

 ceived by small clumps of bacteria which may form spontaneously 

 in salt solution or broth for various reasons and are readily 

 visible under the microscope but do not come together in large 

 enough masses to cause a precipitate visible to the naked eye. It 

 is much simpler to carry out a quantitative titration of the aggluti- 

 native power of the serum by setting it up in test tubes than 

 by preparing hang-drops of each dilution. Furthermore, since 

 the quantities used in these tests are relatively large, slight inac- 

 curacies in measurement do not lead to such gross errors as would 



