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. To Tube 8 add a tube of agar melted and cooled 

 to 42 C. and pour into a Petri plate immediately. Incubate the other 

 tubes two hours at 37 C. Add melted agar to each tube and pour 

 plates. When the plates are hard place them upside down in the incu- 

 bator and examine after 24 to 48 hours. 



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 

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

 up and plated in 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 No. 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 colonies 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. 



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 



