THE TECHNIQUE OF SERUM REACTIONS 251 



on Opsonins, p. 285), or by carefully rubbing the clumps against the 

 watch glass with a stiff platinum wire. In the case of the tubercle ba- 

 cillus not even this suffices, but it becomes necessary to grind the moist 

 bacillary masses in a mortar before emulsifying. With the tubercle bacil- 

 lus, too, it is preferable to use salt solution at 1.5 per cent concentration. 



In preparing cultures of streptococcus and pneumococcus for 

 agglutination tests, it has been found convenient by Hiss to grow 

 microorganisms for about four days in flasks of a one per cent glucose, 

 two per cent pepton meat-infusion broth, to which has been added one 

 per cent of calcium carbonate. (See page 126.) The insoluble calcium 

 carbonate sinks to the bottom, but by neutrahzing the inhibiting acid 

 formed in the broth by the microorganisms, permits the development 

 of a mass culture. The flasks should be shaken thoroughly at least 

 once a day. The broth may be pipetted off and the clumps may be 

 removed by a few revolutions of a centrifuge. Without this technique 

 it is sometimes difficult to get sufficient growths of these bacteria for 

 any quantity of emulsion unless large surfaces of agar are employed in 

 special receptacles or by making many slant cultures. 



The serum dilutions are obtained by first making a one to ten 

 dilution of serum with normal salt solution. The serum used for this 

 purpose may be cleared of red blood corpuscles by centrifugalization. 

 From the one to ten dilution any number of higher dilutions may be 

 made, simply by mixing given parts of the one to ten dilution with 

 normal salt solution; thus one part of a one to ten dilution plus an equal 

 quantity of salt solution gives a dilution of one to twenty. One part of 

 one to ten dilution plus two parts of normal salt solution gives one to 

 thirty, and one part of one to twenty dilution plus one part normal salt 

 solution gives one to forty, etc. It must not be forgotten that, when 

 equal parts of the serum and bacillary emulsion have been mixed, each 

 one of these dilutions is doubled. 



In making the microscopic agglutination test, minute equal quanti- 

 ties of serum dilution and bacterial emulsion are mixed upon the sur- 

 face of a cover-shp. The mixture may be made either by measuring out a 

 drop of each substance with a standard platinum loop, depositing them 

 close together on the cover-sKp, and mixing; or, more exactly, equal quan- 

 tities may be sucked up, each to a given mark, in a capillary pipette, 

 mixed by suction in and out, and then deposited upon the cover-slip. 

 The cover-slip is inverted over a hollow glass slide, the rim of which has 

 been greased with vaseline. The drop is then observed, preferably 

 through a (Leitz) No. 7 lens, ocular No. 3. 

 17 



