V. PREPARATION OF BACTERIAL ENZYMES 641 



with a pore diameter of 5.5 to 7.5 m^i are used in a specially designed 

 filter of stainless steel. Filtration is carried out at a pressure of 50 

 pounds per square inch. To minimize contamination by air bacteria and 

 particles of dust, the air is filtered through Seitz asbestos pads in- 

 serted between the air line and each individual filter. Chloroform is 

 used as an antiseptic during filtration. At the end of filtration (it may 

 take 5 days to filter to dryness 600 cc. of enzyme solution), the mem- 

 branes are washed several times in distilled water and all the enzymatic 

 activity is thus recovered in solution. After filtration through Berkefeld 

 (V) candles, the preparation is frozen and in this state evaporated to 

 dryness in a vacuum. The final product is used dissolved in water. 



VI. Serological Reactions 

 Agglutination 



Since different lots of serum may exhibit different agglutinin titers, it 

 is advisable to test each batch of serum in varying dilution against a 

 culture of known type-specificity. It may occasionally be necessary, 

 also, to employ dilutions of the material (suspensions of living or killed 

 pneumococci) used as antigen, since zonal effects may inhibit complete 

 agglutination. As a rule, an 18-hour broth culture, containing approxi- 

 mately one billion organisms per cubic centimeter serves as a satisfac- 

 tory suspension for routine tests. 



For the test, it is convenient to mix 0.5 cc. of serum dilutions with an 

 equal amount of the culture. The serum dilutions may range from un- 

 diluted, for certain types, to 1 to 5 through 1 to 80 or 160. With un- 

 known types, it may be well to perform a bile-solubility test at the 

 same time. The mixtures are incubated in a water-bath at 37° to 40° 

 and examined at frequent intervals up to two hours. Those tubes show- 

 ing no agglutination, or an indefinite reaction, should be placed at 4° 

 overnight, and if the reaction is still negative, the tubes are then dis- 

 carded. 



With cultures of strains known to be closely related, it is well to per- 

 form tests for heterologous reactions. Thus, a Type V culture should be 

 tested with both Type V and Type II serum, and likewise a Type II se- 

 rum should be tested against both Type II and Type V cultures. 



Antipneumococcic rabbit serums exhibit a higher degree of type- 

 specificity than do horse serums, and for this reason are of value in case 

 of doubt regarding the type-specificity of recently obtained strains. 



