364 PATHOGENIC MICROORGANISMS 



has taken place. Dilution with water until the solution equals 0.85 

 per cent NaCl now prepares the emulsion for inoculation. Whichever 

 of the various methods is adopted, the intervals of injection should not 

 be shorter than a week, preferably ten days. The animals so immunized 

 will at the end of six or more weeks withstand an inoculation with many 

 times the fatal dose of virulent pneumococci. The sera of animals 

 immunized with pneumococci contain active bacteriolytic and bacteri- 

 cidal substances, easily demonstrable in vivo and in vitro. 



Specific afiffifZwiinins in pneumococcus immune sera were first thoroughly 

 studied by Neufeld ' and since then have been made the subject of ex- 

 tensive studies by Wadsworth,^ Hiss,' and many others. In the sera of 

 normal animals and man, pneumococci are rarely agglutinated in dilu- 

 tions higher than one in ten. In the serum of patients suffering from 

 lobar pneumonia, pneumococci agglutinate in dilutions ranging any- 

 where from one in ten to one in fifty. In the sera of immunized rab- 

 bits, readings up to one in 800 are not rare. Such specific agglutinating 

 sera are most reliable in differentiating between pneumococci and 

 closely allied bacteria and in identifying all pneumococci. 



The table on page 365 illustrates this uniformly high agglutinative 

 power of various pneumococcus-immune sera upon several races of this 

 microorganism, and shows the value of such sera for biological differenti- 

 ation. The table, furthermore, records the peculiar fact that pneu- 

 mococci are agglutinated in high dilution by sera obtained by immu- 

 nization with Streptococcus mucosus, a fact which argues strongly 

 in favor of classifying Streptococcus mucosus more intimately with 

 the pneumococci than with the Streptococci of the pyogenes group. 



To overcome the difficulties often attending agglutination tests 

 with pneumococci, Wadsworth ^ has proposed centrifugalizing young 

 broth cultures and shaking up the sediment with small quantities 

 of isotonic salt solution. Hiss recommends ^ cultivation in glucose- 

 calcium-carbonate broth in small flasks containing 100 to 150 c.c each. 

 After three or four days at 37° C, the growth is usually at its optimum 

 for agglutination work. The flasks should be thoroughly shaken at 

 least once in twenty-four hours. About one hour before use the flasks 

 are again shaken and the calcium carbonate and larger clumps are 

 all owed to settle. ^^ 



^Neufeld, loc. cit. 



2 Wadsworth, loc. cit. 



^Hiss, Jour. Exp. Med., vii, 1905. 



* Wadswcrrth, Jour. Med. Res., x, 1905. 



' Hiss, loc. cit. 



