326 THE STREPTOCOCCUS-PNEUMOCOCCUS GROUP 
Averv'* have found that the common mouth pneumococciis is usually 
the a\irulent type (Type IV) ; convalescents from pneumonia usually 
exhibit the virulent Types I to III, as a rule. These types can be 
identified by agglutination reactions with the specific sera prepared 
by Cole. 
Procedure. — S/j?/^?/?^.— Sputum should be obtained from the deeper 
air passages, as free as possible from mouth contamination. A frag- 
ment of mucus from such a specimen is washed through three suc- 
cessive transfers in sterile saline solution to reduce the surface con- 
tamination. Then grinding is practised in a sterile tube, using a 
sterile glass rod as a pestle, adding small successive amounts of 'sterile 
saline solution to a total volume of about 1 cc. 
One-half a cubic centimeter of the emulsion is injected into the peri- 
toneal cavity of a white mouse; the remainder is stained by Gram's 
method and by a capsule stain. Type III pneumococcus usually 
shows a distinct capsule by this procedure. 
When the mouse shows signs of infection (five to eight hours, usually) 
it is killed and the peritoneal fluid removed with sterile precautions. 
Cultures from the heart blood are made upon blood agar plates. 
The peritoneal fluid is mixed with about 5 cc. of sterile saline solution 
and lightly centrifugalized, to remove clumps. Macroscopic agglu- 
tination tests are made, using the specific type sera. It has been 
found that 0.5 cc. of the centrifuged mouse culture of pneumococci 
added to an equal volume of type serum diluted 1 to 20 will usually 
give clear-cut reactions,^ 
Table Showing Determination of Pneumococcus Types. 
Type serum, Jo dilution, 0.5 cc. 
Pneumococcus, ' ^ 
Mouse exudate 0.5 cc. I. II. III. 
Type I . . + 
"II - + 
"III - - + 
"IV - 
A tube containing 0.1 cc. of sterile ox-bile, and 0.5 cc. of the sus- 
pension is also set up. The solubility of the organism in bile is of 
importance in excluding streptococci. Inoculation at .37° C. is 
practised for one hour. 
An organism not agglutinated by any of the specific sera, soluble 
however, in bile is presumably a Type IV pneumococcus. If the 
Gram stain shows considerable numbers of bacteria not pneumococci 
in the peritoneal fluid of the mouse, plating upon blood agar, and 
subculture in ascitic broth, must be resorted to before performing the 
agglutination tests. 
If mice are not available, the rapid cultural method of Avery^ 
1 Quoted by Cole: Loc. cit. 
2 Avery, Chickering, Cole and Dochez: Monograph No. 7, Rockefeller Institute, 
October 16, 1917. 
= Jour. Am. Med. Assn., 1918, 70, 17. 
