478 Pneumonia 



As much valuable time is lost in the treatment of the case through 

 the delays in making a diagnosis of the type of organism concerned 

 by this method, Mitchell and Muns* recominend a new and 

 quicker method, tried upon 69 sputa from as many cases of pneu- 

 monia, and controlled by the mouse method. The method depends 

 upon obtaining material for specific precipitation from the sputum 

 itself. By this means the identification of the type of pneumococcus 

 may be made in an hour or two. The method is as follows : 



About s cc. of sputum, collected in a sterile container, is pipetted into a small 

 mortar and sufi&cient relatively fine sand added to make a rather stifE mixture. 

 This is ground with the pestle and in about three minutes becomes a thick, 

 tenacious gritty paste. Ten cubic centimeters of normal saline solution are now 

 added, two at a time, the stirring being continued. After thorough mixing, the 

 whole is permitted to stand for a few minutes, that the sand may settle. The 

 fluid is then pipetted into a clean centrifuge tube. Another 10 cc. of saline solu- 

 tion is then added to the sandy precipitate and stirred for a few minutes, fiUowed 

 to sediment and then pipetted into a second centrifuge tube, held in reserve. 

 The tubes are now centrifuged at high speed until the fluid is perfectly clear. 



Into each of three small test-tubes 0.2 cc. of immune sera, type I, t5T)e II 

 and type III, respectively are placed, and to each i cc. of the centrifugated 

 sputum fluid is added, carefully mixed by shaking and stood in a water bath at 

 37°C. Specific precipitation soon appearsr in the tube containing the serum 

 corresponding to the type of pneumococcus occasioning the pneumonia from 

 which the serum was obtained. It sometimes appears at once, sometimes only 

 after an hour or two and is distinctly feathery in appearance. The precipitate 

 sediments after standing in the water-bath. 



Averyf recommends a simple method in which the mouse is elimi- 

 nated and advantage taken of the accelerating influence of glucose 

 and bloOd proteins upon the growth of pneumococcus, and the lytic 

 action of ox bile for pneumococci alone. The method is carried out 

 as follows : 



A kernel of sputum is selected with care, as coming from the deeper portions 

 of the air passages and being as free as possible from admixture of naso-pharyn- 

 geal mucus and saliva. It should be caught in a sterile container and have a 

 bulk about equal to that of a bean. If it cannot be examined at once it should be 

 kept on ice. Such a mass of material should be placed in a centrifuge tube con- 

 taining 4 cc. of a medium specially adapted to the cultural requirements of the 

 pneumococcus and made as follows : A meat infusion broth is made and titrated 

 with phenolphthalein to 0.3-0.5 acid. It is then sterilized on three successive 

 days in the Arnold sterilizer, twenty minutes each time. To each 100 cc. of 

 the broth one adds s cc. of a sterile 20 per cent, solution of glucose and $ cc- of 

 sterile defibrinated rabbits' blood. The medium, thus containing i per cent, of 

 glucose and 5 per cent, of blood is distributed in small centrifuge tubes, about 

 4 cc. in each tube. 



The tube of medium inoculated as directed above is placed in a water-bath 

 and kept at 37°C. for five hours. A blood-agar plate is then streaked with a 

 platinum loop fuU of the culture and set aside for the isolation of the pneumo- 

 coccus, should that prove to be desirable. 



The type determination may now be made either by specific precipitation or by 

 agglutination. 



r. The Method by Specific Precipitation. — The tube is placed in the centrifuge 

 and whirled at low speed to throw out the blood-corpuscles, after which 3 cc. of 

 the supernatant bacterial suspension are .pipetted into a second centrifuge tube 

 containing about i cc. of sterOe ox bile and stood in the water-bath at 37''C. for 



* "Jour. Med. Research," 1917, xxxii. No. 2, p. 339. 

 f'Jour. Amer. Med. Assoc," 1918, lxx, p. 17. 



