STUDIES ON VIRULENCE : II. THE INCREASE IN VIRULENCE IN VITRO OF A STRAIN OF PNEUMOCOCCUS

1. An automatic apparatus is described by which a new supply of food can be furnished actively growing organisms at any desired interval of time-an automatic transferring device. 2. A single strain of pneumococcus, Type I, Neufeld, which had become avirulent for mice, acquired virulence of maximal degree when grown in the described apparatus with skimmed milk sterilized in an Arnold sterilizer as the medium. Transfers were made at intervals of 2, 4, and 8 hours. It is difficult to determine the best interval of transfer, but the 8 hour interval apparently is most suitable, with the 4 and 2 hour following in preference in the order named. 3. Pasteurized skimmed milk from a single dairy, but obtained on different days, when used as medium at a 2 hour interval, varied in effect on the virulence of pneumococci, the results showing that the virulence might be either increased, decreased, or maintained. 4. Skimmed milk heated for varying lengths of time, 30 minutes at 17 pounds pressure, or 60 or 90 minutes at the same pressure, lost its suitability for maintaining virulence of a pneumococcus when transferred every 2 hours, the effect being in direct proportion to the length of time the milk was heated. 5. The H ion concentration of milk had slight effect on virulence. A virulent strain of pneumococcus was grown in milk adjusted to pH = 5, 6, 7, 8, and 9, transfers being made every 2 hours. Virulence was maintained to a like degree on milk titrated to pH = 5, 6, and 7, but when the organism was grown in milk of pH = 8 and 9, virulence decreased, more rapidly at pH = 9 than at pH = 8. 6. A pure line practically avirulent strain of pneumococcus picked by the Barber method, when grown in milk at a 4 hour interval of transfer, increased in virulence 10 million fold; that is to say, until one diplococcus would kill a mouse. Prior to this study, no record has been found in which the virulence of any microorganism has been increased to such a degree by an in vitro method.