OSWALD T. AVERY, COLIN M. MACLEOD, AND MACLYN MCCARTY 141 



found to be present and highly active in the autolysates of a number of different 

 strains. The fact that this intracellular enzyme is released during autolysis may 

 explain, in part at least, the observation of Dawson and Sia (5) that it is essential in 

 bringing about transformation in the test tube to use a small inoculum of young and 

 actively growing R cells. The irregularity of the results and often the failure to 

 induce transformation when large inocula are used may be attributable to the release 

 from autolyzing cells of an amount of this enzyme sufficient to destroy the trans- 

 forming principle in the reaction system. 



In order to obtain consistent and reproducible results, two facts must be 

 borne in mind: first, that an R culture can undergo spontaneous dissociation 

 and give rise to other variants which have lost the capacity to respond to the 

 transforming stimulus; and secondly, that pneumococcal cells contain an 

 intracellular enzyme which when released destroys the activity of the trans- 

 forming principle. Consequently, it is important to select a responsive strain 

 and to prevent as far as possible the destructive changes associated with 

 autolysis. 



Method of Titration of Transforming Activity. — In the isolation and purifica- 

 tion of the active principle from crude extracts of pneumococcal cells it is 

 desirable to have a method for determining quantitatively the transforming 

 activity of various fractions. 



The experimental procedure used is as follows: Sterilization of the material to be 

 tested for activity is accomplished by the use of alcohol since it has been found that 

 this reagent has no effect on activity. A measured volume of extract is precipitated 

 in a sterile centrifuge tube by the addition of 4 to 5 volumes of absolute ethyl alcohol, 

 and the mixture is allowed to stand 8 or more hours in the refrigerator in order to effect 

 sterilization. The alcohol precipitated material is centrifuged, the supernatant 

 discarded, and the tube containing the precipitate is allowed to drain for a few minutes 

 in the inverted position to remove excess alcohol. The mouth of the tube is then 

 carefully flamed and a dry, sterile cotton plug is inserted. The precipitate is redis- 

 solved in the original volume of saline. Sterilization of active material by this 

 technique has invariably proved effective. This procedure avoids the loss of active 

 substance which may occur when the solution is passed through a Berkefeld filter or 

 is heated at the high temperatures required for sterilization. 



To the charcoal-adsorbed broth described above is added 10 per cent of the sterile 

 ascitic or pleural fluid which has previously been heated at 60°C. for 30 minutes, in 

 order to destroy the enzyme known to inactivate the transforming principle. The 

 enriched medium is distributed under aseptic conditions in 2.0 cc. amounts in sterile 

 tubes measuring 15 X 100 mm. The sterilized extract is diluted serially in saline 

 neutralized to pH 7.2-7.6 by addition of 0.1 n NaOH, or it may be similarly diluted 

 in m/40 phosphate buffer, pH 7.4. 0.2 cc. of each dilution is added to at least 3 or 4 

 tubes of the serum medium. The tubes are then seeded with a 5 to 8 hour blood 

 broth culture of R36A. 0.05 cc. of a 10 -4 dilution of this culture is added to each 

 tube, and the cultures are incubated at 37°C. for 18 to 24 hours. 



191 



