178 



mation when large inocula are used may 

 be attributable to the release from auto- 

 lyzing cells of an amount of this enzyme 

 sufficient to destroy the transforming 

 principle in the reaction system. 



In order to obtain consistent and re- 

 producible 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 sec- 

 ondly, that pneumococcal cells contain 

 an intracellular enzyme which when 

 released destroys the activity of the 

 transforming 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 Transform- 

 ing Activity.— In the isolation and 

 purification 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 frac- 

 tions. 



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 pre- 

 cipitated 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 refrigera- 

 tor in order to effect sterilization. The 

 alcohol precipitated material is centri- 

 fuged, 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 redissolved 

 in the original volume of saline. Steriliza- 

 tion of active material by this technique 

 has invariably proved effective. This pro- 



AVERY, MACLEOD, MCCARTY 



cedure 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 de- 

 scribed 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 dis- 

 tributed 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-^ dilu- 

 tion of this culture is added to each tube, 

 and the cultures are incubated at 37°C. 

 for 18 to 24 hours. 



The anti-R properties of the serum 

 in the medium cause the R cells to ag- 

 glutinate during growth, and clumps 

 of the agglutinated cells settle to the 

 bottom of the tube leaving a clear 

 supernatant. When transformation oc- 

 curs, the encapsulated S cells, not be- 

 ing affected by these antibodies, grow 

 diffusely throughout the medium. On 

 the other hand, in the absence of trans- 

 formation the supernatant remains 

 clear, and only sedimented growth of 

 R organisms occurs. This difference in 

 the character of growth makes it pos- 

 sible by inspection alone to distinguish 

 tentatively between positive and nega- 

 tive results. As routine all the cultures 

 are plated on blood agar for confirma- 

 tion and further bacteriological iden- 

 tification. Since the extracts used in the 

 present study were derived from Pneu- 

 mococcus Type III, the differentiation 

 between the colonies of the original R 

 organism and those of the transformed 

 S cells is especially striking, the latter 



