400 S. GARD AND O. MAAL0E 



effect upon the reaction rate, an additional indication that these groups 

 probably are unimportant in the present connection. 



On the assumption of a first-order reaction and from the observation that 

 the regression of log reaction rate on log formaldehyde concentration was 

 rouglily unity, it was concluded that the rate-determining step in inactivation 

 was a single event: the reaction of one molecule of formaldehyde with one 

 essential site. In support of this conclusion the energy of activation was cal- 

 culated at 19,500 cal./mole. 



According to Ross and Stanley, a partial reactivation of the formaldehyde- 

 treated virus could be achieved by prolonged dialysis at pH 3.0. Kassanis and 

 Kleczkowski (1944) could not confirm this observation. Fischer and.Lauffer 

 (1949a), on the other hand, reported a mean ratio of post- to predialysis 

 infectivity of 1.84, possibly significantly different from unity. 



2. Bacteriophage 



In spite of the fact that bacteriophages have to be considered as ideal 

 model viruses in inactivation experiments and have been extensively used 

 in studies of irradiation effects, the chemical inactivation of these agents has 

 attracted little interest. Andrewes and Elford (1933), comparing formalde- 

 hyde-inactivation and neutralization of phage by immune serum, presented 

 curves clearly deviating from the course of a first-order reaction. Eecently, 

 Heicken and Spicher (1956) reported more systematic studies on the coli- 

 phage T3. These authors found inactivation to follow approximately a first- 

 order course down to a survival of about 10~*. In the later stages, however 

 a deviation became apparent, particularly at lower formaldehyde concentra- 

 tions. The phenomenon was supposed to indicate a heterogeneity of the phage 

 population with respect to formaldehyde resistance. Gard (1957) described 

 similar observations but offered a different explanation (see Section IV, B, 5). 



Heicken and Spicher reported a considerable reactivation of form aldehyde - 

 treated phage after addition of various amino acids, serum, or bisulfite. 

 Reactivation continued for several days and amoimted to as much as six 

 powers of ten. This observation has been confirmed in the writer's laboratory. 



3. Poxvirus 



The kinetics of vaccinia virus mactivation was studied by Keogh (1937). 

 The reaction was described as being of the first order. The validity of this 

 conclusion appears somewhat doubtful, however, in view of the fact that 4 

 of the 5 experiments reported show a very marked deviation from a first- 

 order course. The author seems to have attributed this phenomenon to a 

 progressive fixation of formaldehyde by nonviral components of the medium, 

 resulting in a successive reduction in the concentration of free formaldehyde. 

 The loss of formaldehyde was estimated at not more than 25 % in 30 mmutes. 



