Inactivation of Phage by Aldehydes and Aldoses and Subsequent Reactivation

Abstract

Summary and Conclusions Inactivation of phage by aldehydes and other substances depends on the concentration of the reacting substances, the time, the temperature and the pH of the substrate. At 37 C and pH = 7.5 a given volume of an undiluted broth phage was completely inactivated by an equal volume of a 0.05 percent solution of formaldehyde in 24 hours. Increasing concentrations of formaldehyde, or increasing dilutions of phage reduce the time of inactivation. The reaction starts immediately and its rate is an exponential function of time. Glyceraldehyde inactivated phage in the same molecular concentration as formaldehyde. Addition of formaldehyde or glyceraldehyde to phage in broth increases the acidity of the solution. Glucose, glucoseamine and fructose also inactivate phage if proper concentrations are used. The process of inactivation of fructose differs radically from that with glucose. Maltose, sorbitol and sucrose do not inactivate phage. On diluting inactivated phage before or shortly after inactivation is completed, in a substrate consisting of a solution of peptone or aminoacids, free active phage is liberated. The reaction, phage-formaldehyde, proceeds apparently in two stages, and is reversible only after the first stage. The amount of phage recovered depends on the types of aminoacids or polypeptide present in the diluent. When pure aminoacid solutions were used, the maximal reactivation was given by diaminoaminoacids, followed by the aliphatic monocarboxylic aminoacids. The least effective were the di-carboxylic aminoacids. The group attached to the NH2 apparently influences the reactivating capacity. When phage is inactivated by other aldehydes or by aldoses the reactivation is slower and less complete than when inactivated with formaldehyde. Reactivation after inactivation with glucose or glyceraldehyde is much less complete, while with glucoseamine and acetaldehyde it occurs only when the reaction of inactivation is interrupted before it is completed. Evidently there is a difference in the intensity of action of the various substances. The phenomenon of reactivation may be accounted for on the basis of dissociation. It is assumed that in the first phase, at least, the aldehyde combines with the NH2 group of the phage (or virus) and that on dilution this compound dissociates, liberating both aldehyde and phage. By introducing appropriate aminoacids into the diluting substrate, the equilibrium is disturbed, aldehyde is bound by the NH2 group of the aminoacid and more phage dissociated. This process continues until a new equilibrium is reached or until all the phage is liberated. The reaction thus seems to correspond with that between the aldehydes and polypeptids.