A Visible Method for Titration and Neutralization of Viruses on the Basis of pH Changes in Tissue Cultures. ∗

The advantages of determining virus potency and of demonstrating neutralizing antibodies of the Western strain of equine encephalomyelitis (W.E.E.) virus in tissue culture as presented in previous papers 1 , 2 include (a) reduction of cost of experiments; (b) removal of the variable of individual animal reactivity; and (c) the possibility that the tissue culture method is a more sensitive indicator of the presence of virus than animal inoculation. However, the application of the method to other viruses was limited, since the titration and neutralization of the W.E.E. virus depended upon growth, or absence of growth of tissue. Such a test could not be applied to many viruses because of the low susceptibility of the fibroblasts to virus. Thus, the St. Louis encephalitis virus and the Jungeblut-Sanders mouse virus 3 could not be titrated in chick embryo tissue culture and a more complicated procedure had to be developed when embryonic mouse brain, the most susceptible tissue, was used. 4 This complicated procedure was introduced because of the difficulty of growing the mouse brain tissue in plasma patched preparations. To obviate the complex procedure, a simplified method has been developed.

During an investigation concerned with metabolism of infected and non-infected tissue, it was found 5 that the degree of glycolysis in the tissue infected with the W.E.E. virus as measured in the respirometer of a type described by Victor, 6 was much lower than that of non-infected tissue. On the basis of this observation, the present method for titration and neutralization of several viruses has been developed. The method does not require transfer of tissue into Carrel dishes and the whole experiment can be carried out in the same tube. The method is based on the fact that the products of glycolysis are acid in reaction. In preliminary experiments, it was found that when non-infected tissue was present, the pH of the culture medium shifted to the acid side and in the presence of phenol red, the color of the medium changed from red to yellow within 2 hours at 37.5 ° C in partial vacuum (a desiccating jar evacuated for one hour by water suction). Conversely, when tissue was heavily infected with virus for a sufficiently long period of time, there was either very little change or no change in the pH of the medium.