A Test for the Detection of Lactic Bacteriophage Using 2,3,5-Triphenyltetrazolium Chloride

Abstract

Summary In this study, 2,3,5-triphenyltetrazolium chloride (TTC) was used to measure inhibition of lactic cultures by bacteriophage. The procedure follows: Place 8.7ml. of 10% sterile reconstituted nonfat dry milk (NDM) in each of two sterile test tubes. To each tube add 0.2ml. of a lactic culture diluted 1:1 with sterile 10% NDM. Add 1ml. of serially diluted phage nitrate to one tube. To the other tube, remaining as the control, add 1ml. of sterile distilled water. Place sterile rubber stoppers in the tubes and invert twice to mix the contents. Incubate the tests for 4 hr. at 32° C. in a water bath. Add 0.1ml. of 1:25 aqueous solution of TTC per tube. Invert the tubes twice and incubate again for 30min. at 32° C. Compare the color in the tube with added filtrate to the control tube. If less color is developed than in the control, inhibition has occurred. Results of this test are available in 4.5 hr. Its sensitivity for bacteriophage is comparable, and in some cases better, than a litmus milk test (8) requiring from 12 to 16 hr. With mixed-strain lactic starter cultures, the TTC test indicates some cases of phage inhibition in 4.5 hr. that are not evident after extended incubation in the litmus milk test. By using unheated filtrate and filtrate heated to 80° C. for 10min., this test can be used to distinguish between inhibition of lactic cultures caused by heat-stable inhibitors and bacteriophage.