Improved 18-hour methyl red test.

Standard methods for the methyl red (MR) test are not practical for routine use in clinical laboratories because of the necessarily prolonged incubation period. When read after overnight incubation, the usual MR test is often equivocal or falsely positive. The present study demonstrates the importance of standardizing the total volume of broth, the size of the vessel in which the cultures are incubated, and the density of the inoculum. In very small volumes of broth, cultures are better exposed to atmospheric oxygen, and thus MR-negative organisms tend to revert the initial acidic pH much more quickly than in deeper, large-volume broth cultures. In the proposed technique, a single colony was inoculated into a 0.5-ml amount of MR-Voges Proskauer (VP) broth (13- by 100-mm tube), and, after 18 to 24 hr at 37 C, one drop of MR was added. With this technique, the broth cultures produced either a definite red (positive) or yellow (negative) color, whereas various shades of orange were frequently observed when larger volumes of broth were tested after only 1 to 2 days of incubation. With 6.0-ml broth cultures, 18-hr MR tests were totally unreliable, but 18-hr tests in 0.5 ml of broth were comparable to the standard MR test performed after 5 days of incubation and superior to those performed after 48 hr in 6.0-ml broth cultures. With the proposed technique, the MR test can be incorporated readily into the routine scheme for identification of Enterobacteriaceae.