The Effect of Concentration of Dyes on Differentiation of Enteric Bacteria on Eosin-Methylene-Blue Agar

Abstract

Holt-Harris and Teague introduced eosin-methylene-blue as an indicator for differentiation of the colon from the typhoid-dysentery group of bacteria in 1916. Their medium consisted of 1 per cent Witte's peptone, 0.5 per cent beef-extract (Liebig's), sodium chloride, sucrose and lactose, solidified with 1 per cent agar. The reaction was adjusted to + 0.8 per cent normal acid. The medium was cleared with egg whites, filtered, and sterilized in the Arnold for 30 minutes at 100°C. on three successive days. They recommended, as an indipator, 2 ml. of a 2 per cent yellowish eosin and 2 ml. of a 0.5 per cent methylene blue per 100 ml. of the medium. The medium was very satisfactory for its purpose, but the details of preparation made it rather cumbersome and the necessitated change of peptones after the war, coupled with resultant differences in reaction of the medium adjusted on a total acidity scale, tended towards variable results in the hands of different investigators. Levine, in 1918, suggested a simplified medium in which Difco peptone was employed as a base, di-potassium phosphate (0.2 per cent) was utilized as a buffer, to automatically adjust the reaction of the medium to a desired point, and sterilization at 15 pounds for 15 minutes in the autoclave was substituted for the more cumbersome Arnold intermittent sterilizing process. To this basal medium was added 5 ml. of a 20 per cent sterile lactose solution, 2 ml. of 2 per cent yellowish eosin, and 2 ml. of a a per cent methylene blue per 100 ml. of melted basal medium. This medium was designed primarily for the purpose of differentiation of Escherichia coli from Aerobacter aerogenes, but was later found to be equally serviceable for differentiation of the Salmonella-typhoid-dysentery from the colon group of bacteria. The foregoing ratio of eosin to methylene blue was based upon the prewar Grubler dyes. The crude ratio of 2 per cent eosin to I per cent methylene blue is 4 to 1, but considering that the concentration of dye in eosin was about 85 per cent and the dye concentration in the methylene blue was only about 60 per cent, the actual ratio of eosin to methylene blue was approximately 5.8 to 1. In the postwar period, variable results were obtained in different laboratories employing eosin-methylene-blue as an indicator. In some studies for the biological stain commission, Levine found that these could be attributed primarily to variability in methylene blue available on the market. Many of the samples of methylene blue submitted contained approximately 90 per cent dye, which would necessitate reducing the volume of 0.5 per cent methylene blue by about j if the ratio of eosin to methylene were to be maintained at 5.8, and avoid