Gradient Agar Plates

Within the past few years a simple technique has been developed that increases tremendously the amount of information that can be obtained from a single petri plate culture. The technique is termed the gradient plate method and can be performed easily by anyone who can grow bacterial cultures. The method involves solidification of a layer of agar medium while the petri dish is in an inclined position. The angle of inclination is such that the agar layer diminishes to nothing at one edge of the plate (see A in figure 1). An equal amount of agar medium is then added which is allowed to solidify while the plate is level, thus forming two complementary layers (see B in figure 1). The types of gradients that can be established depend on the chemicals added to one or both portions of the agar medium before the portions are poured into the petri plates. Two very common types of gradient plates are those of pH and of antimicrobial compounds. The pH gradient plates are prepared by adding 1.0 ml. of a sterile one-molar solution of KH2PO4 to 15 ml. of the melted cooled agar that is to be poured as the first layer, and 1.0 ml. of a sterile one-molar solution of K2HPO4 to the second 15 ml. portion of melted cooled agar that is to be poured as the second layer. We have found that 15 ml. quantities of sterile nutrient agar contained in tubes are convenient sources of the medium. The solutions of phosphates as well as the nutrient agar are previously sterilized by autoclaving. After solidification of the plates, the establishment of a pH gradient can be demonstrated by placing a filter paper strip that had been soaked in a pH indicator such as brom thymol blue on the surface of the plate in the direction of the gradient (see A in figure 2). Two of the common uses of the pH gradient plate are: a) inocula of various microbial cultures can be streaked in the direction of the gradient to ascertain the optimum pH reactions for growth or pigment production (see B in figure 2), and b) cells of a single microbial culture that grows at all pH reactions in the plate can be spread over the entire surface of the plate and filter paper strips containing various antimicrobial substances can be placed on the plate in the direction of the gradient (see C and D in figure 2).