Diffusion of Chromogenic Inductors of Serratia Marcescens by M. T. M. Rizki*

Spontanieous anid induced color varianits ill the red HY straini of Serratia marcescens have beeii reported by Butiiilgl and Labrum anid Buntinig.2 Malny of the color variants are stable, but unstable types have also beeii isolated. The expressivity of color in these variants as well as in the wild-type strain is influenced by environmental conditions, such as temperature and nutritional factors.3 When three stable color variants, orange, pink, and white, were streaked side by side on a Petri dish merely to demonstrate the variety of mutations found in Serratia, it was noted that the margin of the white culture growing next to the streak of orange had turned red, and similarly the margin of orange facing the pink eulture had become red. Apparently a diffusible factor produced by the orange variant had induced red pigmentation in white, and, similarly, a factor from the pink culture had caused the production of red pigment in the orange streak. Subsequent plating of the colorinduced organisms showed that this induction is a phenotypic change and not permanent. In order to investigate the extent of this phenomenon in Serratia marcescens, a large number of colored mutants were tested for interaction type to determine whether a giveni variant was able to produce a factor which could induce color ini other variants (i.e., was an inductor) or could react to an inductor by expressinig a color change (i.e., was a reactor) or could be qualified both as an inductor and as a reactor or as nieither. All mutants used in the present investigation were induced by ultraviolet irradiation of the HY strain obtained from Dr. M. I. Bunting's laboratory. The mutants are divisible into three color types: white (W), pink (P), and orange (0). Within each of these classes there are differences in shade of color, surface texture, and general appearance of the culture on aging. Of the different variants, only those which can be characterized by the inductor-reactor system of red pigment are included in this report. These twelve mutants were chosen from a preliminary test of twenty-six varianits by streaking different combinations of variants on Petri dishes in the form of a Punniett square. Petri dishes were prepared with a peptone-glycerol agar medium, accordingil to Bunting,' with 2.0 per cent agar. Similar results were also obtained writh the synthetic ammonium-citrate-glycerol agar used by Bunting. A triangular piece of cardboard was used to draw two divergent lines on the bottom of each Petri dish with a wax marking pencil. The smallest distance between these guide lines measured approximately 2-3 mm., while the opposite sides were 40 mm. apart. Each variant of Serratia was suspended in a 0.9 per cent saline blank at approximately 108 concentration, and the suspensions were smeared on the Petri dishes using sterile cotton swabs. Each Petri dish was streaked with two variants, one on either side (Fig. 1). This pattern of streaking is of special advantage for visualizing the diffusion gradient of color inductors. When the Petri dishes were incubated at 250 C., color interaction between mutants was apparent within 36 hours of plating. The observations were recorded on the third and fourth days of incubation.