EFFECTS OF HYDROLOGICAL DYES ON MICROORGANISMS

Dye tracers have been used for decades to characterize hydrological properties of subsurface features (caves, aquifers, and rock fracture environments). However, compared to the large number of studies describing these techniques, the biological effects of dyes are almost uncharacterized. Our ability to decide when, where, or whether to use dyes in microbiologically or macrobiologically sensitive environments is therefore poorly understood. In this pilot study, we challenged two different types of bacterial samples with various commonly used dyes to determine whether any of them had discernible effects on the test organisms. Clearly, the most advantageous dyes to use for hydrological purposes would be those having the least effect (either positive or negative) on the growth or other measurable properties of microbial communities.

Two bacterial sample types — a pure laboratory strain of Bacillus subtilis and an uncharacterized, mixed culture from a microbial mat community isolated from Four Windows Cave in El Malpais National Monument, NM—were grown on two different media (commercially prepared R2A and dilute nutrient broth) spotted with dyes at 100% and 10% concentrations. We used 4 dyes (pyranine, rhodamine, eosin, and uranine). As controls, the same organisms were grown on the same media with no dye. We found that B. subtilis grows slightly better with the 100% compared to the 10% concentration of dye, and both grow better compared to the no-dye control group, implying that there is some nutritive or other stimulatory effect of dye. In contrast, all dyes inhibited the natural cave mixed microbial community relative to their controls. These inocula grew most poorly in rhodamine and best in pyranine. All organisms showed color change as they progressively absorbed dye, implying continuous uptake from the medium. In some environmentally contaminated plates (not used for bacterial growth assessment), the uncharacterized contaminant fungi also appeared to be inhibited by the dye compared to undyed portions of the plates. Based on these findings, we tentatively suggest that rhodamine might be the least desirable dye to use in hydrological studies, while pyranine might be preferable. This study is a first step in helping to inform management decisions on hydrological dye tracing in the subsurface.