Detection and enumeration of faecal indicator bacteria in water
This study investigated the effects of various factors, namely exposure to brass/copper, sunlight, high temperature, chlorine, low pH, or starvation, on the enumeration of faecal indicator bacteria in water, performed using non-selective & selective media under conventional aerobic conditions & under conditions designed to neutralise reactive oxygen species e.g. by the addition of 0.05% w/v sodium pyruvate as a scavenger of peroxides &/or anaerobic incubation, to encourage fermentative metabolism. These methods were compared with standard US & UK methods for the recovery of injured bacteria. The information gained was used for the development of a novel broth-based medium for the enumeration of sub-lethally injured Escherichia coli under field conditions. The novel broth-based field medium ('Coliblack') was tested against a currently used method (H2S test) & evaluated under field conditions with unskilled personnel in rural locations in India. The results showed that growth conditions designed to neutralise reactive oxygen species could enhance the colony count of faecal indicator bacteria, whether enumerated using a non-selective medium, a selective medium, a resuscitative medium, or a chromogenic growth medium, following exposure to stressors such as storage of water in a brass container, sunlight, high temperature & chlorine. Such conditions may induce sub-lethal injury in E. coli & E. faecalis, inactivating the bacterial cells under conventional aerobic enumeration conditions as a result of their oxygen-sensitivity. However, exposure to some other stressors namely low pH & starvation was not strongly oxygen-sensitive, as no substantial differences between counts were observed on various growth media or enumeration conditions. The results from enumeration experiments were used to develop a field-based broth medium with a peroxide-neutralising resuscitative agent (sodium pyruvate), a selective agent showing minimum inhibition (Tergitol 7) & a chromogenic diagnostic agent based on defined substrate technology (8 hydroxyquinoline-glucuronide). The Coliblack medium indicates the presence of Escherichia coli in the drinking water by a colour change of the medium to black. Furthermore, the preliminary trial carried out for the evaluation of the novel broth-based field medium gave positive feedback from users. In this respect the results obtained in the present study represents an advancement in understanding of how to maximise the enumeration of faecal indicator bacteria in water & how to apply this information to improve detection under field conditions.