环境生物学

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a leading causative pathogen for food-borne gastroenteritis. During its course of infection, it confronts myriads of physiological barriers inside the host, such as nutrient deprivation, low micronutrient availability, and toxicity from bile salts, to promote bacterial survival and infection inside the host. The ability of the pathogen to overcome these stressful conditions determines the degree of virulence in the host. Therefore, assessment of the survival of a pathogen during different stress conditions, like glucose starvation, magnesium starvation, and bile stress, are important parameters to assess the virulence of the pathogen. Here, we describe protocols for estimating the survival of the pathogen during the above-mentioned stress conditions. We culture S. Enteritidis in an appropriate growth medium to a required O.D.₆₀₀ and treat it with glucose starvation (M9 minimal culture medium containing 0.03% glucose), magnesium starvation (M9 minimal culture medium containing 20 µM MgSO₄), and bile stress (bacterial cells treated with 15% bile salts in Luria Bertani (LB) culture medium) conditions. The number of surviving bacteria is obtained after the treatment by calculating the colony-forming units (CFU) of the surviving pathogen obtained on LB agar plates at relevant time intervals. The experiments are performed in biological replicates, and statistical analysis is performed to validate the experimental findings. The methodology of these stress response assays is simple and can be adapted to study the pathogenesis and stress response in other relevant and culturable enteric pathogens.

Unculturable bacteria are those bacteria which proliferate in their native habitat but unable to grow or thrive in the normal laboratory media and conditions. The molecular techniques have revealed the significance of these uncultured bacteria in terms of their functional diversity and potential to produce secondary metabolites. To achieve these benefits, scientists have attempted to isolate and cultivate unculturable bacteria in the laboratory using transwell plates, optical tweezers, laser microdissection, microbioreactors, and diffusions bioreactors. However, these techniques are still inadequate to resolve the difficulties of cultivating unculturable bacteria. Therefore, it is essential to develop new cultivation method that enables growth of diverse range of bacteria in the laboratory conditions. Diffusion bioreactor is a membrane bound chamber which allows microbes to proliferate in their native environment by providing the excess to naturally occurring nutrients and signaling compounds. This paper presents efficient and reliable protocol to construct a diffusion bioreactor and its utilization to isolate and cultivate unculturable soil bacteria in laboratory.

The protocol separates bacteria from atmospheric particles, obtaining with greater precision their abundance in the atmospheric deposition. This procedure is similar to the one used to separate bacteria in streambed sediments. The detachment procedure consists of a chemical treatment with sodium pyrophosphate and Tween 20 and a physical treatment with agitation and ultrasonic bath to disperse the bacteria in the liquid sample. We recover the total (free and attached) bacteria by generating a density gradient with Nycodenz by centrifugation. The techniques prior to this procedure do not include the microorganisms that are attached to the aerosol particles and, therefore, considerably underestimate the total load and deposition of airborne microorganisms.