Microbiologists have been using agar growth medium for over 120 years. CFU on PS medium than on PT medium using or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the initial community framework than those cultivated on PT moderate. Moreover, even more hitherto-uncultivated microbes grew on PS than on PT moderate. Intro An enigma termed the great plate count anomaly is a central issue in microbiology; only a small proportion Rabbit Polyclonal to ARRB1 of viable cells in microbial communities can be grown using agar (or other gelling agents) growth media (1). Investigators have been attempting to identify factors limiting cultivation of most microbes on agar plates for a long time (2, 3), but more research is necessary since numerous phylogenetic groups of environmental microbes still lack cultivated representatives. With the advent of state-of-the-art sequencing technologies (4, 5), researchers have been trying to circumvent the limitation and laboriousness of isolation work by sequencing genomes of entire communities to reveal potential novel functions and phylogenetic relationships (6,C8). Nonetheless, the isolation of microorganisms is still necessary for microbiologists to study organisms genetically and physiologically in depth in the laboratory. A number of different strategies have been used to successfully increase the number of cells cultivated from environmental samples as well as to isolate novel bacterial taxa. Some examples of strategies that have been successfully used to improve cultivation efficiency include mimicking the natural environment (9, 10), physically separating cells to decrease competition or growth inhibitors (11, 12), and modifying growth media to be more reflective of the natural environment (12, 13). In some cases investigators have subsequently identified specific factors required to cultivate previously uncultivated taxa (14, 15). A number of 31430-18-9 supplier studies have empirically shown increases in CFU counts when catalase or pyruvate was added to the medium (16,C22), indicating that oxidative stress can inhibit colony growth. The variety of factors that may be limiting laboratory cultivation is vast, making it a challenge to identify specific growth medium components required for, or inhibitory to, cell growth. In addition to chemical components and growth conditions, the method used for medium preparation can also impact cultivation. Detrimental and beneficial chemical 31430-18-9 supplier reactions between medium components have been found to occur during autoclaving of liquid medium. Early studies demonstrated both growth inhibition (23) and stimulation (24) of some bacterial species when phosphate and glucose were autoclaved together. Maillard reaction products have also been shown to inhibit bacterial growth (25). More recently, furan-2-carboxylic acids, which can inhibit bacterial swarming on agar surfaces, were found in various amounts in commercial agars (26). But studies on specific factors in agar leading to bacterial growth inhibition are limited. T-27T was isolated in 31430-18-9 supplier our laboratory as the first cultivated representative of 31430-18-9 supplier the phylum (27). This organism had eluded cultivation for many years because it grew poorly (very low CFU counts and slow growth) on agar plates, but it was finally found to grow well on medium solidified with gellan gum (28). Since the only difference between the growth media was the gelling agents used, we hypothesized that the agar or products produced from the agar interacting with some other medium components were inhibiting.