Prescreening bacterial colonies for bioactive molecules with Janus plates, a SBS standard double-faced microbial culturing system

Abstract

Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method’s utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s10482-012-9746-7) contains supplementary material, which is available to authorized users.

Assessing Bacterial Diversity in the Rhizosphere of Thymus zygis Growing in the Sierra Nevada National Park (Spain) through Culture-Dependent and Independent Approaches

“…The taxonomic identities of isolates were assigned by 16S rRNA gene sequence analysis. Bacterial genomic DNAs were extracted by microwave lysis [ 14 ] by suspending a few colonies from each strain in 750 μL of MilliQ water in a microcentrifuge tube and irradiation at maximum power in a microwave oven in three sessions of alternating 45 s pulses with 30 s recovery intervals. For recalcitrant bacteria, DNAs were alternatively isolated by using a GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).…”

Antimicrobial Activity of Heterotrophic Bacterial Communities from the Marine Sponge Erylus discophorus (Astrophorida, Geodiidae)

“…This double-faced plated assay optimized by Fundación MEDINA [37] is based in exclusively designed plates by Nunc with two sides, which allow culture growth on the opposite layers in solid media. In order to maximize the number of potential active secondary metabolites produced, 5 different media were chosen to carry out the miniaturized fermentations: Marine Broth (2216 Difco), Medium F (0.015 g K 2 HPO4; 0.2 g CaCl 2 ; 0.75 g KCL; 23.4 g NaCl; 7 g MgSO4; 1 g Mannitol; 1 g Yeast extract; 1 g Peptone; 16 g Agar; 1 ml Hutner's basal salts [38] and 999 ml ddH 2 O), R2A (218263 - Difco) and saline (supplemented with 3% of SeaSalts of Sigma) R2A and Starch Agar (Difco).…”

“…Subsequently, the inoculated media with the target microorganisms were added on the opposite side thus forming the assay layer. The double-faced Janus plates were incubated at 37°C for 20–24 h. A search of inhibition zones indicative of antibacterial or antifungal activity was then carried out [37] . This in vitro screening assay was performed against a panel of Gram positive bacteria ( Bacillus subtilis (ATCC6633) and Staphylococcus aureus MRSA), Gram negative bacteria ( Acinetobacter baumannii ) and the yeast Candida albicans .…”

“…Pre-inoculum and inoculum medium used to grow A. baumannii and B. subtilis was Luria Broth (LB) (Miller's, Invitrogen). For C. albicans the pre-inoculum medium was Sabouraud Dextrose Broth (SDB) and the inoculum medium was YNBD [37] . The pre-inoculum and inoculum medium prepared for the overnight growth of S. aureus MRSA was Brain Heart Infusion (BHI).…”

How to Translate a Bioassay Into a Screening Assay for Natural Products: General Considerations and Implementation of Antimicrobial Screens

“…Since the assay endpoint relies on a visually measurable inhibition zone, the assay requires large amounts of reagents and laboratory space, and suffers from a slow and labororiented readout because of the need for manual determination of the zones of inhibition. Nevertheless, the assay can be miniaturized and automated by using image-based screening platforms capable of quantifying the bacteria-free zones on a much smaller scale [78,79].…”