Growth and Multiplexed Analysis of Microorganisms on a Subdivided, Highly Porous, Inorganic Chip Manufactured from Anopore

Abstract

A highly porous inorganic material (Anopore) was shown to be an effective support for culturing and imaging a wide range of microorganisms. An inert barrier grid was printed on the rigid surface of Anopore to create a "living chip" of 336 miniaturized compartments (200/cm 2 ) with broad applications in microbial culture.There is a pressing need in microbiology for the development of automated, miniaturized, and multiplexed growth formats that improve on the petri dish and the microtiter plate (1, 3). One approach to microbial culture has been to use flexible organic membranes as a support, permitting growth and imaging in situ (2, 10). We suggest in this work that an inorganic and rigid porous surface may be a better choice for many applications. Anopore is an inert ceramic, an aluminum oxide that is formed in sheets by a high-pressure and etching technique (4,6,8), creating a uniquely porous planar material; up to 50% of the volume is pores. Anopore has been identified as a superior surface for the imaging of microorganisms compared to flexible organic membranes (8, 9), in part due to its flatness and limited autofluorescence. The motivation for this work was to test the suitability of Anopore in the creation of versatile new growth formats or "living chips." Anopore strips (8 by 36 mm; 60 m thick; 0.2-m-diameter pores; 3 ϫ 10 9 pores cm Ϫ2 ) were a gift from PamGene International ('s-Hertogenbosch, The Netherlands). A latex solution (masking fluid 052; Royal Talens, The Netherlands) was applied to one surface in 1-mm-thick lines using a mapping pen and allowed to polymerize at room temperature for 20 min. The strips were then washed with distilled water and twice with 96% (vol/vol) ethanol and air dried. The grid formed a surface barrier 0.5 mm wide and 0.4 mm high that delineated eight culture areas of 7 by 4 mm. The polymerized latex was not strongly autofluorescent: illumination in the 515-to 730-nm range required exposures of Ͼ5 seconds to saturate the chargecoupled device camera. Anopore strips were placed on an appropriate nutrient agar base (5), inoculated on the upper surface at a density of 100 to 2,000 CFU/mm 2 , and then incubated. Microcolonies were stained by transfer of these strips right-side up to a microscope slide covered with a 1-mm-thick film of 1% (wt/vol) solidified low-melting-point agarose (Sigma, The Netherlands) containing 10 M Syto-9 dye plus 40 M propidium iodide (PI) or 5 M hexidium iodide (HI) for bacteria and 20 M Fun-1 for yeasts (7) (all dyes were from Invitrogen, The Netherlands). Staining was for 20 min at room temperature for bacteria and 30°C for yeasts. These procedures allowed staining of the organisms on the Anopore surface through the pores without disruption of the microcolonies. Strips were then imaged directly (without coverslip, immersion oil, or fixative) using an Olympus BX-41 fluorescence microscope equipped with U-MWIBA filters (excitation spectrum of 460 to 490 nm, dichroic mirror splitting at 505 nm, and an emission spectrum of 515 to 550 nm, used for Syto9 an...

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

“…The regions in which rounded cells formed were targeted and recovered with a sterile toothpick guided by bright-field light microscopy. The recovered cells were resuspended in MH broth and double stained with Syto 9 and propidium iodide using dye concentrations described previously (10), with incubation for 20 min at room temperature (Live/Dead staining kit; Invitrogen, Carlsbad, CA). Stained cells were visualized by fluorescence microscopy of wet mounts and by phase-contrast light microscopy to detect any poorly stained cells.…”

“…Strains were cultured in shake flasks containing MH broth with 100 mg/liter ampicillin. Spots of the cultures (1 l) were placed on Anopore strips (obtained from PamGene International, The Netherlands, and manufactured by Whatman UK) resting on MH plates without ampicillin and incubated (10). After 3 to 4 h, if swarming was observed, the Anopore strips were transferred (bacterial inoculum up) to MH agar plates containing 3% (wt/vol) glutaraldehyde (Sigma, The Netherlands) and fixed for 3 h. Anopore is an inert ceramic, an aluminum oxide that is formed in thin sheets by a high-pressure and etching technique that creates a porous planar material; up to 50% of the volume is pores.…”

Central Role of the Cell in Microbial Ecology

“…(117). As a response, several techniques that use low concentrations of nutrients for isolation and cultivation efforts have emerged (10,59,83,103,112,114,170,249). However, growing bacteria at a low concentration of nutrients will logically result in low biomass gains.…”

Two Homologous Agr-Like Quorum-Sensing Systems Cooperatively Control Adherence, Cell Morphology, and Cell Viability Properties in Lactobacillus plantarum WCFS1

“…Cell morphology was examined immediately. Cells were visualized by staining with the fluorogenic dye Syto 9, followed by imaging with an Olympus BX-41 fluorescence microscope (15). Data were captured using an 8-bit Kappa chargecoupled device camera, and cell lengths were determined using TIFF images and ImageJ software (http://rsb.info.nih.gov/ij/).…”

“…Colonies of the wild-type strain and the ⌬lamA, ⌬lamR, and ⌬lamA ⌬lamR mutants were grown for 30 h on strips of Anopore placed on MRS agar and were then stained by transfer of the strips to microscope slides with a 1-mm-thick film of 1% (wt/vol) low-melting-point agarose containing the fluorogenic dyes Syto 9 and propidium iodide. This stained the colonies from beneath, essentially as previously described (15). Imaging and data capture were performed as described above using a ϫ4 Fluorotar objective lens (Olympus, The Netherlands), and images were merged using Photoshop software (Adobe).…”