Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The overall goal of this method is high throughput parallel analysis of multi-member microbial communities in confined environments using silicon micro well arrays. The main advantage of this technique is that it allows for a high throughput highly parallel screening of finely localized microbial interactions that are significant to industry, medicine, and the environment. This method can help answer key questions in the biomedical and microbial ecology fields such as how do spacial constraints inherent at fine scales drive deterministic and stochastic parameters of community development and what are they key effects on microbial member abundance and organization?

Begin with preparing the micro well arrays. First, cut individual micro well array chips from silicon wafers printed with multiple arrays using a diamond scribe. Each individual chip contains sub-arrays of wells with diameters ranging from five to 100 microns at three different spacing densities.

On each chip, the complete motif of wells is also repeated four times. Next, make a humidified chamber using a pipette tip box and a PBS soaked wipe. Then apply a 150 microliter droplet of BSA solution to each chip and incubate the chips in the box for an hour at room temperature.

Meanwhile prepare the bacteria. Spin down a culture and re-suspend it in 500 microliters of fresh R2A medium with two percent glycerol. Then measure the culture density at 600 nanometers and adjust the concentration to an optical density of 0.02.

After the chip incubation, remove the BSA solution and rinse the chips three times with PBS. After the rinses, dry the chips with nitrogen gas and return them to the humidified chamber. Next, add 150 microliters of culture suspension to each dry chip and incubate the chips for an hour at four degrees Celsius so that the bacteria will have time to adhere to the wells, but the cell division of any possible contaminate is slow.

To prepare a chip for imaging, first make an agarose-coated cover slip for each chip. Liquefy a small volume of agarose, about five milliliters is needed for each cover slip. Next, wash one side of a cover slip with ethanol and center it onto a glass slide with the washed side up.

Then position two one millimeter thick PDMS spacers along the long edges of the cover slip and shift the cover slip so one millimeter hangs off the slide. Now, pour enough agarose onto the cover slip to completely cover it. Then use a second glass slide to flatten the agarose into a uniform thickness.

Prepare several such slide assemblies in parallel. When the agarose begins to solidify, transfer the assemblies to four degrees Celsius. After 15 minutes of cooling, trim off the excess agarose around the cover slips.

Then, put the assemblies in petri dishes and store them at four degrees Celsius. After the chips have incubated with the bacteria, dip the chips in ultra pure water one at a time for 10 seconds each. Then set the wet chips on their edges over a tissue until most of the liquid has drained off.

Now, remove the bacteria that did not settle into the wells. Cut a piece of tape the length of the silicon chip and adhere it to the parylene coat on the silicon. Then peel off the tape to remove the parylene coat and immediately prepare to invert the chip.

Now, place the chip onto a slide assembly such that the micro wells are in contact with the agarose. Be very careful not to move or shift the chip once in contact with the agarose. Now, transfer the complete assembly into the slide holder of a stagetop environmental control chamber and acquire time-lapse images over the next 24 hours at the desired interval and under 10x magnification.

Process the image stacks using conventional, freely available software. First, import the image sequence. For averaging, load the correction or the dark field images.

Then perform a background subtraction. Provide a radius value such as 135 pixels and select sliding paraboloid. Next, remove the average dark field image from the average illumination field image by selecting the two images and using the subtract operation.

Now, apply an illumination correction as follows. Set the operation to divide, set i1 to the well image, set i2 to the correction image, set k1 to the correction image mean, and set k2 to zero. Then click Create New Window.

To quantify the bacterial growth in the micro wells, first select the regions of interest with the micro array plug in. In the map menu, click reset grid and specify the rows, columns and well diameters. Then from the ROI shape menu, select circle.

To adjust the ROI array to the image, the ROI array can be moved by pressing the ALT or ALT+SHIFT key and selecting the top left ROI with the mouse. To resize the ROIs, press the shift key while selecting the bottom right of the ROI array. To adjust the spacing between the ROIs, press the shift key while dragging the top or bottom side of the array.

Once the ROI array fits over the wells of the image, click Measure RT.Then a table with all the measurements for each image or time point will be outputted and it can be copied into a spreadsheet for analysis. The growth of two strains of Pseudomonas aeruginosa was compared. One strain constitutively expresses toxic effector proteins associated with type six secretions.

The other is susceptible to type six pathogenesis due to loss of function. Both express a different fluorescent protein. Co-cultures were studied longitudinally and in different well sizes.

The bacteria were cultured individually and as a co-culture. 20 hours of growth was imaged in 30 minute intervals. After software corrections were performed on the imaging data, quantitative growth trajectories were plotted.

In the co-culture, the data does not suggest too much of a change in growth. To advance the analysis, each trajectory was fitted to a modified logistic function with three parameters, maximum signal, maximum rate, and lag time. This analysis suggest that the co-culture of these species had a negligible effect on their overall growth and their variability seen in the growth curves is most likely due to environmental factors.

Once mastered, the set up can be executed in just a few hours if it is performed properly. While attempting this procedure, it is important to have robustly growing cells to properly adjust the final cell OD values to have uniform auger layers in the chip assemblies and to be mindful during the parylene peel off step. Following this procedure, questions like how does gene expression within each community change as a function of community composition and organization, can be addressed by genetic material analysis.

Don't forget that working with Pseudomonas aeruginosa can be potentially hazardous, and precautions such as gloves, jacket, goggles, and proper aseptic techniques should always be used while performing this procedure.