Photodegradable Hydrogel-Based Isolation of Bacterial Colonies in Microwell Arrays

Begin with a microwell array coated with an inert layer across its flat surface.

Add a suspension of bacterial cells onto the array, allowing them to settle into the wells.

Peel off the coating to remove surface-bound cells, retaining those localized within the wells.

Then, secure the microwell array between spacers.

Take a slide with a non-adhesive coating and deposit a hydrogel precursor solution containing a synthetic polymer and a crosslinker dissolved in a buffer onto it.

Invert the slide onto the array and incubate. The crosslinker reacts with the polymer to form a porous hydrogel that entraps the cells.

Remove the slide and place the array in a medium that supports bacterial growth and contains antibiotics to prevent contamination.

The confined bacteria proliferate into spatially distinct colonies.

The hydrogel contains photodegradable cross-links that break upon exposure to ultraviolet light, enabling targeted hydrogel degradation for selective extraction of bacterial colonies.

Seed 700 microliters of bacterial cell suspensions with OD 600 of 0.1 over the microwell array substrate and make the hydrogel precursor solution as demonstrated previously using 12.5 microliters of phosphate buffered saline ATGN of pH eight. Pipette 12.5 microliters of the precursor solution on a non-reactive perfluoroalkylated glass slide and place two 38 micrometer steel spacers on two opposing sides of the microwell array substrate innoculated with cells. Then invert the perfluoroalkylated glass slide with the precursor solution droplet and place a droplet in the middle of the microwell substrate.

When the hydrogel is formed after an incubation of 25 minutes at room temperature, gently remove the glass slide from the microwell substrate and place the substrate in a Petri dish containing ATGN media supplemented with antibiotic.