This study outlines a method for assessing bacterial attachment to polymer microarrays using fluorescence microscopy. The approach involves incubating mixed bacterial cultures on agarose-coated slides to evaluate the effectiveness of different polymer spots in promoting or resisting bacterial colonization.
Place a sterilized, agarose-coated microarray slide with multiple polymer spots into a multiwell plate. The agarose minimizes non-specific bacterial binding.
Add a mixed bacterial culture to the slide and incubate it with gentle agitation.
Bacteria secrete distinct adhesion proteins that bind to favorable polymer spots, resulting in stronger bacterial attachment and colonization over time.
Conversely, repellent polymers resist protein adsorption and reduce bacterial attachment.
Rinse the slide with phosphate-buffered saline or PBS to remove loosely bound bacteria.
Add DAPI to stain the DNA of surface-attached bacteria.
Wash the slide with PBS, dry it, and seal it with an adhesive coverslip.
Sterilize the outer slide surface with ethanol.
Capture bright-field images to locate the polymer spots. Then, switch to fluorescence mode to detect the fluorescence on the polymer spots.
Polymer spots that exhibit reduced fluorescence indicate minimal bacterial attachment and are identified as bacteria-repellent polymers.
After culturing and preparing inocula for the microarrays, according to the text protocol, place the UV sterilized polymer microarrays in rectangular four-well plates and add six milliliters of mixed bacterial cultures. Incubate the plates at 37 degrees celsius with gentle agitation for five days. After the incubation, use PBS to gently rinse the microarrays twice.
Then add one microgram per milliliter of DAPI in PBS and incubate for 30 minutes. Next, transfer the microarrays to a fresh four-well plate, cover the microarrays with PBS then gently swirl. Then change the PBS once and repeat the wash.
After rinsing, dry the microarrays under a flow of air then apply sealing film and affix glass cover slips to the microarray slides. When the glue is dry, use 70% ethanol to sterilize the outer surface. Using a fluorescence microscope, fitted with a 20x subjective, capture single images for each polymer spot in bright field and DAPI channels.
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