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1. Inoculation of Polymer Microarrays with Bacteria
NOTE: Ensure good aseptic technique. All the handling of cultures should be performed in a sterile environment: either using a Bunsen burner or in a flow hood. Cultures should be grown with oxygen availability, growth medium, and temperature adjusted to the requirements of each species.
- Prepare overnight cultures by inoculating 5 mL Luria-Bertani broth (LB: 10 g/L Bacto-tryptone, 5 g/L sodium chloride, NaCl, and 10 g/L yeast extract) with a colony from a plate. Incubate overnight at 37 °C, shaking at 200 rpm (revolutions per minute).
- Prepare freezer stocks of overnight cultures by the addition of glycerol (10% concentration overall) and store the stock at -80 °C.
- In order to determine the cell numbers from samples of the thawed bacterial stocks, perform serial dilutions of each stock and grow the bacteria overnight on solid media. Accurate determination of cell numbers in stocks ensures appropriate inoculation of each species.
- Prepare inocula for microarray. Single-species cultures are grown as above and applied directly to the microarrays. BacMix-1 is a bacterial mixture of Klebsiella pneumoniae (K. pneumoniae), Staphylococcus saprophyticus (S. saprophyticus), and Staphylococcus aureus (S. aureus). BacMix-2 is a bacterial mixture consisting of Streptococcus mutans (S. mutans), S. aureus, K. pneumoniae, and Enterococcus faecalis (E. faecalis).
- To produce either mixed culture, mix overnights in equal volumes (3 ml each) and dilute four times with fresh LB (to around 50 ml final volume).
- Place ultraviolet-sterilized polymer microarrays in rectangular 4-well plates and incubate them with 6 ml of mixed bacterial cultures at 37 °C with gentle agitation (30 rpm) for 5 days.
- After incubation, gently rinse the microarrays twice with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM potassium chloride, KCl, 10 mM disodium hydrogen phosphate, Na2HPO4, 1.8 mM potassium dihydrogen phosphate, KH2PO4) and incubate with a solution of 1 µg/ ml of 4',6-diamidino-2-phenylindole (DAPI) stain in PBS for 30 min.
- Wash the stained microarray slides in a fresh well plate by covering with PBS and swirling gently, changing the PBS once. Dry under a flow of air. Apply a glass cover slip to the microarray slide and seal in place with glue. Once dry, sterilize the outer surface with 70% (v/v) ethanol.
- Safely dispose of cultures according to their containment level.
2. Microarray Imaging and Analysis
- Capture single images for each polymer spot in brightfield and DAPI (Excitation/Emission, Ex/Em = 358 nm / 361 nm) channels. Perform this manually with a fluorescent microscope or using an automated fluorescence microscope (with an X-Y-Z stage controlled by an image acquisition software) fitted with a 20X objective.
NOTE: Adjust the microscope to obtain images using brightfield and DAPI channels. Ensure that the gain and exposure time for image capture for all the microarrays are the same. - Obtain the average fluorescence intensity of each spot in the DAPI channel by choosing the spot area and quantifying the fluorescence with an image processing software.
- For the background fluorescence of each spot, obtain images of the polymer spots on a replicate microarray incubated only with media with no bacteria. Deduct the background fluorescence from the intensity of the spot to obtain fluorescence from bacteria.
- Calculate the average normalized value from the four spots, representing each polymer, used for comparing bacteria binding of various polymers.
- Identify the polymers that exhibit the least bacterial binding (lowest fluorescence) as 'hit' polymers.