1. Confocal Laser Scanning Microscopy of A. baumannii S1 Biofilm
1. Grow a 5 ml culture of A. baumannii S1 in LB at 30 °C in a shaking incubator (220 rpm) for 16 hr.
2. Adjust the culture of A. baumannii S1 to a desired OD600 of 0.8. Using a 35 mm glass-bottomed µ-Dish, inoculate the bacteria culture (1:100 dilution) into fresh LB containing 30 µl of purified GKL enzyme (40 mg/ml); the new culture's final volume is 1 ml.
3. Cover the µ-Dish with a lid and place it in a sealed 10 L plastic container. Incubate the µ-Dish at 30 °C for 3 hr before gently removing the media. Add 30 µl of purified GKL enzyme and fresh medium, bringing it to a total volume of 1 ml. Incubate for another 21 hr at 30 °C.
4. Repeat step 1.3 and incubate the µ-Dish for another 24 hr at 30 °C. Then, remove the media gently.
5. Add 500 µl of 5 µg/ml Alex Fluo 488-conjugated wheat germ agglutinin (WGA) dissolved in Hank's balanced salt solution (HBSS) to the µDish and incubate at 37 °C for 30 min; this will stain the formed biofilm. Remove the staining solution and wash the µ-Dish with 2 ml of HBSS. Repeat the wash step one more time.
6. Add 500 µl of 3.7 % formaldehyde dissolved in HBSS and incubate at 37 °C for 30 min; this will fix the biofilm onto the µ-Dish. Wash the µDish once with 2 ml of HBSS and then remove the solution completely. The µ-Dish fixed with biofilm can be stored in the dark at 4 °C prior to CLSM imaging.
7. For CLSM imaging and analysis, use 63 times magnification to generate 97 stacks per image with an interval of 0.21 µm per stack.
