This protocol presented an easy and reliable method to measure alkalized phosphatase activity in S.aureus biofilm culture. It will help us to understand the function of ALP in biofilm formation. The major advantage of this technique is high throughput.
It is very sensitive and easy to perform. Demonstrating the procedure will be Kevin Danikowski, a former student from Harper College. To prepare TSB, add pancreatic digest of casein, papaic digest of soybean meal, sodium chloride, dextrose, and dipotassium phosphate into distilled water.
Cover the flask with aluminum foil and put it in an autoclave to sterilize before use. To prepare a TSA, add agar to TSB and put it in the autoclave for sterilization. Then let it cool to room temperature.
Next, pour it into the Petri dish at a ratio of 20 milliliters per dish. To prepare a biofilm culture medium, add glucose to autoclaved TSB to achieve the concentration of 10 grams per liter. Using a vacuum filtration kit, filtrate the solution with a 0.2 micrometer pore sized filter.
To inoculate, use a sterilized inoculation loop to pick one individual colony of Staph aureus from the Petri dish and place it in the culture tube filled with 10 milliliters of TSB. Put the tube in the incubator to grow overnight at 37 degrees Celsius. With a micropipette, draw 100 microliters of overnight culture and transfer it to a culture tube filled with 10 milliliters of TSB with glucose for a 1:100 volume ratio dilution.
Vortex gently to mix for a consistent concentration. Use a micropipette to transfer 200 microliters of diluted culture into a total of 18 wells from one 96 well plate. Incubate the 96 well plate at 37 degrees Celsius for 24 hours for biofilm formation.
For the best biofilm formation of S.aureus, it is better to pick a flat or round bottom 96 well plate. After that, slightly tilt the plate to decant the supernatant by aspiration from the edge of each well. Wash each well with 1X PBS.
Put the plate in the centrifuge to spin for five minutes at 8, 000 revolutions per minute and decant the supernatant by aspiration. Add 75 microliters of buffered ALP substrate consisting of commercially available PNPP to each well and use a timer to record each time point in triplicate. After each incubation time point, add 75 microliters of five molar sodium hydroxide to each of three wells to stop the reaction.
Centrifuge the plate for five minutes at 800 revolutions per minute. Use a pipette to transfer 100 microliters of supernatant into a new 96 well plate for calorimetric measurement. Use a 96 well plate reader to measure the absorbance of each well at 405 nanometers.
Use the 30-minute time point wells to control for background noise. Repeat the entire experiment in three 96 well plates. This protocol develops a quick and reliable assay to measure ALP activity in S.aureus biofilm that does not require protein isolation.
The representative result of ALP activity shows an increased trend for up to 75 minutes. After 75 minutes, no increase of the ALP activity was observed. It is very important to establish a good biofilm culture and it may take quite a few trials to master.
You can confirm biofilm formation by performing crystal violet assay as previously reported by us and others or you can pre-treat the plate with human plasma to facilitate biofilm formation. After its development, you can screen potential ALP inhibitors that might interfere biofilm formation. The result will provide important information toward biofilm management in medical field.
Five molar sodium hydroxide might be hazardous in case of contact. Be sure to wear personal protective equipment when handling.
