Determining biofilm growth amount (absorbance)
An.Huang
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Abstract
This protocol describes a method to determine the growth amount of biofilm at the early stage of biofilm formation by measuring absorbance. Here we use our own engineered bacteria, and it requires induction of IPTG and cultured with silver ion. You may use a different kind of bacteria and different culturing medium.
Steps
IPTG induction
Escherichia coli grown overnight was diluted by LB to OD600=0.4-0.6.
IPTG was added to cell culture to 1mM IPTG finally, and incubated 3h at 171 rpm, 37℃ in orbital shaking incubator.
Sample preparation
Preparing several 100mL flasks by filling the flasks with MBBR carrier K1. Autoclave all the flasks and dry them in an oven at 60s℃.
Cells cultures and silver nitrate (to 6μM) solution were mixed in advance. Add the mixture into each bottle.
Incubate all bottles in a biochemical incubator at37°C .
Absorbance measurement
At the day of measuring the absorbance, take the flasks to be tested out from incubator.
Using a pair of forceps, carefully pick out the carriers that has not been submerged into the culture and discard.
Randomly pick five carriers out from culturing flask and put into a 50 mL centrifuge tube. Label this tube as "Washing tube".
Wash these five carriers using 1X phosphate-buffered saline (PBS) for three times. Collect all eluents.
Use a dropper, add 2-3 mL 1X PBS into the Washing tube. Cap the tube and rinse every corner of the inside wall of tube using the PBS added by rotating the tube. Collecting the eluents by pouring these PBS into Eluent tube. Repeat these steps for three to five times.
Centrifuge the Eluent tube at10000rpm .
Discard all supernatants.
Add 3µL 1X PBS into Eluent tube. Resuspend the pellet.
Measure the absorbance of the liquid at OD600. Record the absorbance.
Repeat steps from Step 7 to Step 14 for three times. Calculate the mean value for the three measurements. A growing curve of biofilm can be generated from the data collected.
