Biofilm Formation

Introduction

To generate biofilm in order to study the effects of the generated bacteria on the intensity of the dye use to detect the biofilm. Proctocol adapted from: Here Here

Materials

Bacterial strains of interest (SRB culture)
70% ethanol
LB broth media
0.1% (w/v) crystal violet in water
Artifical Sea Water Solution composed of (per liter of distilled water): : 24.6g NaCl, 0.67g KCl, 1.36g CaCl2•2H2O, 6.29g MgSO4•7H2O, 4.66g MgCl2•6H2O, 0.18g NaHCO3, 3g peptone, 1 L distilled water (pH 7.5-8).
Plate reader or spectrophotometer
Small trays (e.g., large pipet tip boxes) sufficient in size to hold 96-well microtiter plates
96-well microtiter plates, not tissue culture–treated (Becton Dickinson catalog no. 353911) with lids (Becton Dickinson catalog no. 353913)
Solvent (e.g., 30% v/v acetic acid in water; see Table 1B.1.1 for other options) for solubilizing dye and biofilm biomass

Procedure

Inoculation of the SRB culture

Inoculate SRB of interest in a 3-to-5-ml culture media and grow to stationary phase (check using Klett OD reader)

Inoculation in different Salt concentrations and incubation

Dilute cultures 1:100 in the desired media.

Pipette 100 μl of each diluted culture into each of four wells in a fresh microtiter plate which has not been tissue culture treated

Add 100 μl of LB and Sea salt solution with varying degrees of NaCl

Make solutions of 2X LB or sea water with NaCl concentrations 0.05 M, 0.1 M, 0.2 M, 0.4 M, 0.8 M and 1.6 M

NaCl Final Concentration (M)	2x LB broth or Sea Water without NaCl (mL)	5 M NaCl (mL)
0	10	0
0.05	9.9	0.1
0.1	9.8	0.2
0.2	9.6	0.4
0.4	9.2	0.8
0.8	8.4	1.6
1.6	6.8	3.2

Make solutions of 2X LB with NaCl concentrations 1.7 M, 1.9 M, 2.1 M, 2.3 M and 2.5 M

NaCl Final Concentration (M)	2x LB broth(mL)	5 M NaCl (mL)
0	10	0
1.7	6.6	3.4
1.9	6.2	3.8
2.1	5.8	4.2
2.3	5.4	4.6
2.5	5	5

Cover plate and incubate at optimal growth temperature for the desired amount of time (48 hours).

Removal of Planktonic cells

Set up four small trays in a series and add 1 to 2 inches of tap water to the last three. (The first tray is used to collect waste, while the others are used to wash the assay plates.)

Remove planktonic bacteria from each microtiter dish (step 2a or 2b) by briskly shaking the dish out over the waste tray. To wash wells, submerge plate in the first water tray and then vigorously shake out the liquid over the waste tray. Replace water when it becomes cloudy.

Add 125 μl of 0.1% crystal violet solution to each well. Stain 10 min at room temperature.

Shake each microtiter dish out over the waste tray to remove the crystal violet solution. Wash dishes successively in each of the next two water trays (i.e., the two not used in step 4), and shake out as much liquid as possible after each wash.

Shake each microtiter dish out over the waste tray to remove the crystal violet solution. Wash dishes successively in each of the next two water trays and shake out as much liquid as possible after each wash.

This step will remove any crystal violet that is not specifically staining the adherent bacteria. The wash trays can be reused for a number of plates, but the water should be replaced when its color becomes dark or when the efficiency of the washes is observed to decrease.

Invert each microtiter dish and vigorously tap on paper towels to remove any excess liquid. Allow plates to air-dry

At this stage, the staining is stable and the dried plates may be stored at room temperature for at least several weeks.

Measurement of the optical density (OD)

Add 200 μl of 30% acetic acid (or another appropriate solvent) to each stained well. Allow dye to solubilize by covering plates and incubating 10 to 15 min at room temperature.
Briefly mix the contents of each well by pipetting, and then use a plate reader to measure OD @ 500-600 nm.
Generate standard curve (OD vs NaCl concentration) to observe the relationship between Salt concentration and biofilm production.

Team:CMUQ/Biofilm Formation Lab

Biofilm Formation

Biofilm Formation