Biofilm Formation

C1: 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 (E.Coli-GFP tagged)
• Appropriate media for bacteria under study (LB Ampicilin Arabinose Broth (50ml) and Plates (30 plates) for E.Coli)
• 70% ethanol
• 0.1% (w/v) crystal violet in water
• Solvent (80% ethanol/20% acetone; see Table 1B.1.1) for solubilizing dye and biofilm biomass
• 96-well microtiter plates, not tissue culture–treated (Becton Dickinson catalog no. 353911) with lids (Becton Dickinson catalog no. 353913)
• 96-prong inoculating manifold, sterile (DanKar Scientific)
• 4 Small trays (e.g., large pipet tip boxes) sufficient in size to hold 96-well microtiter plates
• Optically clear flat-bottom 96-well plates, nonsterile
• Plate reader or spectrophotometer
• Bioflux 2000

Procedure

Preparing Inoculating Culture

1. Inoculate E.Coli by subculture overnight cultures of the strain of interest and grow to midlog phase (OD600∼0.5 (50 on the Klett)). Dilute subcultures again to OD600= 0.2 (20 on the Kett)
2. Next day, OD reading. Dilute it down to prefered OD (4 diff; 0.1, 0.2, 0.3, 0.5), vialble count by spreading into LB plates.

Inoculation and Incubation

3. Dilute cultures 1:100 in the desired media. Pipet 100 μl of each diluted culture into each of four wells (4 replicates of strian) in a fresh microtiter plate which has not been tissue culture treated. Cover plate* and incubate at optimal growth temperature for the desired amount of time (48 hrs)**

*Lids for the microtiter dishes may be reused. Clean lids with 70% (v/v) ethanol and air-dry prior to each experiment.

**The time course for attachment varies depending on the organism and must be determined empirically, although when using this system, many organisms commonly studied will form a biofilm within 48 hr.

Removing Planktonic (floating) Cells

4. Set up 2 small trays in a series and add 1 to 2 inches of deionized water to the second tray. (The first tray is used to collect waste, while the other is used to wash the assay plate)
5. Remove planktonic bacteria from each microtiter dish by briskly shaking the dish out over the waste tray. To wash wells, submerge plate in the water tray and then vigorously shake out the liquid over the waste tray. Replace water when it becomes cloudy. Keep waste tray.

*Staining of Adherent Cells

6. Add 125 μl of 0.1% crystal violet solution to each well. Stain 10 min at room temperature.
7. Prepare 2 wash trays with 1 to 2 inches of deionized water.
8. 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.

9. 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.

*Measuring Dye Absorbed by Adherent Cells and Matrix

10. Add 200 μl of 80% ethanol/20% acetone* to each stained well. Allow dye to solubilize by covering plates and incubating 10 to 15 min at 25°C (see Table 1B.1.1)

*for SRB, different solvents must be tested; start with 30% acetic acid as it covers a wide range of microbes.

11. Briefly mix the contents of each well by pipetting, and then transfer 125 μl of the crystal violet/acetic acid solution from each well to a separate well in an optically clear flat-bottom 96-well plate. Measure the optical density (OD) of each of these 125-μl samples at a wavelength of 500 to 600 nm (560 nm).
12. Determine μ and δ of OD of biofilm.

SRB Biofilm

13. Thaw cell, at rtp on shaker overnight.
14. Good OD: Plate / Bad OD: Let incubate again overnight; maybe split and incubate at different temperatures (37 and rtp).
15. Tag SRB with GFP (find protocol)