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Latest revision as of 01:53, 2 November 2017
Biofilm Formation
Biofilm Formation
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
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 |
Removal of Planktonic cells
- 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.
- 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.