Ethanol Resistance Assay to Evaluate Biofilm Matrix Integrity

Abstract

Source: Bucher, T., et al. Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp. (2016).

This video demonstrates how biofilm matrix integrity influences bacterial resistance to ethanol by comparing colony-forming units in untreated and inhibitor-disrupted biofilms. Reduced CFUs in inhibitor-treated samples demonstrate that the extracellular matrix is essential for protecting biofilm-embedded cells from antimicrobial stress.

Protocol

1. Ethanol Resistance Assay

1. Grow biofilms and after 68 hr of growth at 30 °C, cut the biofilm colonies into two equal parts with the help of a razor blade and the template.
2. Carefully lift each half of the biofilm colony from the agar plate with a small spatula and move it to a 1.5 ml microcentrifuge tube containing 500 µl of phosphate-buffered saline (PBS). If necessary, scrape the remaining cells from the plate and transfer them to the microcentrifuge tube as well.
   NOTE: The second half of the biofilm colony is treated differentially, depending on whether it is the control or to test the resistance to sterilizing agents.
3. For the control, incubate the second half of the biofilm colony in 500 µl PBS. To assess resistance to sterilizing agents, transfer the second half of the biofilm colony to 500 µl 50% (v/v) ethanol.
   NOTE: Alternative sterilizing agents such as sodium hypochlorite can be used. For all sterilizing agents used, determine the active concentration and incubation time in a preliminary experiment.
4. Incubate the biofilm colonies for 10 min on the bench-top at room temperature.
5. Centrifuge the biofilm colonies for 5 min at 18,000 x g and carefully remove the supernatant with a pipette. Add 300 µl of PBS.
6. Sonicate the cells mildly (amplitude 10%, pulse 5 sec) with the microtip of a sonicator.
   NOTE: The sonication energy must be sufficient to separate biofilm aggregates. However, too harsh sonication may lyse the cells. Confirm in advance by light microscopy that the sonication energy used does not lyse the cells and that all aggregates are dissolved.
7. Add 700 µl of PBS to a final volume of 1 ml. Perform a serial dilution (to 10^-7) in PBS and spread 100 µl of 3 dilutions on a LB-1.5% agar plate using sterile glass beads.
   NOTE: The optimal dilutions to be plated should be determined in a preliminary experiment, as this depends on the amount of cells in the biofilm colony of interest and the survival rate of the cells in response to the sterilizing agent.
8. Incubate the plates overnight at 30 °C, count the CFU and determine CFU/ml. From the final CFU/ml of each the half biofilm colonies, calculate the percentage of survivors.
   NOTE: When performed and analyzed as described, the two halves of the control biofilm colony and the untreated versus ethanol-treated half of the untreated biofilm colony should yield differences below 10% in viable cell counts, verifying the symmetry or the resistance of the colony, respectively. Alternatively, the results can be represented in total CFU. The cell counts of the control and untreated biofilm colony should remain in the same order of magnitude. In contrast, the cell counts of the ethanol-treated half of a small molecule-treated biofilm colony are expected to drop by a minimum of two orders of magnitude to claim for an increased sensitivity to the sterilizing agent.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Luria Broth, Lennox	Difco	240230
Bacto Agar	Difco	214010
Potassium phosphate monobasic	Sigma, 136.09 g/mol	P0662-500G
Potassium phosphate dibasic	Fisher Scientific, 174.18 g/mol	BP363-1
3-(N-morpholino)propanesulfonic acid	Fisher Scientific, 209.27 g/mol	BP308-500
Magnesium chloride hexahydrate	Merck, 203.30 g/mol	1.05833.0250
Calcium chloride anhydrous	J.T. Baker, 110.98 g/mol	1311-01
Manganese(II) chloride tetrahydrate	Sigma, 197.91 g/mol	31422-250G-R
Iron(III) chloride hexahydrate	Sigma, 270.30 g/mo)	F2877-500G
Zinc chloride anhydrous	Acros Organics, 136.29 g/mol	424592500
Thiamine hydrochloride	Sigma, 337.27 g/mol	T1270-100G
L-tryptophan	Fisher Scientific, 204.1 g/mol	BP395-100
L-phenylalanine	Sigma, 165.19 g/mol	P5482-100G
L-threonine	Sigma, 119.12 g/mol	T8625-100G
Glycerol anhydrous	Bio-Lab Itd	712022300
L-glutamic acid monosodium salts hydrate	Sigma, 169.11 g/mol	G1626-1KG
D-leucine	Sigma, 169.11 g/mol	855448-10G
Ethanol anhydrous	Gadot	830000054
Razor blade	Eddison	NA
Circular cellulose filter papers	Whatman, 90 mm	1001-090
Glutaraldehyde	EMS (Electron Micoscopy Science), 25% in water	16220
Paraformaldehyde	EMS, 16% in water	15710
Sodium cacodylate	Merck, 214.05 g/mol	8.2067
Calcium chloride 2-hydrate	Merck, 147.02 g/mol	1172113
Shaker 37 °C	New Brunswick Scientific Innowa42	NA
Centrifuge	Eppendorf table top centrifuge 5424	NA
Digital Sonifier, Model 250, used with Double Step Microtip	Branson	NA
Incubator 30 °C	Binder	NA
Incubator 23 °C	Binder	NA
Filter System, 500 ml, polystyrene	Cornig Incorporated	NA
Rotary Shaker - Orbitron Rotatory II	Boekel	NA