Measurement

1. Background & Principle

Biofilm is a three-dimensional structure formed when microorganisms attach to solid surfaces and secrete extracellular polymeric substances (EPS). In marine environments, biofilms accumulate on ship hulls, water pipelines, and offshore equipment, resulting in increased drag, higher fuel consumption, and accelerated corrosion.

Our project aims to enhance the production of Zosteric Acid (ZA) in Escherichia coli and validate the anti-biofilm efficacy of the resulting fermentation product for antifouling applications. To assess the performance of a silicone-based coating incorporating the ZA-rich fermentation extract, we utilized the crystal violet staining method.

This assay quantifies biofilm formation by leveraging the specific binding of crystal violet to the biofilm matrix, which generates a distinct purple color. As a natural and eco-friendly antifouling agent, ZA operates through non-biocidal mechanisms such as interfering with microbial adhesion and signaling pathways, thereby minimizing environmental impact while also effectively inhibiting biofilm development.

2. Assay Design Overview

To systematically assess antifouling performance, we designed three complementary assays, each representing a distinct level of environmental realism:

1. 96-well Crystal Violet Assay – standard and high-throughput quantification of biofilm biomass under uniform conditions.
2. Tube Assay – simulates the liquid–air interface, where biofilms typically form in static aquatic systems.
3. Slide Biofilm Assay – mimics real coating conditions using galvanized metal slides to simulate ship-hull surfaces.

These multilevel approaches enable us to evaluate Fer’s antifouling efficiency across laboratory-standard conditions to ocean application-oriented environments.

Two coating formulations were tested: a control consisting of pure silicone elastomer (SYLGARD 184, referred to as the 184 coating) and an experimental coating (Fer) incorporating a ZA-enriched fermentation product. All assays were conducted using the marine bacterium Vibrio natriegens ATCC 14048, a fast-growing Gram-negative model organism relevant to marine biofilm research. Bacterial cultures were grown in LB medium supplemented with v2 nutrients and inoculated at an initial OD₆₀₀ ≈ 0.1 to ensure consistent and reproducible biofilm formation.

1. Coating Preparation

Experimental Materials and Equipment: Silicone elastomer base (SYLGARD 184A), silicone elastomer curing agent (SYLGARD 184B), 96-well plate, 5 mL tube, 20×20 mm slide, fermentation product of AIS-S24.

1-1. Preparation of ZA-Free Silicone Coating

1. Combine 0.90 g of SYLGARD 184A with 0.09 g of SYLGARD 184B (10:1 mix ratio).
2. Mix vigorously using a stirring rod.
3. Apply one drop (~0.05 g) of the mixture to slide / culture tube / 96-well plate.
4. Cure the coated substrates in a 65°C oven for 4 h.
5. Sterilize by autoclaving at 121°C for 30 min, or by UV irradiation for 30 min.

1-2. Preparation of a silicone coating containing the ZA-enriched fermentation product

1. In a 2 mL microcentrifuge tube, aliquot 500 µL S24 strain fermentation solution.
2. Dry the 500 µL fermentation product completely in a 65°C oven for 48 h.
3. Combine 0.90 g of SYLGARD 184A with the dried powder.
4. Add 0.09 g of SYLGARD 184B and mix thoroughly.
5. Mix vigorously using a stirring rod.
6. Apply one drop (~0.05 g) of the mixture to slide / culture tube / 96-well plate.
7. Cure the coated substrates in a 65°C oven for 4 h.
8. Sterilize by autoclaving at 121°C for 30 min or by UV irradiation for 30 min.

2. Crystal Violet (CV) Detection

Experimental Materials and Equipment: 96-well flat-bottom polystyrene plate, LB + v2 medium, Crystal violet solution (0.1%), Acetic acid solution (30%), ddH₂O, Vibrio natriegens ATCC14048, square culture plate.

2-1. 96-well Format

1. Prepare an overnight culture of Vibrio natriegens ATCC14048 in LB + v2 medium (30 °C, 220 rpm).
2. Dilute culture to OD₆₀₀ ≈ 0.1. Inoculate 100 µL per well.
3. Incubate statically at 30 °C for 48–72 h.
4. Remove planktonic cells and wash wells 2–3 times with ddH₂O. Dry at 65 °C for 2 h.
5. Add 125 µL 0.1% crystal violet per well. Stain at room temperature for 10 min.
6. Remove the stain, wash 3–4 times with ddH₂O, and air-dry overnight.
7. Add 200 µL of 30% acetic acid. Incubate 10 min at room temperature with gentle pipetting.
8. Transfer eluate to a fresh plate and measure OD₅₉₀.

Figure 1. 96-well Crystal Violet (CV) Detection Workflow. (1) Prepare an overnight culture of Vibrio natriegens ATCC14048 in LB + v2 medium at 30 °C and 220 rpm, and dilute to OD₆₀₀ ≈ 0.1. (2) Inoculate 100 µL of the bacterial suspension into silicone-coated 96-well plates containing either 184 (control) or Fer (ZA-containing) coatings. (3) Incubate statically at 30 °C for 48–72 h to allow biofilm formation. (4) Remove planktonic cells, wash wells 2–3 times with ddH₂O, and dry at 65 °C for 2 h. (5) Add 125 µL of 0.1% crystal violet, stain for 10 min at room temperature. (6) Remove excess dye and air-dry overnight. (7) Elute the bound dye using 200 µL of 30% acetic acid for 10 min. (8) Transfer eluate to a fresh plate and measure absorbance at OD₅₉₀ nm. Fer coatings showed significantly lower OD₅₉₀ values than 184 coatings (p < 0.05), confirming their antibiofilm activity.

2-2. Tube Format

1. Prepare an overnight culture of Vibrio natriegens ATCC14048 in LB + v2 medium (30 °C, 220 rpm).
2. Dilute the overnight culture to OD₆₀₀ ≈ 0.1. Add 0.5 mL to each tube.
3. Incubate statically at 30 °C for 48–72 h to allow ring biofilm formation at the air–liquid interface.
4. Remove planktonic cells and wash 2–3 times with ddH₂O. Dry at 65 °C for 2 h.
5. Add 0.65 mL of 0.1% crystal violet. Stain for 10 min at room temperature.
6. Remove the stain, wash 3–4 times with ddH₂O, and invert the tubes to drain.
7. Add 0.8 mL of 30% acetic acid. Incubate 10 min at room temperature with pipetting.
8. Transfer 200 µL eluate to a flat-bottom 96-well plate and measure OD₅₉₀.

2-3. Slide Format (OD + ImageJ Quantification)

1. Prepare an overnight culture of Vibrio natriegens ATCC14048 in LB + v2 medium (30 °C, 220 rpm).
2. Place coated slides into square culture plates containing 20 mL inoculated culture (OD₆₀₀ ≈ 0.1).
3. Incubate statically at 30 °C for 72 h.
4. Remove planktonic cells and wash 2–3 times with ddH₂O. Dry at 65 °C for 2 h.
5. Add 0.65 mL of 0.1% crystal violet. Stain for 10 min at room temperature.
6. Remove the stain, wash 3–4 times with ddH₂O, and dry at 65 °C for 2 h.
7. Photograph slides under uniform light conditions.
8. Elute dye with 30% acetic acid for 10 min. Transfer 200 µL eluate to a 96-well plate and measure OD₅₉₀.
9. Analyze biofilm coverage (%) = (stained area / total area) × 100% using ImageJ (Otsu threshold method).

3. Data Analysis

1. The biofilm inhibition rate was calculated as the difference between the OD₅₉₀ value of the control (set at 184 OD₅₉₀) and the OD₅₉₀ value of the fermentation product (Fer OD₅₉₀).

   biofilm inhibition rate (%) = 184 OD₅₉₀ − Fer OD₅₉₀ 184 OD₅₉₀ × 100%

2. The biofilm coverage area percentage was determined by analyzing images with ImageJ, defined as the ratio of the stained area to the total surface area.

   biofilm coverage (%) = stained area total area × 100%

3. Statistical analysis. All data were shown as mean ± SEM from three parallel replicates. Statistical analysis was performed using multiple t-test. Significance indicates as p < 0.05(*), p < 0.01(**). Created by GraphPad Prism 8.0.2.

OD₅₉₀ Quantification of Biofilm Biomass

Figure 2. Crystal violet detection. Biofilm formed by Vibrio natriegens ATCC 14048 on different coatings in 96-well plate, tube and slide assays. 184 (blue) represents the coating without ZA; Fer (orange) represents the coating with ZA-enriched fermentation product. Bar chart (top) with representative photographs (bottom). Data are mean ± SEM, n = 3. Statistics by multiple t-tests; significance: p < 0.05 (*), p < 0.01 (**). Created with GraphPad Prism 8.0.2.

Based on the crystal violet assay for biofilm formation, the OD₅₉₀ value—proportional to biofilm biomass—was consistently lower in the Fer group than in the 184 group across all three substrates (96-well plate, tube, slide), corresponding to inhibition rates of 48.63%, 14.82%, and 26.75%, respectively. This quantitative result corroborates the qualitative observation that the 184 group exhibited darker purple staining than the Fer group after staining, indicating significant antibiofilm activity of the Fer coating under multiple substrate conditions.

ImageJ Surface Coverage Analysis

Figure 3. Biofilm surface coverage analysis of the slide by ImageJ. (A) Actual photographs and corresponding ImageJ-based segmentation. (B) Comparison of biofilm coverage (%) with different coatings on slides. Data are mean ± SEM (n = 3). Statistics by t-test; significance: p < 0.05 (*). 184 (blue): coating without ZA; Fer (orange): coating with ZA-enriched fermentation product.

We performed ImageJ analysis on bright galvanized slides (Fig. 3A). Using a standardized enhancement protocol (contrast uniformly increased by 0.3; threshold range 0–100), the average biofilm coverage area was quantified (Fig. 3B).

The results showed coverage of 48.27% for the 184 group and 29.92% for the Fer group, representing a significant reduction of 37.99% (p < 0.05) in biofilm attachment area for the Fer coating compared to the 184 control.

Segmentation was performed using the Otsu threshold method. Slight deviations (e.g., sample 184-2) were attributed to lighting artifacts rather than true biomass variation. Overall, ImageJ analysis confirmed the OD₅₉₀ trend, demonstrating lower biofilm biomass on Fer coatings.