Materials and Reagents

10 ml Borosilicate glass tube (ISOLAB, catalog number: 077.02.003)
14 ml sterile tube (SPL Lifescience, catalog number: 40014)
Filter (PALL Lifesciences, catalog number: AP-4219)
Strains P. syringae pv. phaseolicola 1448A (Psph) (Xiao et al., 2007) and rhpS deletion mutant (ΔrhpS) (Xie et al., 2019)
NaOH (UNI-CHEM, catalog number: 1310-73-2)
MgSO₄·7H₂O (Aladdin, catalog number: 10025-84-0)
K₂HPO₄ (Aladdin, catalog number: 7758-11-4)
Bacto^TM Proteose peptone No.3 (AOBOX, catalog number: 01-049)
Rifampin (Aladdin, catalog number: 13292-46-1)
Agar (MP Biomedicals, catalog number: 9002-18-0)
Crystal violet (Beijing Dingguo, catalog number: 548-62-9)
Glycerol (Beijing Bailingwei, catalog number: 262536)
100% ethanol (Honeywell, catalog number: 32221-2.5L)
King's B (KB) (see Recipes)

Equipment

1 ml pipette (Eppendorf, catalog number: 3123000063)
Benchtop shaking incubator (Labwit Scientific, model: ZWYR-240)
Constant temperature incubator (Labwit Scientific, model: ZXDP-B2120)
Evolution^TM 350 UV-Vis Spectrophotometer (Thermo Fisher Scientific, catalog number: 912A0959)
Synergy^TM 2 Multi-Mode Microplate Reader (BioTek)
Test tube stand (ISOLAB, catalog number: 079.01.005)
-80 °C freezer (Thermo Scientific, catalog number: 5IDTSX)

Software

Microsoft Office Excel 2016 and GraphPad Prism 8.0.2.

Procedure

Notes:

Select wild-type P. syringae pv. phaseolicola 1448A (Psph) as the model strain (Xiao et al., 2007) and rhpS deletion mutant (ΔrhpS) (Xie et al., 2019) as the test strain.
Perform the step-by-step protocol described in Figure 1.
Perform the entire procedure gently to avoid damaging the biofilm.
Collect the liquid crystal violet and ethanol waste liquor in specially labeled containers for professional disposal by trained staff, as per the safety regulations at City University of Hong Kong.

Figure 1. Schematic step-by-step protocol for visualizing P. syringae biofilm formation. A. The Psph strain was activated on King’s B (KB) plate and cultured in liquid medium. B. Then the cultures were inoculated at 1:500 dilutions into KB liquid medium and incubated statically to sampling points. C. Then stained the biofilm by using 0.1% crystal violet. D. Measure the biofilm production at OD_590nm. Two biological replicates were showed.

Bacterial growth
1. Collect a Psph colony from the glycerol stock culture frozen at -80 °C and inoculate on a King’s B (KB) plate supplemented with rifampicin (25 μg/ml). Incubate the KB plate at 28 °C for 36 h in a constant temperature incubator.
2. Collect a single colony from the cultured plates and inoculate into a sterile 10 ml tube containing 2 ml KB liquid medium supplemented with rifampicin (25 μg/ml). Incubate the tube in a benchtop shaking incubator at 28 °C for 12 h with constant shaking at 220 rpm.
Biofilm formation
Inoculate the Psph culture (1:500 dilutions) into 16 sterile 10 ml borosilicate glass tubes containing 2 ml KB liquid medium (supplemented with rifampicin) and ensure consistent initial dose. Incubate the Psph-containing glass tubes at 28 °C in a constant temperature incubator without shaking.
Biofilm visualization
1. Harvest the biofilm samples at different time points. For Psph in our study, the biofilms were harvested at 24, 48, 72 and 96 h. Gently discard the planktonic cells with a 1 ml pipette and wash the tubes three times with sterile distilled water. Avoid damaging the biofilm formed on the tube wall.
2. Stain the biofilm forming bacteria with 2.5 ml 0.1% crystal violet for 20 min without shaking. Discard the dye and wash the tubes with sterile distilled water to remove the unbound dye. Dry the tubes and take photographs (Figure 2A).
Biofilm measurement
1. Elute the biofilm with 2 ml 100% ethanol and shake the tubes at 220 rpm for 20 min to ensure that the dye has dissolved completely. Take photographs (Figure 2B).
2. Measure the eluted samples at OD_590nm using a spectrophotometer (2 ml) or Synergy 2 Plate Reader (BioTek) (100 μl). If the sample concentrations are too high, diluted them before measuring. Use an equal volume of 95%-100% ethanol as the blank control. Psph biofilm formation is shown in Figure 2C. The ΔrhpS produces lower biofilm compared with Psph wild-type strain (Figure 3).
  
  Figure 2. Visualization and quantification of biofilm in Psphwild-type strain. A. Biofilm samples were grown from 24 to 96 h. Biofilm adhered to borosilicate glass tubes at different time points were stained with crystal violet. B. The crystal violet bound to the biofilm on the wall of the tubes was eluted by ethanol. C. The elution samples were measured at OD_590nm by using spectrophotometer or Synergy 2 Plate Reader (BioTek). Psph wild-type strain produced more biofilm at 96 h than 24 h. * represents P-value < 0.05. *** represents P-value < 0.001. Error bars indicate S.D. among four biological replicates. Two biological replicates were shown.
  
  Figure 3. The ΔrhpS strain produced less biofilm than did that in Psph wild-type strain. A. Biofilm produced by the Psph wild-type and the ΔrhpS strain were visualized using borosilicate glass tubes and stained with crystal violet at 96 h. B. The crystal violet bound to the biofilm on tube wall was eluted by ethanol. C. The Psph wild-type strain produced more biofilm than the ΔrhpS strain (P-value = 0.000136). *** represents P-value < 0.001. Error bars indicate S.D. among four biological replicates. Two biological replicates were shown.

Data analysis

Student’s t-tests were performed using Microsoft Office Excel 2016. Quantitative data (OD_590nm) were collected from four biological replicates in the figures and tables (Tables 1 and 2).

Table 1. Biofilm production of Psph wild-type strain presented in Figure 2C. Two-sample equal variance was calculated by the following one-tailed Student’s t-test formula in Excel = TTEST (array1, array2, tails, type). Array 1 is the first data set. Array 2 is the second data set. Tails show the number of distribution tails (1 for the one-tailed distribution, 2 for two-tailed distribution). Type is the kind of t-test to perform (1 for paired, 2 for two-sample equal variance, and 3 for two-sample unequal variance. For example, we used TTEST (B2:E2, B3:E3, 1, 2), TTEST (B2:E2, B4:E4, 1, 2) and TTEST (B2:E2, B5:E5, 1, 2) for biofilm production at 48 h, 72 h, 96 h, compared to 24 h respectively. The P-values were showed in Excel H3, H4 and H5 respectively. * represents P-value < 0.05. ** represents P-value < 0.01. *** represents P-value < 0.001. Means and S.D. are shown in columns F and G. All experiments were repeated four times.

Table 2. Biofilm production of the Psph wild-type and the ΔrhpS strain presented in Figure 3C. Two-sample equal variance was calculated by the following Two-tailed Student’s t-test formula in Excel = TTEST (B2:E2, B3:E3, 2, 2). The P-values were showed in Column H3. * represents P-value < 0.05. ** represents P-value < 0.01. *** represents P-value <0.001. Means and S.D. are shown in Column F and G. All experiments were repeated four times.

In sum, the results showed that the wild-type Psph strain produced more biofilm in 48 h (P < 0.05), 72 h (P < 0.001) and 96 h (P < 0.001) than at 24 h (Figure 2 and Table 1). Besides, the ΔrhpS strain produced less biofilm than Psph wild-type strain (P < 0.001) at the same time point (96 h) (Figure 3 and Table 2).

Recipes

King's B (KB) (King et al., 1954)
Bacto^TM Proteose peptone No.3
20.0 g/L
K₂HPO₄
1.5 g/L
Glycerol
15 ml/L
Dissolve in 993.75 ml ddH₂O and adjust the pH to 7.2. Add 15 g/L agar for solidified media and autoclave
Dissolve 24.637 g MgSO₄·7H₂O in 100 ml ddH₂O and sterilize this stock solution (1 M) using sterile filter (0.45 µm)
Then, add 6.25 ml MgSO₄·7H₂O (1 M) (final concentration 1.5 g/L) into the autoclaved King's B medium when the temperature drops to 40 °C-50 °C

Acknowledgments

This project has been funded in Health Medical Research Fund [17160022 to X.D.]; National Natural Science Foundation of China Grants [31670127 to X.D.].
This protocol was adapted from O'Toole et al. (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Molecular Microbiology, 30: 295-304.

Competing interests

Conflict of interest statement: None declared.

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