Team:NTNU Trondheim/Measurement

Development of a biofilm measurement protocol

Our measurements of bacterial biofilms to test our luxS-targeted CRISPRi-system were not possible without designing an organized and comprehensive experimental setup. We tested and evaluated E. coli’s biofilm formation in 18 different LB and M63B1 media, with different pH and glucose levels. We also tested a broad range of incubation time points for our bacteria and also added adjustments to optimize the conditions. All our observations were thoroughly evaluated and implemented in our well-designed model for the acquired data and observations.

As an extra feature, we implemented two sets of negative controls for our CRISPRi system. We grew DH5α and TG1 without the inducer for expression of the CRISPRi system as a negative control. In addition, we considered DH5α as an extra negative control, due to its low biofilm formation in the first place.

Testing of biofilm promoting mediums

At the beginning of the project, we found a protocol for measuring biofilm formation [1]. Then we had a meeting with Kåre Bergh, who pointed out weaknesses with the current protocol and showed a protocol he had used on a different kind of bacteria [2]. Later we talked to Sven Even Burgos, who also talked to his colleague Anne Tøndervik. They advised us to look at an article [3] that discussed different kinds of methods for quantification of biofilm formation. We had been informed by Kåre Bergh and Sven Even Borgos (see Human Practices for more information about these interviews) that the bacteria’s ability to form biofilms is highly dependent on their growth conditions, and that they are especially sensitive to glucose concentrations and pH levels. We wanted to test our CRISPRi system in a medium that promotes biofilm formation because it would make it easier to see a noticeable effect if the system works as expected.

In order to investigate which medium gives the highest degree of biofilm formation, we made nine variants of LB and M63B1 medium varying in pH levels (4.5, 7.2 and 9.2) and glucose concentrations (0%, 0.4%, and 0.8%). E. coli TG1 cells were inoculated in each medium and incubated. Table 1 shows the measured OD values for the overnight TG1 cultures. We observed a higher planktonic growth in LB than in M63B1. This is consistent with the fact that M63B1 is a minimal medium with a limited amount of nutrition, mimicking natural conditions [4]. LB, on the other hand, is a nutrition rich medium which provides good conditions for rapid and numerous growth [4]. Generally, the highest growth in both LB and M63B1 were observed in mediums with a pH level of 7.2 supplemented with glucose, either 0.4%, and 0.8%, with only slight differences between the two.

Table 1: Measured OD600 values in overnight E. coli TG1 cell cultures grown in LB and M63B1 mediums with different pH levels (4.5, 7.2 and 9.2) and supplemented with different glucose concentrations (0%, 0.4% and 0.8%).
pH, glucose OD600
LB M63B1
4.5, 0% 1.790 0
4.5, 0.4% 0.023 0.002
4.5, 0.8% 0.600 0.001
7.2, 0% 1.697 0.002
7.2, 0.4% 1.200 0.201
7.2, 0.8% 1.255 0.228
9.2, 0% 0.006 0
9.2, 0.4% 0.005 0.002
9.2, 0.8% 1.366 0.002

To measure the biofilm formation capabilities of the TG1 cells in the different mediums, we plated each culture on microtiter plates and incubated them for 24 hours. The amount of biofilm adhered to the walls of the wells was then measured by a Crystal Violet Assay. Even though M63B1 gave a low degree of planktonic growth compared to LB (see Table 1), the M63B1 seemed to have a strong biofilm promoting effect in mediums with pH 7.2 and glucose concentrations of 0.4% and 0.8%. The biofilm formation in LB followed the same trend as the planktonic growth. We concluded that the best biofilm conditions were in M63B1 with a pH level of 7.2 and a glucose concentration of either 0.4% or 0.8%. For comparison purposes, we also decided to do measurements in LB medium having the equivalent pH level and glucose concentrations as the optimal biofilm promoting M63B1 mediums.

Improving the Crystal Violet Assay for quantitative biofilm measurements

The next step was to test if we were able to reduce biofilm formation in bacteria using our CRISPRi system targeting the luxS gene in E. coli. We inoculated the chosen LB and M63B1 (pH 7.2 and glucose concentrations of 0.4% and 0.8%) with DH5α and TG1 cells transformed with pdCas9 and pgRNA with the anti-luxS sequence. After an overnight incubation, the cultures were diluted and plated on microtiter plates. Tetracycline was added to the mediums of our test groups since it is the inducer for the transcription of dCas9. Our control groups lacked this addition.

During the first run, we observed that there was some variation between replicates. We therefore decided to run it several times to perfect the protocol to measure biofilm formation using Crystal Violet. In total, we ran the experiment several times before we were satisfied. For each run, we did some slight changes to the protocol:

  • Biofilm measurements with Crystal Violet after only 24 hours of incubation: The first time we performed the Crystal Violet assay we measurement only after an incubation time of 24 hours.

  • Biofilm measurements with Crystal Violet Assay conducted after 24, 48 and 72 hours of incubation.

  • We used our model to investigate what effect removal of the step to change medium after 4 hours of adhesion, would have on the biofilm formation. After studying the model we desided to remove the 4 hours of adhesion step. This also helped to reduce the possibility to get contamination between the wells.

  • Biofilm measurements with Crystal Violet Assay conducted after 3, 5, 8, 24 and 72 hours of incubation.

  • Biofilm measurements with Crystal Violet Assay conducted after 3, 5, 8, 24 and 30 hours of incubation.

  • Tried with parafilm over the plates to reduce evaporation, which may be the reason for the variability observed between the replicates – especially the measurements after 24 and 30 hours.

  • Created a humidity chamber to even further reduce the evaporation.

Final Biofilm Measurement Protocol

Preparation: day 1

  • Grow bacteria overnight in liquid medium until OD600 reaches a minimal value of 0.1.

  • Make media: LB and M63B1(0%, 0.4%, 0.8% glucose of total volume and a pH of 7.2)

  • Prepare solutions: Physiological saline (PS), 30% acetic acid, 99% methanol, 0.1% crystal violet.

Biofilm formation: day 2/3 .

  • From the liquid culture, dilute until the OD is approximately 0.1:

    1. Pipette 3-4 mL overnight culture to sterile tubes (total of 16 tubes)

    2. Centrifuge the cultures for 10 min at 2000 rpm at 9 ℃.

    3. Remove the supernatant.

    4. Add 3-4 mL media (LB/ M63B1, with 0.4%/0.8% glucose, with/without TET) to tubes, after the supernatans are removed.

    5. Resuspend/vortex the cultures, and measure OD600.

    6. Dilute the cultures until target OD600=0.1, and store the cells on ice until they are added to the wells.

  • For each of the diluted cultures, inoculate 100 ul to each well in a 96-well microtiter plate (4 replicates). One plate for each medium (LB/M63B1). Make two replicates of sterile medium for each medium (total of 36) for control. Remember to resuspend/ vortex the cell cultures before incolation!

  • Incubate for 0, 3, 5, 8, 24, and 30hrs at 37 degrees.

Figure 1: Set up for the 96 well plate

Crystal violet assay at room temperature: day 3

  • Remove supernatant, and rinse with 100μL PS

  • Add 100 μL of 99% methanol and wait for 15 minutes while biofilm fixates.

  • Remove the supernatant, airdry the plates.

  • Add 100 μL of CV-solution to all the wells and wait 20 minutes. Wash away excess CV with dH2O

  • Add 150 μL of the acetic acid-solution to each well to dissolve the biofilm and CV.

  • Shake the plate for 5min, and measure the absorbance at 590 nm by plate reader.

  • [1] Microtiter dish biofilm formation assay. J Vis Exp. 2011 Jan 30;(47). pii: 2437. doi: 10.3791/2437.
  • [2] A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods. 2000 Apr;40(2):175-9.
  • [3 ]Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods. 2008 Feb;72(2):157-65. Epub 2007 Nov 21. J Vis Exp. 2011 Jan 30;(47). pii: 2437. doi: 10.3791/2437.
  • [4] Miller, Jeffry H. (1972) "Experiments in molecular genetics."

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