Name: Quantification of Violacein in Chromobacterium violaceum and Its Inhibition by Bioactive Compounds (Video) | JoVE Journal
Uploaded: 2025-08-08T14:10:00.000+00:00
Duration: 7 min 13 s
Description: Learn to quantify violacein production in Chromobacterium violaceum ATCC 12472 using spectrophotometric microplate assays. This method screens bioactive compounds for quorum sensing inhibition without affecting bacterial growth.

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5 minutes

We would like to thank the National Conseil for Scientific Development (CNPQ) through grants #403661/2023-4 and #444794/2024-7 and scholarship #306685/2022-1, and the Sao Paulo Research Foundation (FAPESP) for the grants #2024/05158-6 and 2023/17090-4 and the financial support to the Food Research Center (FoRC) #2013/07914-8.

1. Culture preparation (Figure 2)
<ol>
	<li>Grow in a test tube a 5 mL culture of C. violaceum ATCC 12472 in Luria Bertani broth (LB: tryptone 10 g/L, yeast extract 5 g/L, 5g/L NaCl) at 30 &#176;C in a shaking incubator (220 rpm) for 24 h.</li>
	<li>Adjust the culture to an OD595 of 0.1 in LB broth, corresponding to approximately 1 x 108 CFU/mL.</li>
</ol>
2. Compound preparation
<ol>
	<li>In a flat-bottom 96-well plate, add LB broth and the bioactive compound according to a serial dilution calculation, following the description below. Depending on their solubility, bioactive compounds are typically diluted in pure DMSO or a DMSO: water (1:1) solution. 
	NOTE: In methodologies that assess QSI, it is crucial to ensure that the concentrations used are below the minimum inhibitory concentration (sub-MIC), meaning that they do not affect bacterial growth. Therefore, it is recommended to perform growth curve assays at different concentrations to confirm that the compound specifically inhibits cell-to-cell communication rather than microbial viability. Please see Supplementary Figure 1 for growth curve assays of the tested bioactive compounds used in this protocol.</li>
	<li>To calculate the bioactive compound concentration in the well, considering a stock solution of farnesol and linalool at 20 mg/mL and a target concentration of 200 &#181;g/mL in the well, as well as resveratrol at 2.5 mg/mL in the stock solution and 25 &#181;g/mL in the well, perform the calculation as follows: 
	C1V1 = C2V2 
	20,000&#160;&#181;g/mL x V1 = 200 &#181;g/mL x 200 &#181;L 
	V1 = 40,000/20,000 &#181;L 
	V1 = 2 &#181;L 
	where C1 = Stock solution concentration (20 mg/mL, which is equivalent to 20,000 &#181;g/mL); V1 = volume of stock solution to be added; C2 = desired final concentration (200 &#181;g/mL); V2 = final volume in the well (200 &#181;L).</li>
</ol>
3. Serial dilution20 and plate preparation (Figure 3)
NOTE: Serial dilution is used for quickly evaluating a range of concentrations of a tested compound. In this process, the compound is successively diluted at a constant ratio, in this case 1:2. Each dilution reduces the concentration of the compound by half compared to the previous concentration, resulting in a range of decreasing concentrations20.
<ol>
	<li>In the first column of the serial dilution, add 196 &#181;L of LB broth (in our example, columns 3, 5, and 7). On the following wells of the serial dilution, add 100 &#181;L of LB broth (in our case, columns 4, 6, and 8).</li>
	<li>Then, add 2x the amount of bioactive compound calculated in the previous step (2.2) to perform serial dilution. The calculation resulted in 2 &#181;L of each tested compound to achieve the final concentration in the first well, so add 4 &#181;L of the compound (columns 3, 5, and 7).</li>
	<li>Transfer half of the well&#39;s total volume (100 &#181;L) to the next column (in our case, columns 4, 6, and 8), and so on until the last desired concentration, if doing a serial dilution. Remember to thoroughly mix the contents before the transfer. 
	NOTE: In this experimental setup, we have only used two concentrations for simplicity and all compounds fit into just one plate.</li>
	<li>Perform the experiment in at least triplicate per concentration of each compound. Three biological replicates are also recommended. Change the tip and repeat the procedure until the last desired concentration. In the last column (in our case, 4, 6, and 8), take 100 &#181;L from it and discard.</li>
	<li>Add 80 &#181;L of LB broth to all the wells, totaling 180 &#181;L (rows B to D). Then, add 20 &#181;L of the C. violaceum culture standardized in step 1.2, ensuring a final volume of 200 &#181;L per well (rows B to D). Make sure to change pipette tips every time when changing wells to avoid carryover of higher concentrations between the wells or contamination.</li>
	<li>If the compound is dissolved in any kind of solvent, make sure to prepare the control with that solvent. We used DMSO. In this case, prepare a solvent control, which will be the untreated control - UC (without bioactive compound; column 9). Add 2 &#181;L of DMSO per control well. This gives a maximum concentration of 1% DMSO in the test. 
	NOTE: DMSO at 1% does not affect bacterial growth21,22.</li>
	<li>Prepare the blank, or color control if using pigmented compounds, without bacteria. After adding 80 &#181;L of LB broth to all the wells, totaling 180 &#181;L (rows B to D, as previously described), add 20 &#181;L of LB broth to the corresponding blank wells, totaling 200 &#181;L (rows E to G). This is done because there is some background staining from crystal violet that needs to be subtracted from the OD readings, as will be described in step 5 of this protocol.</li>
	<li>If any column remains empty, fill it with 200 &#181;L of LB broth or sterile distilled water (columns 1, 2, 10, 11, and 12). This step is important to prevent excessive drying of the medium in the incubator.</li>
</ol>
4. Incubation and extraction of violacein
<ol>
	<li>Cover the plate with a lid or a sealing film (as specified in the Table of Materials) to prevent evaporation, depending on the spectrophotometer used.</li>
	<li>Incubate the plate at 30 &#176;C with agitation at 130 rpm for 24 h.Then, remove the plate from the incubator, place it in a drying incubator at 60 &#176;C, remove the lid, and allow the plate to dry completely (approximately 8 h).</li>
	<li>After the plate has dried, remove it from the incubator and add 200 &#181;L of pure DMSO to each well, then cover it. Place the plate in an incubator at 25&#176;C while shaking at 130 rpm for 30 min to dissolve the violacein pigment.</li>
	<li>After this time, remove the plate from the incubator and transfer 100 &#181;L to another microplate, avoiding contact between the tip and the walls of the well.</li>
</ol>
5. Quantification of violacein production
<ol>
	<li>Quantitate the amount of violacein production by measuring the absorbance at 595 nm using an appropriate plate spectrophotometer (Table of Materials). The inhibition of violacein pigment is proportional to lower absorbance readings.</li>
	<li>Quantify violacein production by using a negative control (untreated control) as the reference for 100% violacein production (Table 1). First, subtract the absorbance value of the blank from each treatment replicate to obtain the absorbance corresponding solely to the violacein pigment (excluding interference from the broth or compound color). Then, calculate the percentage of violacein production for each treatment, using the following equation. 
	%violacein production = (OD of the bioactive compound / OD of untreated control) x 100</li>
	<li>Calculate the statistical analyses in three replicates. Results can be submitted to ANOVA, followed by Tukey&#39;s test using the GraphPad Prism 8.0 software. The value of p &#60;0.05 was considered statistically significant.</li>
</ol>

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<li>Mattio, L. M., Catinella, G., Pinto, A., Dallavalle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+and+nature-inspired+stilbenoids+as+antiviral+agents%5BTitle%5D)+AND+%22Eur+J+Med+Chem%22%5BJournal%5D)">Natural and nature-inspired stilbenoids as antiviral agents.</a> Eur J Med Chem. 202 (112541), 112541(2020).</li>
<li>Duarte, A., Alves, A. C., Ferreira, S., Silva, F., Domingues, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resveratrol+inclusion+complexes%3A+Antibacterial+and+anti-biofilm+activity+against+Campylobacter+spp.+and+Arcobacter+butzleri%5BTitle%5D)+AND+%22Food+Res+Int%22%5BJournal%5D)">Resveratrol inclusion complexes: Antibacterial and anti-biofilm activity against Campylobacter spp. and Arcobacter butzleri.</a> Food Res Int. 77, 244-250 (2015).</li>
<li>Martinelli, D., Grossmann, G., Séquin, U., Brandl, H., Bachofen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+natural+and+chemically+synthesized+furanones+on+quorum+sensing+in+Chromobacterium+violaceum%5BTitle%5D)+AND+%22BMC+Microbiol%22%5BJournal%5D)">Effects of natural and chemically synthesized furanones on quorum sensing in Chromobacterium violaceum.</a> BMC Microbiol. 4 (1), (2004).</li>
<li>Tan, L. Y., Yin, W. F., Piper Chan, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((nigrum%2C+Piper+betle+and+Gnetum+gnemon-natural+food+sources+with+anti-quorum+sensing+properties%5BTitle%5D)+AND+%22Sensors%22%5BJournal%5D)">nigrum, Piper betle and Gnetum gnemon-natural food sources with anti-quorum sensing properties.</a> Sensors. 13 (3), 3975-3985 (2013).</li>
<li>Tan, L. Y., Yin, W. F., Chan, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silencing+quorum+sensing+through+extracts+of+Melicope+lunu-ankenda%5BTitle%5D)+AND+%22Sensors%22%5BJournal%5D)">Silencing quorum sensing through extracts of Melicope lunu-ankenda.</a> Sensors. 12 (4), 4339-4351 (2012).</li>
<li>Oliveira, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antioxidant%2C+antimicrobial+and+anti-quorum+sensing+activities+of+Rubus+rosaefolius+phenolic+extract%5BTitle%5D)+AND+%22Ind+Crops+Prod%22%5BJournal%5D)">Antioxidant, antimicrobial and anti-quorum sensing activities of Rubus rosaefolius phenolic extract.</a> Ind Crops Prod. 84, 59-66 (2016).</li>
</ol>

The authors declare that they have no competing financial interests.

The first method for extracting and characterizing violacein from C. violaceum emerged in the 1990s, focusing on identifying suitable carbon sources for pigment synthesis. In this process, violacein -- along with the cells and culture medium -- was dried, resuspended in ethanol, and then separated by supernatant extraction. The violacein, now dissolved in ethanol, was subsequently dried again, yielding a pure, dry form of the pigment27. In 1998, the protocol was modified...

Bacteria can communicate through a system known as QS, which enables coordinated modulation of their behavior in response to changes in cell density. This process involves production, secretion, and detection of small extracellular signaling molecules, also called autoinducers, which allow communication both between different species and within the same species1,2.
This communication is capable of regulating genes and functions such as biofilm formation and dispersion, motility and adhesion, cell proliferation, synthesis of exoenzymes and siderophores, sporulation, acid resistance, horizontal gene transfer, antibiotic resistance, bioluminescence, production of exopolysaccharides, among others3,4,5. Given the relevance of QS-regulated phenotypes, there is great interest in developing systems capable of interfering with this cellular communication. The inhibition of QS signaling is also termed quorum quenching (QQ)6. QQ can occur in several ways, including inhibitors of the synthesis of autoinducers, enzymatic inactivation of these signals, and molecules with an antagonistic effect on these molecules4,7.
The growing threat of antibiotic resistance has driven research into alternative therapies, including quorum sensing inhibitors (QSIs), which disrupt bacterial communication without directly killing pathogens. These compounds may show synergistic effects when combined with antibiotics, enhancing bacterial susceptibility and allowing reduced antibiotic doses, which is a key strategy to slow resistance development. For example, resveratrol and kanamycin together delay Aeromonas hydrophila growth8, while N-acetylcysteine synergizes with tobramycin to disrupt Pseudomonas aeruginosa biofilms5.
With antibiotic overuse and stagnant drug development, antivirulence approaches like QS inhibition offer a paradigm shift9. Instead of targeting viability, they target virulence factors, rendering bacteria less harmful and more vulnerable to host immunity or antibiotics5. This strategy imposes weaker selective pressure compared to traditional antibiotics, minimizing resistance and preserving beneficial microbiota10,11. Despite promising lab results, real-world validation through clinical trials remains underexplored12. QS inhibition represents a promising complement to antibiotics, but its translation to practice requires further evidence of efficacy and safety in complex biological environments.
C. violaceum is a beta proteobacterium that forms violet colonies with a smooth and shiny surface. They are facultative anaerobic bacilli and do not form spores13. The mechanism of QS in C. violaceum involves a positive feedback loop based on the gene system analogous to the lux model of Aliivibrio fisheri, which contains proteins&#160;CviI (homologous to LuxI) and CviR (homologous to LuxR; Figure 1). Violacein exhibits biocidal activity against various organisms, including fungi, bacteria, nematodes, and viruses, particularly during the microbial stationary growth phase, when population density is extremely high, and nutrients are nearly depleted. Consequently, violacein production can be regarded as a competitive strategy to prolong the survival of the microbial colony14.
Plants produce compounds originating from secondary metabolism, which play a crucial role in increasing their ability to adapt to unfavorable environments. These metabolites, such as alkaloids, phenols, flavonoids, quinones, tannins, terpenes, and lectins, are widely recognized as defense mechanisms against herbivores and microorganisms15. The potential of plant-derived compounds as QSI has attracted special attention due to the dynamic interactions between plants and microorganisms. Studies on plant-pathogen interactions in the rhizosphere have demonstrated that plants can respond to bacterial autoinducers by secreting bioactive compounds capable of acting as inhibitors against pathogens16.
In this study, we describe a practical method aimed at quantifying violacein production by C. violaceum, focusing on its inhibition by bioactive compounds. This method serves as an initial screening tool for selecting compounds with potential QSI activities. It provides a rapid and practical approach for future therapeutic development against Gram-negative pathogenic bacteria. This standardized protocol will be useful because it provides a detailed description that has not been done in previous studies and will facilitate comparison among different studies in the future17,18,19.

<table><tbody><tr><td>DMSO&amp;nbsp;</td><td>Synth</td><td></td><td></td></tr><tr><td>Farnesol</td><td>Sigma-Aldrich</td><td>F203</td><td>95%</td></tr><tr><td>Linalool</td><td>Sigma-Aldrich</td><td>L2602</td><td>97%</td></tr><tr><td>Luria-Bertani Medium</td><td>Difco</td><td>244620</td><td></td></tr><tr><td>Optical Adhesive Film</td><td>Thermo Fisher</td><td>4311971</td><td>MicroAmp</td></tr><tr><td>Resveratrol</td><td>Sigma-Aldrich</td><td>R5010</td><td>&amp;ge;99% (HPLC)</td></tr><tr><td>Synergy HTX multi-mode reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Tissue Culture Plates 96 well</td><td>Kasvi</td><td>K12-096</td><td></td></tr></tbody></table>

quantification of violacein in chromobacterium violaceum and its inhibition by bioactive compounds

Quantification of Violacein in Chromobacterium violaceum and Its Inhibition by Bioactive Compounds

Bacteria communicate through a system known as quorum sensing (QS), which allows them to coordinate their behavior in response to changes in cell density. This process involves the production, secretion, and detection of small extracellular signaling molecules, usually called autoinducers (AI). QS regulates a wide range of genes and functions, including biofilm formation and dispersal, swarming motility, production of virulence factors, antibiotic resistance, and bioluminescence, among others. QS inhibition has become a promising antivirulence strategy against antibiotic-resistant bacteria as it exerts a lower selective pressure compared to traditional antibiotics. In the bacterium Chromobacterium violaceum ATCC 12472, the QS proteins CviI/CviR, which are homologous to the LuxI/LuxR-type QS proteins of Aliivibrio fisheri, control many genes, including those responsible for producing the purple pigment violacein. C. violaceum has been widely used as a biosensor strain in QS inhibition studies, particularly the inhibition of violacein production. Many compounds have the potential to inhibit QS by binding to QS proteins, either LuxI or LuxR homologues, disrupting QS circuits. This protocol describes the method for quantifying violacein produced by C. violaceum ATCC 12472 and its inhibition by bioactive compounds at concentrations that do not affect bacterial growth. The assay described here demonstrates a significant reduction in violacein production by the tested compounds. This study highlights the potential for using this protocol to screen promising candidates in QS inhibition research.

For the results shown here, three bioactive compounds previously studied by our research group8, resveratrol, farnesol, and linalool, were used. Each compound was tested for its ability to inhibit violacein production in C. violaceum ATCC 12472, a phenotype regulated by the QS system over the vioABCDE operon23.
The results validate the efficacy of our protocol in quantifying violacein production and its inhibition by bioactive c...

The objective of this protocol is to quantify violacein production by Chromobacterium violaceum ATCC 12472 as a proxy for assessing quorum sensing inhibition by bioactive compounds.

Bacteria communicate through a system known as quorum sensing (QS), which allows them to coordinate their behavior in response to changes in cell density. ...

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Source: Tay, S. B., et al. Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases. J. Vis. Exp. (2016)The video demonstrates a method to quantify biofilm biomass in a pathogenic bacterium, comparing untreated controls with samples treated with a quorum-quenching enzyme. The enzyme disrupts bacterial signaling during early incubation, reducing cell adhesion. After washing and further incubation, control wells form dense biofilms, while treated wells show...

Quantifying Enzyme-Induced Biofilm Suppression in Pathogenic Bacteria

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 a pellicle formation assay using Bacillus subtilis to evaluate small-molecule inhibitors that disrupt matrix synthesis and biofilm development, providing a model to screen compounds that selectively inhibit surface-associated growth without affecting free-floating cells.

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Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

Antibiotic resistance amongst adherent-invasive Escherichia coli strains poses challenges for assessing bacterial invasion using the traditional gentamicin protection assay. An improved method to evaluate bacterial invasion quantitatively by enumerating colony-forming units and qualitatively through visualization of infected cells by immunofluorescence confocal microscopy is described.

Amikacin Protection Assay for Quantification and Visualization of Escherichia coli Cell Invasion

Lens development involves interactions with other tissues.  Several zebrafish eye mutants are characterized by an abnormally small lens size.  Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Learn to quantify violacein production in Chromobacterium violaceum ATCC 12472 using spectrophotometric microplate assays. This method screens bioactive compounds for quorum sensing inhibition without affecting bacterial growth.

Quantification of Violacein in Chromobacterium violaceum and Its Inhibition by Bioactive Compounds

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