Microtiter Plate Test for Biofilm Assay

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Biofilm formation in microtiter plates is certainly the most commonly used method. In the classical procedure, bacterial cells were grown in wells of polystyrene microtiter plates. At various time points, wells are emptied and washed to remove plankton and then stained for biomass attached to the well surface. In the microtiter plate assay, biofilm biomass is assessed by measuring all attached biomass. Chemical methods use dyes or fluorescent dyes that can bind or adsorb to biofilm components. They are indirect methods that can be used to measure specific biofilm components, such as EPS components. These assays stain both live and dead cells, as well as some components present in the biofilm matrix, and are therefore well suited for quantifying total biofilm biomass.

General Principle

The test uses microplates with flat-bottom or U-shaped wells in which all stages of biofilm formation occur. To detect biofilm production, wells of the samples were stained with specific dyes to quantify the main structure of the biofilm. Colorimetric assays have also been used to assess cellular physiology in biofilms. The basic principle is to convert the cellular metabolic activity of a specific substrate into a colored product that can be measured with a spectrophotometer.

Microtiter Plate Test at Creative Biolabs

  • Crystal Violet

Crystal violet (CV) staining of biofilms formed within the wells of microtiter plates or on other abiotic surfaces is the most commonly used quantitative method to analyze biofilm formation, inhibition, and eradication. It is the most used colorimetric test in the microplate test, making it possible to quantify the entire biomass and matrix of biofilm. This quantification is very useful for the cellular estimation of biofilm growth.

  • Safranin

Safranin is a non-toxic dye, and its use is similar to that of crystal violet. However, it is less used for biomass quantification and results in lower optical densities than crystal violet staining.

  • XTT

The tetrazolic salt XTT is used to evaluate the number of viable cells and the metabolic activity of biofilms. When microorganisms in the biofilm produce metabolites, XTT changes its chemical conformation and converts to formazan, a color change from neutral to orange can be seen in the microtiter plate. This compound is most commonly used to detect and evaluate the metabolic activity of biofilms produced by fungi.

96-well flat-bottom microtiter plate-crystal violet adsorbed biofilm.Fig.1 96-well flat-bottom microtiter plate-crystal violet adsorbed biofilm. (Vijayababu, 2018)


  • High-Throughput.
  • Inexpensive.
  • No need for advanced equipment apart from a plate reader.
  • Versatility (Applicable to a broad range of different bacterial species, as well as eukaryotic cells such as yeasts or fungi.)
  • Dedicated microscopic-grade microplates allow noninvasive imaging.
  • Microorganisms do not need to be separated from those required for the support plate count, avoiding biased estimates of the number of cells in the biofilm due to VBNC status.

Creative Biolabs enables scientific breakthroughs for academic, pharmaceutical, and biotech clients. We are committed to providing researchers with the most comprehensive and cutting-edge high-quality solutions in the probiotic field. If you are interested in our microtiter plate test service, we hope to have the opportunity to work with you, please do not hesitate to contact us, our experts will be very friendly to communicate with you for detailed requirements.


  1. Vijayababu, P.; et al. Patulin interference with ATP binding cassette transferring autoinducer - 2 in Salmonella typhi and biofilm inhibition via quorum sensing. Informatics in Medicine Unlocked. 2018, 11: 9-14.

For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.

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For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.

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