Microbiology and Immunology, Dartmouth Medical School
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O'Toole, G. A. Microtiter Dish Biofilm Formation Assay. J. Vis. Exp. (47), e2437, doi:10.3791/2437 (2011).
Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.
1. Growing a Biofilm
2. Staining the Biofilm
3. Quantifying the Biofilm
4. Representative Results:
Figure 1. shows a representative result for biofilm formation assays performed for Pseudomonas aeruginosa, Pseudomonas fluorescens and Staphylococcus aureus. (A) A side view of the well with a biofilm of P. aeruginosa (8 hrs, 37°C). (B) A side view of the well with a biofilm of P. fluorescens (6 hrs, 30°C). (C) A top-down view of the biofilm formed by S. aureus in a flat-bottom microtiter plate (two wells, 24 hrs, 37°C). P. aeruginosa and P. fluorescens are both motile organisms and form a biofilm at the air-liquid interface. S. aureus is non-motile and forms a biofilm on the bottom of the well.
This method can be modified for use with a wide variety of microbial species. Motile microbes typically adhere to the walls and/or bottoms of the wells, while non-motile microbes typically adhere to the bottom of the wells. The optimal conditions for biofilm formation (i.e., growth medium, temperature, time of incubation) must be determined empirically for each microbe. I recommend performing multiple replicates for each strain or condition (4-8), and including a positive control, and if possible, a negative control on each plate.
No conflicts of interest declared.
My thanks to Sherry Kuchma, Pete Newell and Robert Shanks for providing the images in Figure 1. This work was supported by NIH grant R01AI083256 to G.A.O.
|1 X M63||Prepare as a 5X M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature. Dilute 5X stock 1:5, autoclave, cool, then add the desired components.|
|Magnesium sulfate||Fisher Scientific||M63-500||Add to 1 mM final concentration. Prepare as a 1 M stock in water and autoclave.|
|Glucose||Fisher Scientific||D16-3||Add to 0.2% final concentration. Prepare as a 20% stock in water and autoclave.|
|Casamino acids||BD Biosciences||223050||Add to 0.5% final concentration. Prepare as a 20% stock in water and autoclave.|
|Arginine||Sigma-Aldrich||A5131||Add to 0.4% final concentration. Prepare as a 20% stock in water and filter sterilize. This alternative carbon/energy source can replace glucose and casamino acids|
|Microtiter plates||BD Biosciences||353911||Falcon 3911, Microtest III, Flexible assay plates, 96 well, U-bottom, non-sterile, non-tissue-culture treated.|
|Microtiter plate lids||BD Biosciences||353913||The lids can be reused by cleaning with 95% ethanol in water.|
|Crystal violet||Sigma-Aldrich||229641000||Prepare as a 0.1% solution in water.|