A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

The overall goal of this procedure is to identify and measure changes in biofilm formation by assessing the development of wrinkled colony formation over time. This is accomplished by first growing bacterial strains of interest in liquid culture. The second step is to spot the cultures onto nutrient agar.

Next, the spots are monitored over time to identify the time at which they begin to develop three dimensional structures indicative of the initiation of wrinkled colony formation. Development of colony morphology is monitored until no further changes are visualized. Ultimately, microscopy of the spots is used to show differences in biofilm development over time by specific strains or under specific conditions in a manner that is semi-quantitative.

The main advantage of this technique over existing methods is that it permits a semi quantitative analysis of bacterial biofilm formation using the development of wrinkled colony morphology. Though this method can provide insight into biofilm formation in Vireo fishery, it can also be applied to the characterization of biofilm phenotypes in other bacterial systems such as basc, slus, pseudomonas osa, and vial cholera. We first had the idea for this method when we identified bacterial mutants that exhibited a delay of the development of wrinkled colony formation.

Biofilm formation is generally influenced by cell density and growth rate. Therefore, prior to assessing wrinkled colony morphology, the growth rate and yield of the strains of interest should be determined to determine growth rate. Measure the increase in optical density or OD of each strain over time and plot a growth curve to determine yield or final cell number.

A cell plating assay can be used. The bacterial strains of interest are grown in liquid culture, and then dilutions of the cultures are plated onto agar plates. After incubation, the number of viable cells are calculated and represented graphically as shown here.

It is also important to identify the best conditions for spotting conditions that reveal the most distinct differences between the control and mutant of interest. To accomplish this prior to spotting growth strains in liquid culture under various conditions such as different media or temperatures, and to different stages of growth, such as exponential or stationary phase spot 10 microliters of culture of various cell densities onto the appropriate media and incubated the desired temperature until colony morphologies become apparent. In this way, an optimal starting OD for a given set of strains or conditions can be determined to begin this procedure.

Inoculate v Fisher eye cells from agar plates into five milliliters of LB salt or LBS medium containing any necessary antibiotics incubate with shaking at 28 degrees Celsius overnight on the following morning subculture to the cells with a one to 100 dilution into five milliliters of fresh LBS medium incubate under the same conditions until the cells have reached the desired OD at 600 nanometers. For Vibrio fii, the best results are obtained when spots are generated from cultures with an OD of approximately 0.2. Next pipette, one milliliter of each culture into a micro fuge tube and centrifuge at maximum speed.

For one minute, remove the supinate by aspiration and resuspend the pellet in one milliliter of sterile, 70%artificial seawater or a SW centrifuge. The cells, again, this wash step will remove residual media and extracellular components. After centrifugation, remove the supinate and resuspend the washed pellet in one milliliter of 70%A SW to ensure that each sample contains the number of cells as estimated by the OD.At 600 nanometers.

Make any necessary adjustments by diluting more concentrated samples with additional 70%A SW spot, 10 microliters of each culture onto LBS plates containing any necessary antibiotics. Make sure to study the pipette with your finger just above the agar surface and spot vertically not at an angle. Eject the liquid slowly for uniform distribution of the spot, include the appropriate positive and negative controls on the same plate.

When assessing wrinkled colony formation as minor plates, AFL variation may impact biofilm development. The most difficult aspect of this procedure is identifying the start of wrinkled colony formation. Sometimes it is only apparent after development has ensued.

To catch the start of wrinkle colony development, we take hourly time points within a specific timeframe. This also permits us to go back and make comparisons After development has begun. To ensure that the spotted culture remains evenly distributed, allow the spot to dry before moving the plate to the incubator, invert the plates and incubate them at 28 degrees Celsius.

One way to assess the morphology of the spotted cultures is by an endpoint assay, which involves assessment of wrinkled colony formation and a predetermined time point after spotting. This is a representative result from an endpoint assay in which wrinkled colony formation was assessed. 40 hours post spotting, a non biofilm forming strain of Vibrio FII is shown on the left and a biofilm forming strain is shown on the right.

The endpoint assay is useful for strains that exhibit or a proposed to exhibit severe defects in biofilm formation. Another assay for assessing colony morphology is a time course assay in which wrinkled colony formation is assessed over a period of time post spotting to easily keep track of the time post spotting. Set a timer to count up.

In this demonstration, the morphology of the growing spot will be monitored hourly, beginning at 12 to 15 hours following, spotting a dissecting scope with a camera attachment and image acquisition and image analysis. Software programs are used to observe and document colony morphology and to assess the start and progression of wrinkled colony formation. Before imaging spotted cultures, the lid of the Petri plate is typically removed to provide the clearest image.

Here, the spots are illuminated from underneath through a transparent glass stage with a cold light source while the images are captured from above. This setup is optimal for imaging wrinkled colony development because Vibrio fisher eye colonies are translucent. To best visualize wrinkled colony development, adjust both the lighting intensity and angle of reflection underneath the bacterial colonies such that the three dimensional morphology of the developing biofilms can be discerned.

Determine the optimal lighting conditions that provide the strongest contrast between the spotted colony and the surrounding agar background. It is important to use the same magnification when collecting images throughout the experiment using the lever located on the back of the camera, switch the view from the eyepiece of the microscope to the computer screen, adjust the view, focus accordingly, and then capture the image, capture appropriate images to document the start and development of wrinkled colony formation. The end of the experiment occurs when there is no further development of the biofilm.

At the end of the experiments acquired, images are consolidated into figures to visualize the pattern development over time for each strain or condition. Note how much time elapses from the time of inoculation to the time at which wrinkled colony formation initiates the start of wrinkled colony formation is defined at the time point at which the formation of patterning and 3D structures is first apparent. In this example, initiation of biofilm formation is evident at 12 hours for a biofilm competent control strain of Vibrio FII depicted in the upper panel.

In the lower panel, a representative images of a mutant strain of Vibrio FII that exhibits a four hour delay in the start of wrinkled colony formation over time with biofilm formation initiating its 16 hours post inoculation. Note that while subtle differences between these strains are observed at the earlier time points at 40 hours, the strains look similar in the intensity and patterning of biofilm formation at each time point following the start of wrinkled colony formation. The pattern of wrinkled colony development should be noted.

The architecture may develop from the outside in as illustrated in panel A, or it may develop from the inside out as illustrated in Panel B.This assessment provides a mechanism to distinguish the biofilms formed by different strains or under different conditions. A second semi-quantitative measure of biofilm formation makes use of the changing colony diameter of the developing colony over time. As shown in the upper panel, a representative biofilm competence strain forms, colonies that increase in complexity and diameter.

In contrast, another strain shown in the lower panel does not form biofilms. Finally, this figure shows a graphical representation of the increase in colony diameter over time. By the same two strains, the biofilm competence strain is represented by white circles.

While the strain that does not form biofilms is represented by black squares. This analysis revealed that the size of the biofilm competent colonies increase at a greater rate than the biofilm negative colonies, and at the end time point, the two differed by almost twofold. While performing this procedure, it's important to remember to carefully spot each strain and remember to document the starter wrinkled colony formation for each strain.

Following this protocol, other methods such as a iCal assay or crystal wide staining can be performed in order to answer additional questions about the processes and involved and biofilm formation.