The overall goal of this procedure is to grow bacterial biofilms in the wells of a chamber Slide. Each well is first inoculated with bacteria in growth medium, then cultured at 37 degrees Celsius. Once the biofilms have formed, a vital dye is used to stain the cells.
The biofilms are then washed and fixed, and the wells are sealed using a cover slip. Finally, the cells can be observed by confocal microscopy and the morphology of different biofilms characterized. One advantage of this technique over existing methods such as flow cells, is that the volumes are much smaller and therefore reduce the amount of any experimental compound that will be used.
Also, more variables can be compared at one time in an eight well chamber slide compared to three chamber flow cell. This method could help answer several key questions in the field of microbiology, such as kinetics of biofilm formation. This method can also provide insight into multiple diseases that include a biofilm component such as chronic otitis media, cystic fibrosis, and chronic bladder infections to name a few.
Generally individuals new new to this method will struggle because there are numerous steps where the existing biofilm can be artificially disrupted. So therefore, visual demonstration of this method is critical as a removal and addition of fluid steps are difficult to learn, and the chance of disrupting the biofilm due to sheer forces is greatest at these points in the protocol demonstrating the procedure today will be Liz, a grad student from the laboratory, Inoculate bacteria in liquid culture medium, and incubate the culture until it reaches mid log phase. Dilute the mid log phase suspension at one to 2, 500 in prewarm medium for nontypeable hemophilus influenza, which is being used in this example.
This will provide for 40, 000 colony forming units per milliliter as a starting concentration. Aseptically transfer 200 microliters of diluted culture into each well of a sterile eight well chamber slide. Incubate the slide at 37 degrees Celsius in a 5%carbon dioxide incubator to allow the bacteria to begin forming biofilms 16 to 18 hours later.
Remove the slide from the incubator and aspirate the medium from the corner of each chamber. Then replace it with 200 microliters of fresh prewarm medium. Be sure to dispense the new medium along the wall of the chambers in order to avoid disrupting the biofilms.
Return the slide to the incubator and allow the cultures to grow to the desired density. Replacing the medium every eight to 16 hours to maintain bacterial viability. To visualize the biofilms, first, remove the culture supernatants as described earlier and gently wash the biofilms twice using 200 microliters of sterile saline.
Once the biofilms have been washed, add 200 microliters of backlight live dead stain to each well for one eight well chamber slide. Make up a total of two milliliters of the stain solution. Incubate the slide in the dark at room temperature for 15 minutes.
From this step forward, be sure to protect the slide from light. Incubate the slide in the dark at room temperature for 15 minutes. Once the cells have been stained, remove the live dead stain and wash each well twice with saline.
As demonstrated earlier, add 200 microliters of neutral buffered formin to each well and incubate the slide at room temperature for at least 30 minutes to fix the specimen. Following this incubation, remove and discard the fixative and wash the biofilms twice Using saline, remove the plastic wells from the slide and add approximately 150 microliters of saline to each. Well cover the wells by placing a cover slip on the gasket, making sure no bubbles are present and seal the edges of the cover slip with mounting medium.
Alternatively, nail polish can be used to seal the cover slip. In this example, we are using nail polish For better visualization of the procedure, allow the sealant to dry at room temperature for at least one hour before examining the biofilms under a microscope. This image is a three dimensional reconstruction of Zack images of a live dead stained H influenza biofilm collected using a confocal microscope.
The biofilm shown here is an example of a biofilm formed by NTHI after 24 hours of growth in the chamber slide. Using this technique, the bacteria were viable at the time of fixation as indicated by the green fluorescence and the absence of red fluorescence. The architecture of the biofilm can be seen quite clearly with numerous towers within the biofilm structure and many water channels present.
The side view in panel B demonstrates the relative height of the biofilm formed at this time point. While attempting this procedure, it's important to remember to add and remove the fluids from the well slowly to minimize the generation of sheer forces that may disrupt the biofilm present in the well. By following this procedure, other methods such as immuno labeling for specific proteins of interest or lectin labeling can be performed in order to answer additional questions regarding biochemical components present within the matrix formed by the bacteria in the biofilm.
After watching this video, you should have a good understanding of how to use an eight well chamber slide to study the formation of bacterial biofilms.
