IMAGING BIOFILMS
	Department of Biology	Technology Facility
I & C Technical Guides

Many devices have been developed to grow biofilms in the lab for different purposes. For our purpose, it was essential to use material that permits fluorescent light penetration and does not have autofluorescence, i.e.  glass with high optical properties. The next point to consider is whether you require a static biofilm or a biofilm grown in a flow cell. For mutagenesis screens for example the static biofilm will be more suitable. In contrast, to analyze biofilm formation the flow cell may provide valuable additional information. For our long term goals, we wanted to be able to analyze biofilms in a flow cell in real time.

Factors governing flow cell choice

• Bacterial attachment surface
  • Glass enables imaging of the biofilms using fluorescent microscopy. We chose a flow cell that uses coverslips, and found we could use the thinner, higher optical quality #1.5 coverslips (0.17-0.19mm thick). These proved strong enough to use in the flow cell yet were of high optical quality and allowed direct imaging.
• Cost
  • The flow cell had to be reusable and also had to be autoclavable. In addition to providing significant advantages for the experimental set up, a flow cell with dual chambers is also more cost effective.
• Design
  • A flow cell with sufficient depth (z-axis) to allow significant biofilm growth was desired.  In addition, for our goals, we needed to be able to add dyes directly to the biofilm with minimal disruption so a flow cell with injection ports was most appropriate.

Based on these requirements, we chose to use the FC-281 flow cell from Biosurface Technologies shown below.

Modifications to the flow cell
The microscope stage-mounting tab was removed and modified with a widened viewing window (see figure below) to facilitate access of the flatter, wider Plan-Apochromat 63x /1.4NA oil DIC objective and other high NA lenses and retain space for movement in the X,Y plane. Biosurface Technologies carried out this modification so any similar adaptation of the product is accessible to all research groups. 

Original window	Modified window

Set up
Our set up involves placing the flow cell in an incubator, either on the microscope stage or as shown here in the lab during biofilm establishment (left hand image). The media was supplied at a controlled temperature using a water bath. A bubble trap (centre image) was used to limit air bubbles entering the flow cell. A highly accurate low speed Watson-Marlow 250U pump was used with 3.2mm diameter tubing (1.6mm bore, 1.6mm wall) for the whole system.

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