Preparation

In this step, we prepared the necessary materials needed to conduct our experiment. These included the bacteria culture for the well diffusion test and for the establishment of biofilm in the microtitre plate assay. We also prepared the extracts to see if they could inhibit microbial biofilms

Preparing the bacteria culture

E. coli, S. epidermidis and P. fluorescens were inoculated into 20 millilitres of LB broth and were grown at 30 degrees Celsius overnight in a shaking incubator as seen in Figure 2.

Figure 2: The bacteria culture being incubated in a shaking incubator

Preparation of extracts

The samples were firstly rinsed with saline. Then, 4 grams of each sample was added to 20 millilitres of 50 percent ethanol or saline and ground with a mortar and pestle. The extracts were then centrifuged at 7000 rpm for 10 minutes and the supernatants were collected. Lastly, the extracts were filter-sterilised through a 0.45 millimitre microfilter.

Establishment of biofilm

The method used was according to O’Toole (2011), with modifications. 0.1 millilitre of the E. coli, S. epidermidis and P. fluorescens cultures were placed into the wells of a microtitre plate. Then, the plate was incubated for 5 days at 30 degrees Celsius in an incubator without shaking. Five replicates for each bacterium were prepared. A sample of a plate prepared can be seen in Figure 3.

Figure 3: Bacteria culture being added to the wells of the microtitre plate before being incubated so that they can from biofilm