Microtitre plate assay

Removal of established biofilm

After incubation, the planktonic-phase cells were gently removed by washing the wells with phosphate-buffered saline 5 times for 30 seconds each time. 200 microlitre of extract was then added to the wells and left to incubate for another 24 hours at 30 degrees Celsius in an incubator without shaking. As the negative control, 50 percent ethanol and saline were used instead.

The next day, the wells were washed thoroughly with phosphate-buffered saline.

100 microlitre of crystal violet solution was added to each well and incubated for 15 minutes at room temperature. The wells are then washed 3 times with phosphate-buffered saline. After that, the microtitre plate was dried overnight.

The following day, 100 microlitre of 95 percent ethanol was added to each well to solubilise the crystal violet retained by the biofilms and incubated at room temperature for 15 minutes. 100 microlitre of solubilised crystal violet was then transferred to a new microtitre plate. The absorbance was measured at 550 nanometre with ethanol as the blank using a microtitre plate reader.

The percentage inhibition of biofilm formation was calculated as follows:

OD of control – OD of treatment x 100%

OD of control

A sample of a microtitre plate stained with crystal violet can be seen in Figure 5.

Figure 5: A microtitre plate being stained with crystal violet

Inhibition of formation of biofilm

The same procedure was used as in the removal of formed biofilms. However, extracts were added directly to the bacteria in the wells at the start of the experiment and incubated for 5 days under the same conditions as mentioned. After 6 days, the wells were rinsed with phosphate-buffered saline to remove the extracts and bacteria in the planktonic form. The remaining cells (biofilm) were stained with crystal violet solution. The absorbance of the wells were read at 550nanometre the next day. Figure 6 illustrates a flowchart which summarises the two different microtitre plate assay tests.

Figure 6: Flowchart which summarises the two different microtitre plate assay tests