Team:British Columbia/Notebook Biofilm

Biofilm Quantification Protocol

Day 1

1. PIck an isolated colony from a Trypticase Soy Agar (TSA) plate and incubate in 5 mL of Trypticase Soy Broth (TSB) without shaking overnight

Day 2

1. Vortex the overnight culture and dilute 30 uL of culture by 1:100 in a 1% glucose solution diluted with TSB

2. Vortex to mix bacteria sample

3. Innoculate 200 uL of dilute sample into pre-determined wells of a microtiter plate

4. Use 1% glucose solution diluted with TSB, but without bacterial sample, as control and test samples in triplicate

5. Place lid onto microtiter plate and place in a non-shaking incubator for 24 hours at 37C

Day 3

1. Take microtiter plate out of the incubator and decant all liquid from the wells into a biohazard container

2. Wash each well with 3 x 300 uL of Phosphate Buffered Saline (PBS) at room temperature

3. Heat fix remaining bacteria through exposure for 1 hour to 60C air using a heat block

4. Leave plate inverted overnight at room temperature

Day 4

1. Add 150 uL of 0.1% crystal violet dye diluted with deionized water to each well

2. Allow staining to occur for 15 minutes

3. Decant crystal violet dye from each well into a biohazard container

4. Wash wells with deionized water until crystal violet is no longer present in the water

5. Air dry plate

6. Take OD readings at an absorbance of 550 nm on a plate reader

Optimization

Initial experiments were conducted to determine the conditions that were suitable for optimal biofilm growth. The original protocol called for incubation of bacteria in media with a glucose concentration ranging from 0.25%-2%. Through our experiments which tested the OD readings of various glucose concentrations, we concluded that 1% glucose media provided the best environment for biofilm growth of S. aureus strains RN4220 and 8325-4. As well, we determined using identical samples of bacteria and media solution that the outer edges of the Greiner 96-well flat bottom microtiter plate showed abnormal growth possibly due to increased exposure to oxygen when compared to the center wells. Therefore, we decided to use only the center wells from C3 to E10 for our experiments as to avoid extraneous variables. Day 3 of the original protocol was altered as it called for the aspiration of liquid from the wells with a micropipette, whereas we simply inverted the plate into a biohazard container as it was decided that the original method often resulted in biofilm disturbance.

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