- Inoculate the washed cells (25μl) into 10 ml BHI in a sterile
50 ml Falcon tube. - Grow cells at 37C, 200 rpm, and measure optical densities
(OD 600 ) at regular intervals to monitor growth. At selected
time points, remove 1 ml samples and immediately filter
through a 0.22μm filter. - Strains used asagrgroup controls are grown until stationary
phase (6 h in the growth conditions described). Supernatants
should be collected and filtered as described above. - Use supernatants directly (Subheading3.2) or store at 20 C
until required.
3.2 Semiquantitative
Reporter Assay
for AIPs in Culture
Supernatants
- Streak the specificagrreporter strain required onto a BHI agar
plate supplemented with 10μg/ml chloramphenicol, and incu-
bate the plate overnight at 37C. If theagrgroup of the strain
is unknown all fouragrreporters may be used in parallel to
identify the correctagrgroup (seeNote 3). - Inoculate a single colony of the reporter strain in 5 ml BHI
supplemented with 10μg/ml chloramphenicol and incubate
overnight at 37C with shaking, 200 rpm. - Dilute the overnight culture 200-fold in 10 ml BHI supple-
mented with 10μg/ml chloramphenicol and incubate at 37C
with shaking for 1 h. - Thaw frozen test supernatants.
- Prepare a series of AIP-I stock solutions over a range of con-
centrations in BHI. Add 10μl of each concentration in tripli-
cate to a sterile 96-well microtiter plate. Include a negative
control, e.g., 10μl of BHI (seeNotes 4– 6 ). If the appropriate
synthetic AIP is unavailable, supernatant from a strain with a
knownagrgroup can be used as a positive control. - Add 10μl of each supernatant collected from Subheading3.1
in triplicate, into the empty wells of a sterile, black, 96-well
microtiter plate. - After 1 h of growth, dilute the reporter strain tenfold in fresh,
pre-warmed BHI to a final volume of at least 25 ml (see
Note 7). - Use a multichannel pipette to add 190μl of diluted reporter
strain culture to all 96 wells of the microtiter plate (seeNote 6). - Use a suitable multi-mode plate reader to measure the OD 600
and RLU every 15 min for a period of up to 10 h at 37C(see
Note 8). - Use a suitable statistical software package (seeNote 9) to plot
RLU/OD 600 against time so that peak values can be obtained,
Fig.3a.
Detection of AIPs Produced byS. aureus 93