Biology Reference
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5. Dilute the overnight culture 1:100 into fresh medium and aliquot 100 μL into the
wells of the 96-well microtiter plate containing the extracts for testing.
6. Incubate at 37°C and take regular readings for up to 24 h to determine growth
yield and to calculate the MIC values.
3.2.2. The Induction or Inhibition of Elastase Activity in
P. aeruginosa
Performed as Described in ref. (22) with Some Modifications
1. Grow the
P. aeruginosa
in ABt at 37°C with shaking (200 rpm).
2. The following morning, dilute the culture 1:100 into fresh medium, either add
extract at half the MIC dose, as determined above or 200 μg/mL (
see
Note 8
), or
add an equal volume of solvent to the control culture and incubate at 37°C with
shaking (200 rpm).
3. At hourly intervals, collect 1.5 mL of supernatant and also determine the OD
600
.
Filter out the cells from the 1.5-mL aliquot using a 0.2-μm filter (Pall Acrodisc,
0.2-μm Supor Membrane, Pall Life Sciences, East Hills, New York, USA) and
store at -20°C (
see
Note 9
).
4. Once samples have been collected, thaw on ice. Whilst thawing, prepare tubes
with 1 mL of Elastin-Congo red buffer and 20 mg of Elastin-Congo red.
5. Add 100 μL of sterile supernatant to the Elastin-Congo red tubes and incubate at
37°C for 18 h with agitation.
6. Stop the reaction by the addition of EDTA to a final concentration of 12 m
M
.
Centrifuge at 16,000 ×
g
to remove insoluble Elastin-Congo red.
7. Collect the supernatant and measure the OD
495
. A blank, with Elastin-Congo red
and buffer, but without sample, should be used to subtract out the background.
Results should be normalized (
see
Note 10
) to correct for differences in cell
density.
Fig. 2
presents representative data on elastase repression for a QSI at
two concentrations.
3.3. Detection of AI-2 Activity or Inhibition
AI-2 activity is commonly detected by the use of AI-2 bioassay developed over
a decade ago.
Vibrio harveyi
BB170
(23)
(sensor 1
−
, sensor 2
+
) has a nullmutation
in thegene for sensor 1and thus is abioluminescent reporter strain forAI-2activity.
V. harveyi
BB152
(23)
(autoinducer 1
−
, autoinducer 2
+
) has a null mutation in the
gene for autoinducer 1 synthase (AI-1) and produces only AI-2, and thus serves
as a positive control for assays of AI-2 production. For the generation of cell-
free supernatants,
V. harveyi
strains were grown in AB medium
(16)
overnight
at 30°C with shaking at 200 rpm and cell-free supernatants prepared as below.
3.3.1. Collection of AI-2 Signal
1. For generation of cell-free supernatants, test cultures are inoculated at a 1:100
dilution and the cultures grown to late stationary phase (
see
Notes 11
and
12
).
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