Biology Reference
In-Depth Information
150
1
125
100
0.1
75
50
0.01
0
5
10
15
20
25
30
Time (Hours)
Fig. 3. Production of autoinducer 2 (AI-2) signal by
Vibrio vulnificus
during different
phases of growth. Supernatant samples from
V. vulnificus
were collected at different
time points, and the percent AI-2 activity compared with that of positive control was
determined (left axis). The growth of the culture, as determined by optical density at
610 nm (OD
610
), is plotted on the right axis and is used to show that AI-2 production
peaks during early stationary phase and decreases after 24 h.
Subheading 3.1.2
to avoid effects on growth. Representative data are shown
in
Fig. 3
, which show the timing of AI-2 production during the growth of
V.
vulnificus
.
3.4. Inhibition of Hemolysin Assay with Signal Antagonist
To test for inhibition of hemolysin activity by signal antagonists, routinely
grow the test cultures overnight. The next morning, 10 mL of fresh medium
is inoculated with a 1:100 dilution of the overnight culture in a sidearm flask
and incubated at 37°C with shaking (200 rpm) and growth measured at OD
610
.
Antagonists are added to the cultures during log phase (OD
610
is approximately
0.3). DMSO is added to control samples to account for the solvent volume
added, and growth should be followed spectrophotometrically to ensure the
concentrations of antagonist compounds used are not growth inhibitory. Cell-
free supernatants should be collected across the growth curve.
1. Wash 4 mL of sheep blood three times with five volumes (20 mL) of PBS (pH
7.2). Centrifuge at 1140 ×
g
for 5 min and resuspend in five volumes (20 mL) of
PBS.
2. Incubate aliquots of cell-free supernatants (200 μL), 600 μL of hemolysin buffer,
and 200 μL washed red blood cells at 37°C for 30 min.
3. Remove intact red blood cells by centrifugation and determine the OD
450
of the
supernatant. Results for the effects of an AI-2 inhibitor are shown in
Fig. 4
.
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