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Figure 8.4
Comparison of salicylate-
induced
P. fluorescens
5RL cells with
and without latex encapsulation. Bio-
luminescence from encapsulated cells
appears somewhat attenuated.
fluorescens
5RL, a naphthalene-
lux
bioreporter, bioluminescence production
was compared between immobilized and free cells. Rovace SF-091 (Rohm and
Haas, Philadelphia, PA) was used to determine toxicity effects on
Pseudomonas
fluorescens
5RL. Uninduced, log-phase cells (OD
546
=
0
.
35) were washed with
mineral salts buffer (MSM) and resuspended in MSM
15% glycerol. Cells
were spotted onto duplicate cellulose membranes and excess moisture was
allowed to dry. The cell spots were coated with the latex mixture at full strength
and dried overnight at 5°C and at 40% humidity.
We determined viability of cells by laying the membranes on LB broth sup-
plemented with sodium salicylate (100 ppm) and qualitatively monitoring light
production. Cells coated with Rovace SF-091 had diminished bioluminescence
compared to the control membrane (Figure 8.4).
A growing-cell assay experiment was performed in a microtiter plate using
P. fluorescens
5RL in the presence and absence of Rovace SF-091 to further
quantify the reduction in bioluminescence. The purpose of the experiment was
to demonstrate the inhibitory effect of the latex on the bioluminescent response
of the cells. The cells were immobilized on a nylon membrane (BIOTRANS
Nylon Membranes, ICN Biomedical, Aurora, OH) with a pore size of 0.45
+
m
using a dot-blot filter apparatus (Bio-Rad, Hercules, CA). Cells were placed
in individual wells in the dot-blot apparatus and transferred to the membrane
by applying a vacuum. The number of cells per patch was calculated to be
1
µ
10
7
CFU. The vacuum was maintained until the cell patches appeared dry.
One-half of the membrane was placed on a slanted surface, and the latex was
poured over the membrane until it was covered. Excess latex driped off the
membrane leaving a thin latex film over the membrane. The latex was dried at
5°C and at 40% humidity overnight. The other half of the membrane was stored
overnight at the same temperature and humidity. The next day, the individual
patches were cut and placed on top of a sterile sponge (3M ScotchBrite) slightly
larger than the patches. The sponge pieces and patches were placed in the wells
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