Transcript Slide 1

Rafael Hernández1,2, Yasiel Hernández1,2, Nasly Jimenez2, Helena Solo-Gabriele1,2
1Department
of Biology, University of Miami, Coral Gables, Florida 33124
2Department of Oceans and Human Health, RSMAS, University of Miami, Miami, Florida 33149
Abstract
Methods
Hypothesis
H1: The concentrations of enterococci will increase
with depth.
H2: High enterococci (CFU) concentrations in the
sand will correspond with high moisture content.
Figure: 1
Figure: 2
• Sand was collected 4.19 meters from the reference point in
the experimental transect, using a core 16cm in length (Fig. 2).
• Once homogenized, 10g of each sand layer (.5cm deep) were
placed in individual sterilized bottles with 100mL of PBS and
shaken vigorously for 2 minutes, followed by ≥1 minute for
settling.
• Vacuum filtration, with 45µm membrane filters, was used to
filter 5mL and 50mL of the supernatant in each bottle (Fig. 3).
• Filters were allowed to cultivate for 24 hours on mEI plates at
41.5oC.
• Sand in each layer was measured for water content.
Figure: 3
Date of Sand Sampling
with Core
Core 1 (08-28-2009)
Core 2 (09-25-2009)
Core 3 (10-12-2009)
Core 4 (10-28-2009)
Core 5 (11-02-2009)
Core 6 (11-30-2009)
Core 7 (01-11-2010)
Core 8 (02-19-2010)
Core 9 (05-19-2010)
Core 10 (07-08-2010)
Conclusion and Future Directions
Compilation of Data Collected With 16cm Core
Concentration (CFU/g)
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
10.5
11
11.5
12
12.5
13
13.5
5
10
15
20
25
30
35
40
45
50
55
60
Correlation Between Water Content (%) vs.
Enterococci Concentration (CFU/g) of Sand
R2 Value
0.734
0.038
0.162
0.038
0.509
0.055
0.005
0.114
0.144
0.295
Table 1: Correlation between the concentrations of enterococci vs. the water
content recorded within the range of 0-13cm deep sand.
Results
Depth (cm)
Sand at Hobie Cat Beach (Fig. 1) has been observed to
have high levels of enterococci, the bacteria used by
regulatory agencies to issue beach advisories, despite
the absence of any obvious sewage contamination.
The sand at this particular beach site has been recently
renourished providing the opportunity to evaluate the
rate at which enterococci is re-established in the sand.
In order to understand the growth of enterococci, a
small square in the sand about 25 cm by 25 cm will be
enclosed; sand will be collected by inserting a sterile
16cm core into the sand. The core will be collected in
the upper supra-tidal zone. Only the top 13cm of the
sand will be collected by layers every .5cm. Later, the
enterococci will be extracted through vigorous shaking
for 2 minutes with PBS as the diluents and a membrane
filtration procedure will be used to analyze the number
of bacterium in the sand sediments. Data collected
before the beach renourishment indicates higher levels
of enterococci in deeper layers of sand. Once the new
sand is added the same procedure will be used to study
the re-establishment of enterococci, with the prerenourishment data as a reference to the bacteria’s
previous levels and distribution.
Results
65
70
75
80
85
90
95
• With an increase in depth, the concentrations of
enterococci seem to fluctuate from low to high
numbers, with lower numbers in the 1cm layer (fig.
4).
Core 1 (08-28-09)
Core 2 (09-25-09)
Core 3 (10-12-09)
Core 4 (10-28-09)
Core 5 (11-02-09)
Core 6 (11-30-09)
Core 7 (01-11-10)
Core 8 (02-19-10)
Core 9 (05-19-10)
Core 10 (07-08-10)
Figure 4: Compilation of 10 data sets collected, using 16cm long core, before the renourishment of the
study-transect at Hobie beach. The horizontal axis represents the concentration of enterococci per gram
of dry sand for every .5cm-deep layer extracted from the core.
Figure 5: Colonies of
enterococci on mEI
plates after 24 hours of
incubating at 41.5
degrees Celsius
• The concentrations at each layer varies significantly
between core samples collected on different days.
Nevertheless, each core seems to demonstrate
higher concentrations between .5 and 4.5cm of
depth. There seems to be a general decrease in
concentration between 5-6cm of depth, which is
then followed by peaks from individual cores.
• These fluctuations may be due to the lack of
different nutrients (necessary for the growth of
enterococci) at different depths; each impacting the
growth of the bacteria in a different manner.
• There is no correlation between water content and
CFU concentrations of the sand in terms of depth
(Table 1).
• Continuation of sampling after the renourishment
will allow the understanding of enterococci regrowth
in new sand.
Acknowledgments
Helena Solo-Gabriele
Lora E. Fleming
Athula H. Wikramanayake
Michael S. Gaines
This work was funded by the NSF and NIEHS Research Experience for
Undergraduates Supplement and the NSF and NIEHS OHH Center grants
(NIEHS # 1 P50 ES12736 and NSF #OCE0432368/0911373)