Transcript 05_Piso - Rutgers University

Identification of Activity Characteristics of Bacteria Isolated
from the Continental Shelf in the Middle Atlantic Bight
Katherine Piso, Gary Taghon
Rutgers University, New Brunswick, NJ
INTRODUCTION
METHODS
DISCUSSION
•Each water sample was streaked onto nutrient rich-agar (1.5% agar and 5g peptone/L in filtered
seawater) in petri dishes using the quadrant method of streaking
There were notable differences in growth rates of the strains.
The growth curve of the white strain, White Ln(A(660nm)),
has readings much higher than compared to the other strains.
It is believed that the White strain had already grown up and
the measurements that were taken were late in its growth
cycle when compared to the other strains. The Peach,
Yellow, and Rough strains all showed relatively similar
growth early in their cycles. The Yellow and Rough strains
were notably similar when comparing the growth curves.
The Red strain’s growth was the slowest of all and could not
be accurately measured. Thus, it was taken out of
consideration for comparison.
The microbial life that makes up the ecosystem
on the continental shelf in the Middle Atlantic
Bight is poorly known. As a result, many
microbial ecologists have rarely been able to
formally draw the link between ecological
processes and the bacteria. The lack of a
connection may be in part because many
methods focus on bacteria as a uniform group,
rather than a community consisting of many
different, individual organisms1.
oA duplicate was streaked for each sample in order to obtain the most abundant bacteria present per
sample
•Bacterial strains were isolated based on color and morphology: White, Peach, Yellow, Red, and Rough
•Each isolated strain was then transferred to liquid media (1% peptone in filtered seawater)
•Each strain was again streaked onto agar in petri dishes using the quadrant method in order to ensure
pure cultures
•Each strain was verified to be a pure culture and was then placed into liquid media as a stock of each
strain
•A timed series of each strain was measured for growth using a Spectrophotometer at A(660nm) in order
to obtain an approximate growth curve for each sample
oSacrificial sampling was used in order to avoid contamination of the pure cultures
•Each strain was then freshly inoculated and the growth was followed
OBJECTIVES
oMeasurements and samples were taken for A(660nm), Protein Concentration, and Bacterial Activity
oBCS Protein Assay developed by Pierce Chemical Company, Technical Note 23225 performed
•Isolate strains of bacteria present at the LEO-15 site on the
continental shelf in the Middle Atlantic Bight
oExtracellular Enzyme (Aminopeptidase) Activity was measured using the method of Mayer2
•Establish pure strains of bacteria
•Obtain a growth curve for each isolated strain of bacteria
Isolation of colonies.
•Measure extracellular enzyme activity rates for each strain
Inoculation of
the T-series.
RESULTS
White Ln(A(660nm))
White [Protein]
18
-1.66
0.12
0.1
0.08
0.06
0.04
0.02
0
14
-1.68
15
20
30
40
50
-1
-2
-3
0.04
0.02
10
20
80
-2
-3
-4
0.02
0
25
-4
20
-4
-5
0.4
0.2
20
5
10
15
20
40
Time (Hours)
60
80
3
2.5
2
1.5
1
0.5
0
25
0
5
10
15
20
25
Time (Hours)
When taking into account all of the variations seen, it can be
concluded that the bacterial strains that I was able isolate do
vary in their response to the fluorochrome, L-leucine 7amido-4-methylcoumarin, used to measure their activity,
depending on their stage of growth. The White strain
showed increasing bacterial activity per protein
concentration, even late in its cell growth. This was
different from the Yellow strain, which showed a general
decrease in bacterial activity per protein concentration early
in its growth. Both the Peach and Rough strains had a
general trend of increasing, decreasing, and then again
increasing bacterial activity per protein concentration along
their growth cycles. The goal from this point is to determine
the conditions that are optimal for each strain which can be
considered representative of conditions seen at LEO-15.
All Strains Bacterial Activity
0.12
0.1
0.08
0.06
0.04
0.02
0
20
40
Rough Bacterial Activity
0
0
Time (Hours)
0.6
0
Bacterial Activity
nmoles/mL/min
-3
[Protein] mg/mL
Ln (A(660nm))
The Remote Access
Sampler resting on the
sediment bed.
-2
0.8
All Strains [Protein]
80
50
Time (Hours)
0.02
All Strains Ln (A(660nm))
60
40
1
Time (Hours)
0
30
1.2
80
0.04
Time (Hours)
40
60
0.06
0
20
40
0.08
-5
0
20
0
Bacterial Activity
nmoles/mL/min
20
[Protein] mg/mL
Ln (A(660nm))
15
-3
-1
10
Yellow Bacterial Activity
Rough [Protein]
-2
The Remote Access
Sampler aboard ship.
0
Time (Hours)
0
The location of
LEO-15.
0
Time (Hours)
0.04
Rough Ln (A(660nm))
10
0.5
50
0.06
Time (Hours)
5
40
0.08
0
0
30
0.1
-5
-1
1
Bacterial Activity
nmoles/mL/min
60
[Protein] mg/mL
40
18
CONCLUSION
Yellow [Protein]
0
17
1.5
Time (Hours)
Yellow Ln (A(660nm))
16
2
0
Time (Hours)
20
15
Peach Bacterial Activity
0.06
0
0
14
Time (Hours)
0.08
-4
-1
18
Bacterial Activity
nmoles/mL/min
0
10
17
Peach [Protein]
[Protein] mg/mL
Peach (A(660nm))
0
16
1.72
1.71
1.7
1.69
1.68
1.67
1.66
1.65
1.64
Time (Hours)
Time (Hours)
The Bacterial Activity of all four strains was very variable.
The White strain, which was most likely late in its growth
cycle, showed high activity initially with little variance.
Both the Peach and Yellow demonstrated a steady increase
in activity but the Yellow decreased slightly for the last time
point, whereas the Peach increased greatly. The Rough
strain was observed to increase in its activity significantly
throughout all of the time points.
White Bacterial Activity
Bacterial Activity
nmoles/mL/min
17
[Protein] mg/mL
16
-1.64
Ln (A(660nm))
Water samples were collected off the New Jersey coast at
LEO-15, approximately 1.2 km southeast of Node B, in
the Middle Atlantic Bight. Samples were captured using a
boundary layer sampler, the Remote Access Sampler
(RAS), on June 13th, 2005. The intake was located 5cm
above the sediment bed and was programmed to collect
the samples at various times over an approximately 24hour time period. Water sample collection times were as
follows beginning June13th and ending June 14th:
4:00pm, 5:30pm, 7:00pm, 10:00pm, 1:00am, 4:00am,
7:00am, 8:30am, and 10:00am.
15
-1.62
Ln (A(660nm))
SAMPLING
Ln (A(660nm))
-1.58
-1.6 14
One of many Protein
Assays performed.
The White strain again was different when compared to the
rest of the strains in protein concentration. The White strain
showed a high protein concentration whereas both the Peach
and Rough strains had low concentrations that increased and
then decreased. The Peach continued to again increase after
the decrease in protein concentration but contrastingly, the
Yellow strain began low with a slow but steady increase in
protein concentration.
60
80
ACKNOWLEDGEMENTS
3
2.5
2
1.5
1
0.5
0
0
20
40
60
80
I would like to thank Charlotte Fuller for all the help in my
times of need, and David Gruber for the assistance in the lab
and guidance with my data.
Time (Hours)
Graphical representation of measurements of (left to right): Ln (A(660nm)), Protein Concentration, and Bacterial
Activity for (top to bottom) White, Peach, Yellow, Rough, and all the strains, versus time.
REFERENCES
1.
Azam, F. and Worden, A. Z. (2004). Microbes, Molecules, and Marine
Ecosystems. Science. 303: 1622-1624
2.
Mayer, L.M. (1989). Extracellular proteolytic enzyme activity in sediments of an
intertidal mudflat. Limnology and Oceanography 34:973-981