Molds and excessive humidity in the living environment
Final Biological Science Research Project BHS012-3 (1)
1. Biological Science Research Project: BHS012-3
Benjamin Cordner
The Laboratory Report
Investigating the Effect of Temperature on the Assembly of Microbial
Communities.
Abstract:
The purpose of this research was to see how temperature effects different organisms
at 5 degree Celsius (°C) intervals. It examines bacteria by microscopically viewing
and recording changes to population density in a microcosm that has been held for a
period of time at a constant temperature (15, 20, 25°C). There were two groups of
researchers looking at a variety of monocultures and bacterial communities.
Measurements were notated using their sizes and movements for identification. As the
eukaryotes differed in size, a variety of methods were employed to observe and record
results, serial dilution, spectrophotometer and PCR. The results were tabulated and
compared. The results from this study were considered in the context of the literature
available within this scientific field. This study asks the question: what is the effect of
temperature on organisms such as pseudomonas fluorescens? Is it possible to discover
their optimum temperature and to note how, in bacterial communities, they react to
one another? Pseudomonas fluorescens results were inconclusive due to being out
compete by other organisms in the community. Results still showed a major change in
population size due to temperature variation. In conclusion the change in
environmental temperature will have a impact on the ecosystem.
1
2. Introduction:
There have been progressive evidence suggesting the need to move towards further
research investigating the impact of environmental warming. Climate changes effects
ecosystems in many ways that are only beginning to be understood. The rise in
temperature is particularly worrying, not just because it forces species migration to
aid survival, but because of the impact it is having on every level of species, including
micro-organisms such as the commonly found Pseudomonas fluorescens. It is
important to understand how temperature changes might effect such organisms and
thus effect the humans. Studies such as those which will be considered in the
discussion to this research highlight how Pseudomonas fluorescens may be changing
in how it reacts to the human respiratory system. It will be noted how it is particularly
responsive to temperature changes and thus research is necessary to look, not only at
how it responses as a monoculture, but how it may react as part of a bacterial
community.
Climate changes and environmental warming impact the entire biological community
therefore it is important to consider changes with one part of the system in relation to
how this also impacts the whole system. Evidence of this is shown in a study by
Naidu et al (2015) which notes that in addition to the decrease in rainfall over the last
decade in India, there has also been an increase in temperature and wind, all of which
have worked together to dramatically decrease the moisture of the soil. This in turn
has an impact on the ecosystem which depends on the soil and thus all that survives in
the area will be effected. In a similar fashion studies such as those by Ferrarini et al
(2014) have noted that climate change may be causing more complex issues within
the plant kingdom than has previously been considered. Evidence such as that
provided from studies into climate changes and environmental warming would
suggest that research into the ways in which temperature increases impact organisms
is of vital importance to understanding what may be expected in the future.
This study examines a variety of organisms, but focuses mainly on Pseudomonas
fluorescens, which will be shown to be reactive to temperature changes (see results
and discussion). Pseudomonas fluorescens are rod-shaped bacteria commonly found
in soil and water, but also present in air, dust and vegetables. It is common to also find
it in food products such as meat, fish, eggs, and particularly milk products. Its
optimum temperature for growth is 25-30 degrees Celsius and it proves to have a low
generation time at low temperatures and spoils at 2-5 degrees. This is important for
considering how to treat and store water, vegetables and food products. For example,
although milk is sterilised at high temperatures which degenerate bacterial including
Pseudomonas fluorescens, they can be re-introduced after the processing and lead to
the spoiling of the milk or milk-based product particularly if it is not stored
continuously at a temperature just above freezing (Decoin, 2015).
Pseudomonas fluorescens is an obligate aerobe which can use oxygen rather than
nitrate during its cellular respiration. It is a nonsaccarolytic bacteria that can produce
heat stable lipase and protase, It can break down casein in milk and coagulate
proteins, which therefore make it useful in the production of yogurts. Pseudomonas
fluorescens is not usually found to cause infections in humans however, as
opportunistic pathogens, it can cause infections in individuals with a debilitated or
2
3. compromised immune system and recent reports have found Pseudomonas
fluorescens in pneumonia cases. It has also however been found to protect plant roots
against fungus and can be cultured to help with the treatment of skin and eye
disorders. Pseudomonas fluorescens is the target organism used in this study therefore
monocultures have been used throughout this experiment in temperatures varying 15-
25°C over the course of the research period.
Polymerase chain reaction (PCR) is a technique that is used to amplify trace amounts
of DNA located in or on almost any liquid or surface. As every human, animal, plant,
parasite, bacterium, or virus contains genetic material such as DNA sequences that are
unique to their species. PCR is a method used to amplify (make many more identical
copies) these unique sequences so they can then be used to determine with a very high
probability the identity of the source (a specific person, animal, or pathogenic
organism) of the trace DNA or RNA found in or on almost any sample of material.
(Bartlett et al).
Gel Electrophoresis is a method used to separate and analyse macromolecules (DNA,
RNA and proteins) and their fragments, due to their size and charge. This is because
DNA has a negative charge and the gel uses positive charge to attract the fragments.
Smaller fragments will move further as there is less friction from the gel and that they
have a lighter weight than larger fragments. It utilises a matrix of agarose or other
substances (Nicholas and Nelson, 2013).
The aim of this experiment is to consider the growth of bacterial communities and
how they compete and interact over a prolonged period. Results will be analysed
according to markers such as the size of the populations, and how they may respond
in a pre-predator relationship according to environmental influences such as
temperature and how it responds over time.
3
4. Method:
Six species of bacteria and five protists (3 ciliates and 2 flagellates) were cultured
from a variety of sources. Microcosms were created using 50mL centrifuge Falcon
tubes. Each tube was developed into a microcosm by adding 0.5g of freeze-dried alga
or Chlorella powder to 1 litre of mineral water, which was then sterilised by
autoclaving at 121 Celsius. Tubes were sequentially labelled for individual
identification with the follow information: temperature, name (or initials of
researcher), protist community, bacterial community (or bacteria monoculture) and
replicate number, e.g. 15-NW-PC-1.Bacterial cultures were added to create two
communities, A and B. Group A included Bacillus Subtilis, E-Coli and micrococcus
luteus. Group B had Pseudomonas fluorescens, Serratia marcescens and Bacillus
Cereus. Either A or B were added to 9 microcosms depending on which mixture the
target species was present in. One half of the group made 3 falcon tubes with a
monoculture of their organism. The other half of the group created a bacterial
community of their group in 3 falcon tubes.
This experiment created 9 microcosms by adding 20mL of Pseudomonas fluorescens
(a monoculture) from Group B using a serological pipette. The chosen protists were
then also added to microcosms using a serological pipette: 10ml of Chilomonas,
100ml of Colpidium and 200ml of P. caudatum. Different amounts were added due to
the difference in the population size; those with a smaller population required a larger
amount. (See Figure 1) These 3 were chosen because they compete with each other
and can be more easily measured due to their difference in size. Chilomonas is small
but with a very large population which is visible under the dissecting microscope.
Colpidium are also small but have a small population so more was added to give a
similar population as Chilomonas. P. caudatum is a larger organism but has a smaller
population than the other organisms per volume, therefore a larger volume was
needed to match the population levels of the other organisms.
Bacteria mixture Protists (ul)
Pseudomonas fluorescens 20,000
Serratia marcescens 20,000
Bacillus cereus 20,000
Chilomonas 10
Colpidium 100
P. caudatum 200
Figure 1: showing the population sizes
All 12 tubes were then placed into a polystyrene rack which was labelled with a
specific temperature and put for 3 days into an incubator that had been set at the
required temperature. In this case the Pseudomonas fluorescens was tested at 15, 20
and 25 degrees Celsius. The expectation was that Pseudomonas fluorescens would
develop different levels of growth at different temperatures depending on its optimum
temperature for growth.
Other members of the group who had bacterial communities looked at how the
communities of 3 bacteria (A or B) reacted at the 3 different temperatures. The results
were measured according to the competition of resources or even consumption of
4
5. other bacteria. Those at an optimum temperature could be seen to thrive and out-
compete the others at different temperatures. However the expectation was also that
they could all have an ideal temperature and co-exist together.
Populations of species were sampled over four different sessions using a variety of
techniques appropriate to the different sizes of the species, which reached different
population abundance in their microcosms. Depending on size the bacteria was
sampled using serial dilution, spectrophotometry and PCR. The eukaryotes (ciliates
and flagellates) were sampled using different powered microscopic lens. The four
parts to the sampling involved: counting the number of eukaryotes in a specific
volume of liquid; calculating the bacterial absorbency of light to estimate bacterial
abundance; preparing the sample for the following week and serial dilution of the
bacteria.
The volume of the sample was variable depending on the species as there had to be
between 20-100 individual eukaryotes to be able to make an accurate result. To avoid
effecting the temperature of the microcosms, sampling was done as quickly as
possible. This involved placing 1 ml sample in a curette, leaving for 15 minutes to
settle, then viewing under the dissecting microscope to estimate the number of the
species. Large numbers required taking a smaller sample to reach below 100 per
count. The media was then mixed to ensure a representative sample of perhaps 0.2 ml
of the original sample, which was mixed with 6-9 droplets of water (depending on
size) on a Petri dish.
Sample
Number
Temperature
(°C)
Replica Number
or culture
1 15 1
2 15 2
3 15 3
4 15 monoculture
5 20 1
6 20 2
7 20 3
8 20 monoculture
9 25 1
10 25 2
11 25 3
12 25 monoculture
Figure 2: Order of Samples. Showing how they were pipetted into the curette. A blank
of 1 ml water was also used.
There were 12 curettes and 12 measurements in total (see Figure 2 for labelling
system). Measurements were taken using a blank spectrophotometer (with the clear
side, arrow pointing left to right, used for correct absorbency levels) against the water
and measuring at 6000nm. After these results had been recorded 1ml of each sample
was pipetted into an eppendorf tube and labelled with initials and sample number.
Next the samples were put into a centrifugal tube and spun at 14,800rpm for 1 minute
to remove all the bacteria, and leaving a pellet in the bottom. The sample liquid was
then pipetted back into the curette leaving the pellet behind in the tube. This gave
5
6. optical density before and after. After the sample settled (15mins) absorbency levels
were measured as before. Measurements were calculated to 1 ml. A record was made
of the volume of the sample against the number of individuals in each species. For the
smaller organisms a haemocytometer was used for counting under a powerful
microscope. The haemocytometer grid was divided into counting squares and live
cells in one corner square (0.1x0.1x0.01cm) were counted and added to obtain a cell
count between 20-100.
Next a set of serial dilutions were created by adding 180ul of water into a 12x6
microtiterplate. The samples were pipetted onto agar plates beginning with number 1
through to 12 (see Figure 3). An X was marked on each plate on the same place or
grid. Each agar was labelled with name, date and plate number. Serial solutions make
it possible to see how many bacteria there are in a sample.
6
7. Results:
The results for this experiment are calculated by counting the number of eukaryotes of
each microcosm. After viewing under a dissecting microscope to find an appropriate
volume depending on population size. A haemocytometer was used for high
populations to calculate the amount that would be in 1 ml.
The results from the experiments are shown in the following tables. The tables were
created from results taken over the course of the experiment using sampling and PCR.
Note that day 1 was mainly setting up the sample and preparation.
When measuring the samples, notes were taken of changes to colour as this was an
indicator of the changes to the different cultures. The colours of the different cultures
were checked after mixing in the falcon tube and all were a cloudy green. The 3
monocultures however were clearer than the 9 other tubes. This suggested that there
had been less growth in the monocultures when compared to the 9 mixtures of
organisms.
Tables numbered 1 to 3 shows the absorbency levels of all 12 samples which was
obtained using a spectrophotometer. There are two sets of results for absorbency
levels; before centrifuging and after centrifuging. The results taken before
centrifuging showed the light absorbed by everything present in each sample. By
centrifuging some of each sample, the larger organisms and debris were removed.
The reason for taking two different sample absorbance levels is to give a clearer
accuracy in the end results by taking the after results away from the before.
Table 4 complies the results across the 3 days, (shown in Tables 1-3). It shows the
calculated absorbance levels, which were obtained by taking away the result after
centrifuging from the result before centrifuging. This allows for easier comparisons
between the 3 days of results and will give a calculated absorbency level that is more
precise.
The sample numbers shown throughout the experiment (e.g. in Table 1) refer to the
cultures that were created using the micro-organisms and bacteria shown in Figure 1.
The breakdown of this is shown in Figure 2.
Absorbance
levels
Sample
number
Before
centrifuge
After
centrifuge
1 0.168 0.042
2 0.168 0.023
3 0.153 0.023
4 0.369 0.04
5 0.229 0.04
6 0.268 0.034
7 0.233 0.024
8 0.215 0.029
9 0.186 0.03
10 0.304 0.022
11 0.261 0.027
12 0.154 0.02
8. Table 1: Absorbance levels from day 1 taken before and after centrifuging.
Absorbance
levels
Sample
number
Before
centrifuge
After
centrifuge
1 0.23 0.04
2 0.163 0.024
3 0.143 0.037
4 0.358 0.029
5 0.138 0.03
6 0.183 0.03
7 0.114 0.014
8 0.17 0.025
9 0.075 0.025
10 0.085 0.025
11 0.065 0.026
12 0.185 0.026
Table 2: Absorbance levels from day 2 taken before and after centrifuging.
Absorbance
levels
Sample
number
Before
centrifuge
After
centrifuge
1 0.24 0.071
2 0.164 0.076
3 0.152 0.07
4 0.428 0.099
5 0.124 0.057
6 0.213 0.05
7 0.136 0.068
8 0.226 0.015
9 0.129 0.01
10 0.14 0.066
11 0.127 0.03
12 0.333 0.046
Table 3: Absorbance levels from day 3 taken before and after centrifuging.
Table 4 below shows the calculations for all 3 of the different sets of above results.
Note that day 1 calculations are shown in the first column beside their sample number.
This demonstrates how the calculations were obtained. The end result is obtained by
minus-ing the results of the absorbency level after centrifuging from the absorbency
level before centrifuging. The results of this are shown in the remaining 3 columns.
8
9. By creating this table it is possible to have a more accessible comparison of the 3 sets
of results. This can be seen more clearly in the graph created from table 4 data (graph
1).
Calculated Absorbance
levels
Sample
number
Day 1
calculation
Day 1
results
Day 2
results
Day 3
results
1 0.168 - 0.042 = 0.126 0.19 0.169
2 0.168 - 0.023 = 0.145 0.139 0.093
3 0.053 - 0.023 = 0.145 0.106 0.082
4 0.369 - 0.04 = 0.329 0.329 0.329
5 0.229 - 0.04 = 0.189 0.108 0.067
6 0.268 - 0.034 = 0.234 0.153 0.163
7 0.233 - 0.024 = 0.209 0.1 0.068
8 0.215 - 0.029 = 0.186 0.145 0.211
9 0.186 - 0.013 = 0.156 0.05 0.119
10 0.304 - 0.022 = 0.282 0.06 0.134
11 0.261 - 0.027 = 0.234 0.039 0.097
12 0.154 -0.02 -= 0.134 0.059 0.287
Table 4: Calculated Absorbance levels from days 1-3 including calculations from day
1 absorbance level data using before centrifuge minus after centrifuge.
Graph showing calculated absorbance of all
data
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
1 2 3 4 5 6 7 8 9 10 11 12
Sample number
Calculatedabsorbancelevels
Day 1 results
Day 2 results
Day 3 results
Graph 1: Calculated Absorbance Levels of all 12 samples from days 1-3.
The data in graph 1 give a clear visual comparison of the difference in results over the
3 days of absorbance levels. The difference in the polycultures and the monocultures
can be noted when reviewing sample number 4, 8 and 12.
9
10. To enable a more reliable result, the average calculated absorbance levels of the 3
replica samples (not the monoculture) from each temperature on days 1-3, were used
(as seen below in tables 5).
Average calculated Absorbance levels
Temperature
(°C)
Day 1
results
Day 2
results
Day 3
results
15
0.13866666
7 0.145 0.114666667
20
0.21066666
7
0.12033333
3 0.099333333
25 0.224
0.04966666
7 0.116666667
Table 5: The average calculated Absorbance levels of each temperature on days 1-3.
The data below in table 6 shows the standard deviations of the average calculated
absorbance levels (shown in table 5).
Standard deviation of the average
calculated absorbance levels
Temperature
(°C)
Day 1
results
Day 2
results
Day 3
results
15
0.01096965
5
0.04232020
8 0.047374395
20
0.02254624
9
0.02857154
8 0.055139218
25
0.06359245
2
0.01050396
8 0.018610033
Table 6: The Standard deviation of each average calculated absorbance level.
Using both table 5 and 6 a graph can be made to give a more clear representation of
the results and demonstrate the standard deviation using error bars. This can be seen
in graph 2 which also can be useful when comparing the increase and decrease of
average absorbance over time.
10
11. Graph 2: Average calculated absorbance levels of each temperature on days 1-3
including standard deviation error bars.
Tables 7 through to 10 below are the results from all 4 days of sampling. They show
the effect of the different temperatures (15, 20, 25°C), on 3 different species (P.
caudatum, Colpidium, Chilomonas).
On Day 1 of the experiment samples containing each organism were viewed under a
microscope to allow for familiarity in shape, movement, size and colour. Notes were
taken so that descriptions were available of each eukaryote. This enabled the
recognition of the various organisms in subsequent microscopic analysis.
The next stage of the experiment involved counting the eukaryotes (P. caudatum,
Colpidium, Chilomonas) using a stage microscopes and a dissecting microscope. The
bacteria could not be counted this was as they were too small to be seen using these
microscopes. In order to do this a 1000 ul sample was taken from each falcon tube and
viewed microscopically and the number of each organism were counted. Some of the
1000 ul samples had too many to count so a smaller volume had to be taken until the
number was low enough to count. This number was then calculated to the estimated
number present in 1000uls as shown below in Tables 7-10.
Date:
12.01.15
Temperature
(°C)
Sample
No. Species
Volume
Sampled
(ul)
Number
Counted
Number
Calculated to
Vol. of 1000 ul
15 1 P. caudatum 1000 10 10
15 2 P. caudatum 1000 0 0
15 3 P. caudatum 1000 1 1
20 1 P. caudatum 1000 24 24
20 2 P. caudatum 1000 3 3
11
12. 20 3 P. caudatum 1000 8 8
25 1 P. caudatum 1000 50 50
25 2 P. caudatum 1000 3 3
25 3 P. caudatum 1000 4 4
15 1 Colpidium 10 26 2600
15 2 Colpidium 10 26 2600
15 3 Colpidium 10 22 2200
20 1 Colpidium 5 30 6000
20 2 Colpidium 5 32 6400
20 3 Colpidium 5 25 5000
25 1 Colpidium 5 20 4000
25 2 Colpidium 5 40 8000
25 3 Colpidium 5 32 6400
15 1 Chilomonas 20 20 1000
15 2 Chilomonas 20 25 1250
15 3 Chilomonas 20 22 1100
20 1 Chilomonas 10 21 2100
20 2 Chilomonas 10 23 2300
20 3 Chilomonas 10 26 2600
25 1 Chilomonas 5 31 6200
25 2 Chilomonas 5 36 7200
25 3 Chilomonas 5 42 8400
Table 7: Cell count results on day 1 calculated to 1000ul.
As you can see in the above table there weren’t as many P. caudatum in the first
sampling, at each of the different temperatures, but many more in the other replicates
at 1000ul.
All of the above microcosm populations are seen to increase at a higher temperature
suggesting that their ideal temperature is 25°C or higher.
Date:
14.01.15
Temperature
(°C)
Sample
No. Species
Volume
Sampled
(ul)
Number
Counted
Number
Calculated
to Vol. of 1000ul
15 1 P. caudatum 1000 12 12
15 1 Colpidium 2 35 17500
15 1 Chilomonas 50 21 420
15 2 P. caudatum 1000 8 8
15 2 Colpidium 2 16 8000
15 2 Chilomonas 50 26 520
15 3 P. caudatum 1000 6 6
15 3 Colpidium 2 18 9000
15 3 Chilomonas 20 21 1050
12
14. 25 1 Colpidium 10 35 3500
25 1 Chilomonas 2 24 12000
25 2 P. caudatum 1000 0 0
25 2 Colpidium 2 8 4000
25 2 Chilomonas 2 11 5500
25 3 P. caudatum 1000 0 0
25 3 Colpidium 10 28 2800
25 3 Chilomonas 1 15 15000
Table 9: Cell count results on day 3 calculated to 1000ul.
The above Tables 5-7 show a variety of results which would suggest there may be
some anomalies. The data shown below in Table 8 does not include many samples but
focuses instead on the specific samples to view their differentiation.
Date:
19.01.15
Temperature
(°C)
Sample
No. Species
Volume
sampled
(ul)
Number
Counted
Number
Calculated
to Vol. of 1000ul
15 1 P. caudatum 1000 16
15 1 Colpidium
15 1 Chilomonas
15 2 P. caudatum 1000 16
15 2 Colpidium
15 2 Chilomonas
15 3 P. caudatum 1000 8
15 3 Colpidium
15 3 Chilomonas
20 1 P. caudatum
20 1 Colpidium
20 1 Chilomonas
20 2 P. caudatum
20 2 Colpidium
20 2 Chilomonas 1000 0
20 3 P. caudatum 1000 9
20 3 Colpidium
20 3 Chilomonas 1000 0
25 1 P. caudatum
25 1 Colpidium
25 1 Chilomonas
25 2 P. caudatum 1000 0
14
15. 25 2 Colpidium
25 2 Chilomonas
25 3 P. caudatum 1000 0
25 3 Colpidium
25 3 Chilomonas
Table 10: Cell count results on day 4 calculated to 1000ul.
Below are the tables showing results taken from the Petri dishes all of which were
kept at 30°C for 16 hours (these can be seen in the appendix, image 1-3, with related
information in figure 4-5). The time between sampling and recording results explains
why they have different dates, one being the date they were sampled and the other the
date they were counted. Before the results were recorded the samples were examined
and growth could be seen by the red and white cultures, which showed a smaller
colony count for each dilution.
Tables 11 to 13, in a similar to fashion to the above tables, show the results taken over
3 days from samples kept at 3 different temperatures and how these effect the 3
different samples and the monoculture (sampling as from Figure 2). Each sample was
recorded from 6 different dilutions ranging from strong (1in 10) to weak (1 in 1,000
000).
Date:
12.01.15
Serial dilution
Temp.
(°C)
Sample
No.
Strong - 1
(1/10)
2
(1/100)
3
(1/1000)
4
(1/100000)
5
(1/100000)
Weak - 6
(1/1000000)
15 PC - 1 TMTC TMTC 11 4 4 3
15 PC - 2 TMTC TMTC 10 5 5 3
15 PC - 3 TMTC TMTC 5 3 7 3
15 MC - 1 TMTC TMTC 55 2 0 0
20 PC - 1 TMTC 6 0 2 2 0
20 PC - 2 TMTC TMTC 2 2 0 1
20 PC - 3 TMTC 14 2 2 1 0
20 MC - 1 TMTC 0 0 0 0 0
25 PC - 1 TMTC 12 0 0 1 0
25 PC - 2 TMTC 9 0 0 2 0
25 PC - 3 TMTC 12 2 0 0 2
25 MC - 1 TMTC 0 0 0 0 0
Table 11: Culture colony results from Petri dish counted on day 2(sampled on day 1).
15
16. Table 12: Culture colony results from Petri dish counted on day 3(sampled on day 2).
Date:
16.01.1
5
Serial dilution
Temp.
(°C)
Sample
No.
Strong - 1
(1/10)
2
(1/100)
3
(1/1000)
4
(1/100000)
5
(1/100000)
Weak - 6
(1/1000000)
15 PC - 1 TMTC TMTC 45 2 1 0
15 PC - 2 TMTC TMTC 18 2 1 0
15 PC - 3 TMTC TMTC 11 1 0 0
15 MC - 1 TMTC TMTC TMTC 21 2 0
20 PC - 1 TMTC 10 1 0 0 0
20 PC - 2 TMTC 19 2 0 0 0
20 PC - 3 TMTC 17 2 0 0 0
20 MC - 1 TMTC TMTC TMTC 8 1 0
25 PC - 1 TMTC 10 0 1 0 0
25 PC - 2 TMTC 9 4 0 0 0
25 PC - 3 TMTC 18 5 0 0 0
25 MC - 1 TMTC 29 3 0 0 0
Table 13: Culture colony results from Petri dish counted on day 4(sampled on day 3).
Polymerised Chain Reaction (PCR) was used during week 2 to determine the micro-
organisms present in each sample. This was done using primers that were designed at
a previous bioinformatics session. In order to collect data from PCR reactions using
the cultures from this experiment it was necessary to first test if the primers worked
with the target organism. This was done by testing the primers with a control sample
known to contain the target organism.
Date:
14.01.15
Serial dilution
Temp.
(°C )
Sample
No.
Strong - 1
(1/10)
2
(1/100)
3
(1/1000)
4
(1/100000)
5
(1/100000)
Weak - 6
(1/1000000)
15 PC - 1 TMTC 24 6 3 1 0
15 PC - 2 TMTC 6 2 1 0 0
15 PC - 3 TMTC 5 1 0 0 1
15 MC - 1 TMTC TMTC TMTC 28 14 9
20 PC - 1 TMTC 14 3 1 0 0
20 PC - 2 TMTC 6 4 0 0 0
20 PC - 3 TMTC 6 6 1 0 0
20 MC - 1 TMTC TMTC TMTC 34 12 8
25 PC - 1 TMTC 8 3 0 0 0
25 PC - 2 TMTC 6 3 1 0 0
25 PC - 3 TMTC 9 1 0 1 0
25 MC - 1 TMTC TMTC TMTC 22 6 4
16
17. This was done using gel electrophoresis after PCR and viewing the gel under UV light
to see the florescent markers as bands that had moved due to the charge,
This data can be seen in image 4 which showed bands for the second primer that was
developed during bioinformatics. the band was around 700kb which is correct as the
target size that was provided stated it to be 650kb.
The results also showed that there were no other bands for the other organisms in the
mix used (serratia or B.subtilus), thus no cross reactivity will occur for primer pair 2.
Primer pair 2 was then used in the PCR and electrophoresis with a control these
samples:
Sample Tempurature (°C) Weight of supernatent
(mg
1 15 41
2 15 27
3 15 38
1 20 10
2 20 55
3 20 56
Table 14: PCR samples used with the weight of the supernatant.
A gel electrophoresis was used and the results are shown on image 5 in the appendix.
The control had worked suggesting the sample and procedure had been performed
correctly however no P.flurecence was detected in any of the sample. This was tested
for a second time with the same outcome suggesting that most or all of the
P.flurecence has been eaten or out compete by the other organisms on day 3 at
temperatures 15 and 20 degrees Celsius.
17
18. Discussion:
The above results suggest that temperature variations had a marked effect on the
organisms viewed in this study over the duration of the study. This effect has been
particularly pronounced for Pseudomonas fluorescens, other researched from studies
shown below have considered how it might respond to temperature changes.
An early study by Gill (1975) considered how environmental factors such as chemical
changes and physical properties could effect the growth of Pseudomonas fluorescens.
Gill suggested that batch cultures were an important method for measuring variations
in the organism as they allowed for several parameters to be changed simultaneously.
When Pseudomonas fluorescens was subjected to various temperatures it was proven
that this had a marked effect on its growth. It was also noted that the degree of
saturation was not a continuous curve over the scale of the temperatures. At 10
degrees Celsius Pseudomonas fluorescens saturation variations decreased, which was
supposed to be due to a decrease in the ability to control fatty acids. Once the
temperature was lowered to below 10 degrees however there were few further
changes. Gill concluded that Pseudomonas fluorescens controlled its fatty acid
composition in the lower third of the temperature range (i.e. below 10°C) to allow for
a constant saturation. Gill also noted that other factors, such as changing the growth-
limiting substrate from carbon to nitrogen, had a marked enhancement to these
changes.
Fouchard et al (2013) study on Phospholipid fatty acids (PLFA) considers how
experiments which measure fatty acid changes are particularly useful ways of noting
bacterial growth. PLFA are found in cell membranes, which make direct contact with
the cells environment and therefore signal changes to variants such as pH and
temperature changes. Fouchard et al notes how Pseudomonas fluorescens is highly
versatile as it uses a variety of nutrients, therefore it can be used in a broad range of
growth conditions. In this experiment 2 different strains of Pseudomonas fluorescens
(SF1 and DSM 7155) were grown at 3 different temperatures (16, 26 and 36°C)
before optical density was measured (using similar techniques to this research) and
cell multiplication results were analysed. In the discussion of their results they note
how certain Pseudomonas fluorescens strains ( in this case DSM 7155) did not
produce UV visible spectra and therefore could not be measured for light absorbency.
This is an important point to consider when looking at some of the anomalies in the
above results which were mainly gained from a similar experimental use of counting
population density using a spectrophotometer.
Arana et al (2010) compare how environmental conditions (temperature variation and
nutritional deprivation) effect 2 different bacteria, Pseudomonas fluorescens and
Escherichia Coli. They note that when nutrients were removed the temperature
variations did not create a common pattern. When Pseudomonas fluorescens was
subjected to temperatures between 5-15°C it responded differently than it did at
higher temperatures irrelevant to the nutritional composition, which suggested that
temperature was an important factor for the organism. This makes it an ideal subject
for experiments which are measuring the effects of variations In temperature.
18
19. Recent studies on Pseudomonas fluorescens show it has become increasingly
problematic as a bacteria that is found infecting the human body. Donnarumma et al
(2010) study on Pseudomonas fluorescens suggest there is a need to research into the
ways in which this bacteria exists at different temperatures. They discovered that this
micro-organism could grow at 28 and 37 degrees Celsius. At the lower temperatures
Pseudomonas fluorescens induced proinflammatory cytokines and a bio-film was
formed at 37 degrees Celsius. This suggests it performs as an opportunistic pathogen
and further research needs to be done to discover how it responds to temperature
changes in different environments.
The data shown in table 7 - 10 show a result of 0 P.caudatum for sample 2 at 15°C on
day 1. This is because the falcon tube was not mixed before the 1000ul sample was
taken and view through a dissecting microscope. This will have effected all of the
results for 15°C on day 1 P.cardatum but the falcon tubes were mixed after this first
sample for all of the other results. As they were not mixed for the first results all of
the organisms will have been closer to the bottom of the tube this is why the result is
lower for the first 3 shown in the table and even 0 counted for sample 2. Evidence of
this can be seen on tables 8-10 that do have a population counted for all of the
P.cardatum at 15°C. In relation to this when looking at p.caudatum on these tables it
can be seen that the number is reduced each day at the higher temperatures but
increases at the lower temperature. It can be seen that P.cardatum is not present in any
samples at 25°C after day 2. It also shows that there is a higher population of
p.caudatum in the first replicate of each temperature and when looking at the agar
plates shown in image 1-3 you can see the large red colonies. The monocultures
however show a much larger growth with each dilution having more colonies.
Another eukaryote that had a number count of 0 at 1000ul was Chilomonas at 20°C
on day 2 sample 2. On day 3 and 4 there was also a count of 0 for sample 2 and 3.
This would suggest that another organisms ideal temperature was around 20°C and
out compete Chilomonas. The date shown in table 11 provides evidence that
Chilomonas is eating the bacteria at the higher temperature, which is why there is a
larger count under a microscope of the microcosm. This is also why there is a higher
count at lower temperature on the petri dish. Chilomonas is eating the bacteria
allowing for more growth of the red Serratia at 15°C as less has been eaten. The Petri
dishes on image 1-3 do show some growth at the more diluted samples. This is most
likely down to bad pipetting but there is less of this on the later days.
The absorbance levels for day 1 (table 1) support the results seen on the agar plate
count (image 1) with more growth of the monoculture at 15°C but less at 20°C and
25°C. As there was some contamination with the first 3 samples on the plate it has
given a larger growth for these first 3 but this is not consistent with the data shown
from day 1 absorbance levels. Day 2 absorbance levels also support day 2 agar plate
count. There was more growth at the lower temperatures and less at the higher. This
includes the monoculture although these seem to be a small absorbance at 25°C for
the monoculture but still a larger amount of growth on the agar plate compared to the
communities. Day 3 has similar data to day 2 in that there is more growth at the lower
temperature and less growth at the higher. Although the 20°C monoculture has less
absorbance than the 25°C monoculture but no by a large amount. This does not
support previous days or the data from the agar plate which follows the lower
temperature having more and lower having less.
19
20. As previously mentioned one of the reasons for these changes in population is that the
added organisms that were chosen out compete at the higher temperature. This meant
there was less bacteria which can be seen when looking at the agar plate (image 1-3)
and absorbance levels (table 1-6, graph 1-2) which had removed he larger eukaryotes
by centrifuge. This is supported by the evidence provided by the microcosm plate
counts done on each day that shows more eukaryotes at the higher temperatures and
less at the lower temperatures. Alternative methods could be used instead of
polymerase chain reaction in the study. A recent scientific journal by Fakruddin et al
(2013) gives supporting evidence in the use of nucleic acid amplification.
Some errors were seen throughout this experiment due to contamination and human
error. In order to get a more accurate results this experiment should be repeated and
each sample should be replicated more than 3 times in order to get a more reliable
result and remove any anomalies. Further research should be done into the growth
factors of different organisms and bacteria at a larger range of temperature and over a
longer period of time to give clearer evidence of the impact of environmental
temperature change. There could also be experiments using animals to see the impact
of temperature change on a larger organism. Ethical impact should be considered
when performing animal testing especially involving experimental changes that could
cause pain and death. Environmental impacts by global warming are shown by
temperature variation in this study. All living organisms have a ideal temperature that
they will thrive at and temperature that they will be out compete or die at. This is
shown by the data provided in this experiment and as there is evidence of climate
change the ecosystem will be effected.
In conclusion the evidence provided by this study suggests that there is a major
impact on the eco-system due to temperature changes. The original aim of this study
was to analyse the impact of temperature on Pseudomonas fluorescens. However, due
to evidence that can be seen by the results of the PCR experiment on the target
organism, the Pseudomonas fluorescens was out compete at all temperatures by the
other organisms and bacteria which were present in the community. The impact of
temperature could still be analysed on the community. It was seen that, at lower
temperatures, certain bacteria thrived and at higher temperature other bacteria thrived.
This gives supporting evidence towards the major impacts of environmental change in
connection to changes in the eco-system. The data provided by this research
highlights the need to examine temperature change on organisms as they exist, not in
isolation, but within a community.
20
21. Appendix:
An important part of the study was identifying the eukaryotes by learning the
differences between their size, shape and movement. Below is a table of the notations
taken from close observation of the eukaryotes used in this experiment.
-------------------------------------------------------------------------------------------------------
Euglena:
- on a dissecting microscope (x10)
• small oblong rods
• black (some dark green)
• abundant and clustered together
• slow movement with some turns
- on a stage microscope (x400)
• small, round, green with dark green circles and some light green colouring
Colpidium:
- on a dissecting microscope (x10)
• small oblong rods
• clear with a black outline
• abundant
• fast movement and multiple turns
Blepharisma:
- on a dissecting microscope (x10)
• large oblong rods
• red with white inside
• sparse numbers
• sporadic movements (some fast, others slow)
- on a stage microscope (x100)
• small, oblong rod, red with red circles inside
Chilomonas:
- on a dissecting microscope (x10)
• small round
• white
• sparse
• circular or spinning movements along with up and down
- on a stage microscope (x400)
• small, round, clear with some clear circles inside
Paramecium caudatum:
- on a dissecting microscope (x10)
21
22. • large oblong
• red and white
• medium numbers
• fast movement, straight with some turns
- on a stage microscope (x400)
• small, oblong, red with white cloudy inside
-------------------------------------------------------------------------------------------------------
Figure 3: Observable differences between Eukaryotes as viewed under dissecting
microscope and stage microscope.
Agar dish Sample
4 1 2 3 5 6 Dilution
* * * * * *
* * * * * *
* * * * * *
* * * * * *
* * * * * *
Agar dish Sample
7 8 9 10 11 12 Dilution
* * * * * *
* * * * * *
* * * * * *
* * * * * *
* * * * * *
Figure 4: A serial dilution of and its position on each agar plate.
Solutions were diluted by adding samples from numbers 1 to 12. The above diagrams
show the position of each sample and the dilution on the agar plate. 2ul were pipette
for each and as you can see the first petri dish placed sample 4 first. This figure relates
to the agar plate shown below in Image 1.
22
23. Image 1: Day 1 agar plates.
Agar dish Sample
1 2 3 4 5 6 Dilution
* * * * * *
* * * * * *
* * * * * *
* * * * * *
* * * * * *
Agar dish Sample
7 8 9 10 11 12 Dilution
* * * * * *
* * * * * *
* * * * * *
* * * * * *
* * * * * *
Figure 5: A serial dilution of and its position on each agar plate.
Figure 5 relates to the agar plate shown in Image 2 and image 3.
23
24. Image 2: Day 2 agar plates.
Image 3: Day 3 agar plates.
24
25. Image 4: primer 2 analyses on electrophoresis gel viewed under UV light, the data
used in this experiment was on the top left.
Image 5: day 3 sample 1-3 of 20 and 25°C on electrophoresis gel viewed under UV
light, the data used in this experiment was on the top left.
25
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Ellis, M. (1998) Infectious Diseases of the Respiratory Tract. Canmbridge U P.
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