PROJECT N°40
Sugars’ Effect on Oral Bacterial Growth
Yasmine van Domburg, Marike Gruijters, Heather Russell
European School Frankfurt am Main
Praunheimer Weg 126, 60439 Frankfurt am Main, Germany
S6 EN
Abstract
The modern nutrition consists of a variety of different sugars, and for this reason we decided to further
investigate the correlation between them and the amount of bacteria they help or prevent to grow, in
order to understand more deeply which foods worsen our general dental hygiene. Using both simple
and complex sugars commonly found in our diets, (glucose and fructose; sucrose and lactose
respectively), we designed a protocol which would help us determine which sugar (or kind of sugar) is
most nutritious for oral bacteria. The sugars and bacteria react together and go through a fermenting
process, to ultimately form acids, which attack the teeth. Bearing in mind that complex sugars need to
be broken down before metabolization, we hypothesized that simple sugars would grow more bacteria
than complex ones, even though we are often warned against sucrose, found in products such as
gummy bears, lemonade-drinks, and so on. Our results, to a great extent, confirmed this: overall, the
petri dishes with glucose in the agar grew the most and largest colonies, covering the largest total
surface area compared to the other sugars. However, the petri dishes containing no sugars grew even
more, showing that sugars are actually inhibitors, which proves the effectiveness of traditional folklore
conservation techniques that use sugar for food preservation.
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1. Introduction
The original aim of our project was to test how different types of sugars affect oral bacterial growth.
The final purpose was to raise awareness and discover which sugars to avoid eating in order to give
recommendations to improve oral hygiene, although in the end our results did not quite allow us to
pull a concrete conclusion in terms of dental hygiene.
Sugars and salts have traditionally been used as preservatives for food and home remedies against
infections, despite being considered bad for our teeth. Since childhood, we are taught not to eat too
many sweets, and to thoroughly brush our teeth. What is the underlying science for this very important
practice? And why do some products, such as honey and sugar stay good for years without chemical
preservatives, when they help encourage bacterial growth in our mouths?
Another motivation for this experiment includes the differences in molecular structures of the
different sugars. Glucose and fructose, both monosaccharides, cannot be further hydrolysed, leading
one to believe that the colonies in the petri dishes containing these would grow faster and better,
since there is no need to break them down any more. Disaccharides, on the other hand, consist of
monosaccharides (sucrose is made of glucose and fructose; lactose is made of glucose and galactose),
meaning that they need to be broken down before they can be a source of nutrition for the bacteria.
Enzymes in the saliva ensure that this would happen in the petri dishes when growing oral bacteria
from saliva samples.
However, sugars are often used as preservatives. This is due to their effect on something called the
water activity.
Water activity is the ratio between water vapour around a substance and the substance itself. Scott
(1953) established that water activity determines whether or not microorganisms will grow. Most fresh
foods typically have a water activity between 0.95 and 0.99, which will provide enough moisture to
sustain the growth of microorganisms. This is why our food will rot after time.
Sugars inhibit microbial growth in food because they will draw available water out of the food and
replace it, via osmosis, happening when the concentration of solutes (sugars in this case) is higher in
the medium than in the inside of cells, which will cause the exit of water from cells, and consequently
cell dehydration. This reduces water activity so that bacteria are less likely or take longer to grow.
Inhibition of bacterial growth starts at a water activity of around 0.91 (Parish, 2016).
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2. Methodology
Designing the experiment
We started developing our experiment early November. At first we had a lot to learn on how to grow
bacteria properly, and it took us a while to discover a procedure that was both efficient and hygienic.
After having read multiple simple procedures on the internet (Science Company, Wikihow), we started
designing our experiment in the lab. Firstly we would have to prepare agar. Agar is a jelly-like substance
obtained from algae that provides the nutrients needed for bacterial growth. It is prepared by boiling
a mixture of water, LB salt broth and agar (in a powder form). After the mixture is thoroughly mixed
and boiled for at least one minute, it is put in petri dishes in such a way that the entire surface is
covered. Then the solution is cooled down and let to solidify before the bacteria can be added.
The very first time we prepared agar, we used 250ml of water, 6.25 grams of LB salt broth and 3.75
grams of agar. As this mixture did not fit in the microwave, we attempted to boil it on a hot plate. This
took longer than expected (over half an hour) and even though we were using a magnetic stirrer, some
of the solution gathered at the bottom of the conical flask and formed a solid, crusty layer. During our
second trial we were able to warm the agar in the microwave as we were using only 100 ml of water
(2.5g LB salt broth and 1.5g of agar) and a smaller conical flask. Although we had to pay attention that
the boiling mixture did not overflow, we found this to be more effective.
However, to explore the effect of different sugars on oral bacterial growth, we had to add these to the
agar solution. Through personal communication with Martín-Cereceda (Universidad Complutense de
Madrid), we learned that we should not add the sugars while making the agar solution. This is because
the molecular structure of the sugars can change due to high temperatures and break down to smaller
3
components. Therefore we only added the sugars after the agar had cooled down to a temperature of
30-40 °C degrees.
In the beginning of the project we experimented with different sugar concentrations. We tested with
5% sugar, 2% sugar and 1% sugar, coming to the conclusion that 1% delivered the best results. This
was because when using higher concentrations of sugar, the petri dishes were already mouldy after
the projected time period, so that it was not possible to count the colonies.
After the agar had become a solid gel and the petri dishes were prepared, the next step was to add
the bacteria. We had to choose a way of obtaining the oral bacteria. We experimented with a few
different methods:
- stroking a cotton swab on the teeth;
- stroking a cotton swab under the tongue soaked in saliva;
- spitting in a tube.
The reason we tested both stroking a cotton swab on the teeth and stroking a cotton swab underneath
the tongue was because according to a study by Gibbons (1989), different bacteria colonise in different
parts of the mouth. However, all three methods resulted in entirely covered petri dishes so that we
could not count the colonies. Therefore it was important to dilute the saliva before putting the bacteria
in a petri dish. The only method suitable for dilution is the third method, and for that reason we
continued using it.
In order to decide how much we would dilute the saliva during our actual experiment, we
experimented with both 10x diluted and 100x diluted before starting. In both cases we would ask
someone to spit in a plastic tube. For the 10x diluted we took 10 µm of the spit, using a micropipette.
For the 100x diluted on the other hand, we only took only 1 µm of spit. Then we added water (in the
case of 10x diluted we added 90 µm of water, in the case of 100x diluted we added 99 µm of water).
After having mixed this solution thoroughly using the micropipette, we poured this in the petri dish
and used a cotton swab to spread the mixture out in such a way that the whole area was covered with
an extremely thin layer of diluted saliva (swabbing).Then we left the petri dishes in the incubator for
several days (at a temperature of 37 °C, the temperature of your body).
The profile of our saliva donors includes that they were all in the same age group, and hadn’t eaten
beforehand. Samples were therefore usually taken in the morning.
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The final experiment
Materials needed:
- 6 graduated plastic test tubes
- 5 mini plastic tubes
- spatula
- digital weighing scale
- glass dishes
- disinfectant (propanol)
- gloves
- lab coats
- 0.1 g of fructose
- 0.1 g of lactose
- 0.1 g of sucrose
- 0.1 g of glucose
- 5 conical flasks
- 500 ml of water (100 ml per flask)
- 12,5 g L.B Broth salt (2.5 g per flask)
- 7,5 g of agar (1,5 g per flask)
- microwave
- heat resistant gloves
- glass rod
- refrigerator
- saliva
- 20-200 µm micropipette (air displacement pipette)
- 0.1-10 µm micropipette (air displacement pipette)
- 15 plastic eppendorf tubes
- beaker
- cotton swabs
- tape
- permanent marker
- 15 petri dishes
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Day one: Measuring
Considering the fact that we never had much time in between lessons, we had to make the procedure
as efficient as possible. One way to make sure that we wouldn’t run out of time while preparing the
agar, was to already measure all the quantities beforehand.
1. Clean the working area and make sure that all the equipment (weighing scale, glass dishes, spatula)
is disinfected.
2. Measure 5x 2.5 g of LB. Broth salt and put these quantities in separate graduated plastic test tubes.
3. Measure 5x 1.5 g of agar and add these quantities to the graduated plastic test tubes. Close the
test tubes.
4. Measure 0.1 g of fructose, lactose, sucrose and glucose. Put these different sugars then in different
mini test tubes.
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Day two: Preparing the agar (with the sugars)
1. Add 100 millilitres of water to each of the conical flasks.
2. Then add the prepared agar mixture from the graduated plastic test tubes and stir with an
(disinfected) stirring rod.
3. Put all the mixtures in the microwave and wait for it to boil.
4. In the meanwhile, write the correct date on the petri dishes with a permanent marker.
5. Stop the microwave and quickly stir again with the stirring rod. Put the flasks back in the microwave
and let it boil for one minute. Be careful not the let it boil over.
6. Put the mixtures out of the microwave and let them cool down to a temperature of more or less
40 °C degrees (when you can touch the flask easily). We often speeded up this process by putting
the flasks in the refrigerator for two minutes
7. After having reached this cooler temperature, add one of the sugars (from the mini test tubes) to
each of the agar solutions.
8. Stir (with a clean stirring rod) until sugars are completely dissolved.
9. Slowly pour every agar mixture in three petri dishes (divide evenly!) and write with a permanent
marker the specific sugar for that petri dish.
10. Put the petri dishes in the refrigerator (in order to let the agar become a solid).
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Day three: Adding the saliva
1. Ask a test person to spit into an graduated plastic test tube and close the lid.
2. Prepare the 0,1-10 µm micropipette by setting it on an amount of 10 µm .
3. Extract, using the micropipette, 10 µm of spit and put this in a plastic Eppendorf tube. Repeat this
process fifteen times.
4. Fill a disinfected beaker with water.
5. Take, using the micropipette of 20-200 µm, 90 µm of water and add this to a plastic Eppendorf
tube. Repeat this process fifteen times
6. Mix the saliva and the water by setting the micropipette on 100 µm and taking the mixture out of
the Eppendorf tube and putting it in again, until thoroughly mixed.
7. Then let the pipette absorb the whole mixture (100 µm) and release the mixture in one of the petri
dishes.
8. Take a cotton swab and spread the mixture over the whole surface. Repeat this process for all the
petri dishes.
9. Close the petri dishes with tape and put them in the incubator.
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Day eight: annotating
1. After the dishes are left in the incubator for five days, take the petri dishes out of the incubator.
2. Count the colonies of each of the petri dishes precisely and annotate your data.
Counting colonies:
In order to obtain our results we counted the number of colonies and percentage covered per petri
dish and found the average, which we then used for the data graphical analysis. When the number of
colonies present was so great that we could not distinguish individual colonies, they were annotated
as over two hundred, as this number was still greater than the others, but not big enough to drastically
alter the scale of the graph. In order to then create the graphs, we simply used two hundred. Because
in certain situations counting the colonies was not always representing the amount of bacteria grown
in a fair way, we also estimated the amount of area covered per petri dish and expressed it in
percentages.
Graphical analyses:
The results were analysed graphically with Excel (Microsoft, 2013).
3. Results
Table 1 shows all the average results obtained per sugar of each test subject. As we can see, on
average, excluding negative control, glucose grew the most bacterial colonies with an average of 137.7
colonies per petri dish. Lactose and sucrose grew approximately the same amount of colonies, 111 and
106.8 respectively. Fructose grew significantly less on average with 67.2 colonies per petri dish.
Looking at individual results, we can also see that the results greatly differed between the test subjects.
Test subject H for instance, had 50-100 colonies for each of the sugars, whereas others, test subject E
for example, had over 200 colonies for almost all of the sugars.
Unexpectedly, the petri dishes without any sugar (negative control) grew more colonies on average
than any of the petri dishes with sugar. Looking at all the test persons individually, only test person E
had the least amount of colonies on the negative control dishes and was therefore the only one whose
results matched our hypothesis. With all the other test persons, the petri dishes without the sugars
(negative control) grew the most.
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Test person A does not have a negative control, simply because that was the very first experiment we
performed. As we were still learning how to carry out the experiment correctly, we accidentally did
not make enough agar. Therefore, we did not make a negative control in this first experiment.
Table 1.
Glucose
Fructose
Lactose
Sucrose
Negative control
A
200
23
200
4
B
102
44
48
95
200
C
105
56
119
105
200
D
94
151
122
200
136
E
200
173
197
200
200
F
111
54
94
81
18
G
200
22
46
120
200
H
90
59
62
50
87
Average
137.7
67.2
111
106.8
148.71
10
Glucose
Fructose
200
200
Amount of colonies
Amount of colonies
250
150
100
50
0
150
100
50
0
A
B
C
D
E
F
Experiment
G
H
A
B
C
250
250
200
200
150
100
50
0
A
B
C
G
H
G
H
Sucrose
Amount of colonies
Amount of colonies
Lactose
D
E
F
Experiment
D
E
F
Experiment
G
150
100
50
0
H
A
B
C
D
E
F
Experiment
Negative control
Amount of colonies
250
200
150
100
50
0
A
B
C
D
E
F
Experiment
G
H
Figure 1.
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The same observations can be made by looking at the graphs in Figure 1. However the visualisation
emphasises the differences much better than the numbers do. Again we see that the results greatly
differed between the test-subjects, and that glucose clearly grew the most colonies of all sugars. 0ver
200 in three cases, and relatively high numbers for the rest. Contrasting fructose where all the values
are quite low aside from in two cases, neither of which, however, reach over 200.
The bacteria that were given lactose and sucrose clearly grew less colonies than the bacteria with
glucose and grew more colonies than the bacteria with fructose. With both lactose and sucrose there
were two cases where the bacteria had developed more than 200 colonies. All other petri dishes with
sucrose and lactose had colonies reaching from 50 to 125 colonies. With sucrose we did have one
interesting result with test person H, whose petri dish only had 4 colonies.
Because in certain situations counting the colonies was not always representing the amount of bacteria
grown in a fair way, we also estimated the amount of area covered per petri dish (in percentages).
Table 2.
Glucose %
Fructose %
Lactose %
Sucrose %
Negative
control %
A
8
13
63
4
-
B
11
19
13
16
35
C
22
32
15
13
50
D
35
48
19
33
18
E
18
32
31
37
57
F
13
7
32
14
4
G
3
3
5
10
20
H
10
11
16
11
14
Average
26
20
24
19
28
The results were similar but not quite the same. As expected, the bacteria grew a lot in the petri dishes
with glucose (26% on average) and grew less with fructose (20% on average). However we expected
sucrose’s results to be between these two values, which was not the case. According to these
measures, the bacteria of the petri dishes with sucrose cultivated less than the the bacteria of the petri
dishes with fructose.
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Similarly to our previous results, also with these results the petri dishes with no sugar (negative
control) has the most bacterial growth (28 % on average).
Here are again the same type of graphs (Figure 2 and 3), except now not with the amount of colonies
on the y-axis, but the percentage of petri dish covered by the bacteria.
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Glucose
Fructose
Area of petri dish covered (%)
Area of petri dish covered (%)
Figure 2.
60
45
30
15
0
A
B
C
D
E
F
Experiment
G
60
45
30
15
0
H
A
B
C
Lactose
Area of petri dish covered (%)
30
15
0
C
D
E
F
Experiment
A
G
G
A
60
45
30
15
0
A
A
B
C
D
E
F
Experiment
G
H
D
E
F
Experiment
Negative control
Area of petri dish covered (%)
Area of petri dish covered (%)
45
B
G
Sucrose
60
A
D
E
F
Experiment
60
45
30
15
0
A
B
C
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Figure 3.
Covered
Covered
Clean
Clean
20%
26%
74%
80%
Covered
Covered
Clean
Clean
19%
24%
76%
81%
Covered
Clean
28%
72%
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Glucose
Fructose
Lactose
Sucrose
Negative control
Amount of colonies
200
150
100
50
0
Average
Glucose
Fructose
Lactose
Sucrose
Negative control
Area of petri dish covered %
60
45
30
15
0
Average
Figure 4.
The two final graphs we would like to show (Figure 4) are the final averages of all our experiments for
a last overview. Although we used different measures for each of the graphs (the first method of
measuring being the amount of colonies grown in the petri dish, and the second being the area of the
petri dish covered in percentages).
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Also these graphs clearly show that negative control and glucose have grown the most bacteria. In
both graphs fructose has grown the least. Only sucrose is giving different results.
4. Discussion
Because sugars are commonly known as damaging to our teeth, it may seem contradictory that the
most colonies grew in our negative control, which contained only the agar and LB salt broth mixture,
with the streaked saliva on it. However, it should be noted that sugars traditionally have been used as
treatments for infections, and were also an active inspiration for many modern medicines. “Studies
showed that solutions of appropriate sugar concentration incubated at pH 7.0 and 35 degrees C were
lethal to the bacterial species studied.” (Chirife et al., 1983). This suggests that if we’d used a higher
concentration of sugars, no colonies would have been found after six days. Many student protocols
and forums describe difficulty growing colonies after 15% concentrations (Science Forums, accessed
12.02.17).
Sugars are commonly used for wound treatment and preservation of food. According to Parish (2016),
all curing processes depend heavily on either sugar (primarily sucrose) or salt, which supports our
findings that the percentage covered was least when sucrose was used, and that the average amount
of colonies that grew with sucrose was the second-lowest (after fructose). Sucrose, as a bacterial
inhibitor, is effective in preventing the creation of large colonies, however, over a period of six days, it
does not necessarily prevent small colonies from forming, keeping in mind that six days is already quite
a long time.
With similarities in the results with lactose, perhaps some of the shared characteristics with sucrose
(for example the fact that both are complex sugars) should be noted. Disaccharides take longer to be
metabolized than monosaccharides such as glucose and fructose, and one of the reasons for the slower
growth could be due to the time it takes for the enzymes in the saliva to break it down, hence delaying
the time at which the colonies could start forming. “Growth inhibition due to the presence of lactose
has been clearly documented in literature” (Straight et al., 1988).
Fructose, too, has an inhibitory effect, despite being a monomer. It has been shown to prevent urinary
infections and reduce the general adherence of bacteria (Zafriri et al., 1989). Von Hofsten (1961) also
confirmed that galactosides, as well as certain sugars (including fructose) weakened bacterial growth,
as well as the Food and Agriculture Organization, which stated that fructose and sucrose are very
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effective for preserving food, while glucose is not. Our results show the same result.
“The preferred carbon source for E. coli, as for many other bacteria, is glucose, supporting faster
growth rate compared to other sugars.” (Bren et al, 2015). This is consistent with the results obtained
here, which also show that glucose is the least inhibiting sugar. This is because it is the simplest sugar:
it does not need to break down and is in ample supply of carbon.
All sugars prevented bacterial growth due to their contribution to lowering water activity, which
encourages the growth of microorganisms, as opposed to the petri dishes without any sugars, in which
no such effect could discourage it.
Problems with the methodology of the experiment:
We have found multiple reasons why our experiment may not give clear or accurate results. It is very
likely that we have done something wrong during the experiment itself. Here are several possibilities:
-
Firstly it needs to be said that we were performing our experiment in a school environment.
When working with bacteria, it is extremely important that the working area is clean and
disinfected. Although we tried to work as hygienic as possible by always disinfecting the area
and the equipment before starting, a high possibility of the petri dishes being infected with
other bacteria still remained. We had to work efficiently as well, considering we only had a
limited amount of time for a lot of work. This time pressure also prevented us to work in the
most hygienic way possible.
o
Examples of times we could have worked in a more hygienic way:

We could have been more careful in the process of pouring the agar
in the dishes. Instead of opening the dish halfway, we could have only
opened the dish slightly, reducing the chances of other bacteria (from
the air) entering. In addition, we often held our faces relatively close
to the petri dish while pouring in the agar in order to see if we were
distributing the agar equally. If we would have held our faces further
away, maybe it would have been more difficult for other bacteria
(from our faces) to infect the petri dishes.
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
Sometimes (although not very often) we were in a hurry and forgot to
disinfect the stirring rod before stirring another agar solution.
-
As we could not exactly tell when the agar had cooled down to a temperature of 40-30 °C
degrees, it might have happened several times that the agar mixture was still too hot before
adding the sugars.
-
In addition, we used to close the petri dishes by taping all around the sides of the petri dish.
We did it this way because we had read that bacteria, once grown in such large colonies, are
hazardous and quite dangerous. However, after having done seven experiments already, a
teacher in our school informed us that by taping the petri dishes completely shut, no air would
be able to enter. As a consequence, only anaerobic bacteria (that do not need oxygen) could
grow and aerobic bacteria (that do need oxygen) were not provided with the right conditions
to form colonies.
-
We always had to work in the lab in between lessons. However, on some days we did not have
any free periods and were therefore unable to work on the next step. Because we had divided
the experiment in steps, this was not a problem during the process of preparing the petri
dishes. Afterwards on the other hand, when the annotating of the results had to be done, this
did cause some difficulty. We tried leaving all the experiments in the incubator for five days.
However, limited by the weekend or busy days, sometimes the petri dishes were left in the
incubator for too long (six days).
-
Lastly, it was also extremely difficult to be consistent with our methodology. Because we were
still learning and improving our methodology, we used much more different dishes and flasks
in our first few experiments. Of course, by using more equipment, there is a greater chance of
the final petri dishes being infected by other bacteria. This might be why some of the results
of our earlier experiments differ with the results of our last experiments.
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Also annotating the results came with a few obstacles. Observing the five day old petri dishes turned
out to be more difficult than expected. With the chosen concentration and dilution, we usually had
around 50 colonies. However, the colonies always looked different. Sometimes there were lots of very
small colonies, making it impossible to count. Other times there were only a few large colonies, but it
seemed as if it were multiple colonies that had fused together.
Here are a few examples of times that the colonies had formed in such a way that it had become very
difficult to count.
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5. Conclusion
Dental decay is not primarily caused by sugars, rather by the acids created in the reactions between
sugars and bacteria. Sugars on agar actually decrease microbial activity, by lowering the relative water
activity. Fructose and sucrose are the most effective in doing this, whereas glucose is still relatively
nutritious to bacteria.
6. Acknowledgments
We would like to thank the staff at the European School Frankfurt for all of their help and advice,
specifically Dr. Gisela Oliván, who was our mentor throughout the project, and the technicians, who
supplied all the materials we used.
We would also like to thank our test subjects, without whom the experiment would not have been
possible!
7. References
Literary References, alphabetically ordered
Bren, A; Park, J; Towbin, B; Dekel, E; Rabinowitz, J; Alon, U. 2016. Glucose becomes one of the worst
carbon sources for E.coli on poor nitrogen sources due to suboptimal levels of cAMP, Scientific Reports,
vol. 6.
Chirife, J.; Herzsage, L.; Joseph, A.; Kohn, E. S. 1983. In vitro study of bacterial growth inhibition in
concentrated sugar solutions: microbiological basis for the use of sugar in treating infected wounds.
Antimicrobial Agents and Chemotherapy, vol. 23 no. 5, pages 766-773.
Gibbons, R. J. 1989. Bacterial adhesion to oral tissues: a model for infectious diseases. Forsyth Dental
Center.
von Hofsten. 1961. The inhibitory effect of galactosides on the growth of Escherichia coli. Biochemical
et Biophysica Acta., vol. 48 no. 1, pages 164-171.
22
Microsoft. 2013. Microsoft Excel [computer software]. Redmond, Washington: Microsoft.
Scott, W. J. 1953. Water relations of Staphylococcus aureus at 30 C, Aust. J. Biol. Sci., vol. 6, pages 549564.
Straight, J.; Ramkrishna, D.; Parulekar, S.; Jansen, N. 1989. Bacterial Growth on Lactose:
An Experimental Investigation, Biotechnology and Bioengineering, vol. 34, no.5, pages 705-716.
Zafriri, D.; Ofek, I.; Adar, R.; Pocino, M.; Sharon, N. 1989. Inhibitory activity of cranberry juice on
adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells. Antimicrobial Agents
and Chemotherapy, vol. 33 no. 1, pages 92-98.
Webpage References, alphabetically ordered
Livestrong, accessed 12 February 2017, How Salts & Sugars work to Preserve Foods,
http://www.livestrong.com/article/301425-what-are-some-natural-preservatives/ 
Science Company, accessed November 2016, Bacteria Growing experiments in Petri Dishes,
https://www.sciencecompany.com/Bacteria-Growing-Experiments-in-Petri-Plates.aspx
Science Forums, 14 October 2016, accessed 12 February 2017, What concentration of table sugar
inhibits bacterial growth? http://www.scienceforums.net/topic/99556-what-concentration-of-tablesugar-inhibits-bacterial-growth/
Parish, M, Scientific American, Nature America Inc., 21 February 2016, accessed 12 February 2017,
How do salt and sugar prevent microbial spoilage?,
https://www.scientificamerican.com/article/how-do-salt-and-sugar-pre/
Wikihow,
accessed
November
2016,
How
to
grow
bacteria
in
a
petri
dish,
http://www.wikihow.com/Grow-Bacteria-in-a-Petri-Dish
Personal Communication
Martín-Cereceda, November 2016, indirect contact, Universidad Complutense de Madrid.
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