1.0 Introduction

1.1 Research Question

How does varying the level of sodium chloride (NaCl) concentration (%) within agar impact on the growth of Escherichia coli (E.coli) growth due to water activity (osmosis)?

This investigation seeks to draw conclusions on whether the amount of sodium chloride concentrated in an environment will affect the growth of E.coli K-12 strain and further discusses the possible reasons for the inhibition of growth.

1.2 Background Information

Prior to the advancements of modern day technology, Sodium Chloride (NaCl) was regarded as ‘one of the best methods for inhibiting the growth and survival of undesirable microorganisms.’ (National Academy for Sciences, 2010). Preserving meats and inhibiting growth of bacteria such as E.coli depends on lowering the Aw, (water activity) which can be done by increasing the concentration of NaCl in the substance. Water activity can be described as the availability and movement of water in an environment (Today, 2012).

All organisms depend on H2O to grow and survive. (Todar, 2012). Depending on the NaCl concentration of a substancethe water activity will either be increased or decreased.For example, if the NaCl concentration of a given substance is high it will lower the Aw and vice versa. (Todar, 2012). This is due to the NaCl ability to dehydrate the bacteria as it increases the rate of osmosis from within the bacteria side to the extracellular material. An environment that is high in NaCl will aim for equilibrium and therefore will draw available H20 from inside the bacterial cell. This can be referred to as the bacteria cell being within a hypertonic solution, which results in cellular death due to dehydration of the bacteria cell. This therefore

lowers the water activity within the cell and inhibits the growth of bacteria. (Parish, 2006).

For Escherichia coli (E.coli) the minimum Aw required for growthis .91Aw. (Todar, 2012). Therefore any solution with lowerAw and therefore higher NaCl concentration is going to be likely to inhibit E.coli growth.

The above graph compares the osmolarity and growth rate in three categories of prokaryotes. The growth rate of most bacteria includes nonhalophile prokaryotes such as E.coli. For these organisms, the Aw value, which is commonly91Aw, is important to assure that the environment can still sustain life. In comparison to E.coli and other bacteria, some microorganisms require NaCl for growth andare referred to as Halophiles. An extreme halophile requires as much as 15-30% NaCl for growth, whilst some of the less extreme strands require just 1-6%. (Todar, 2012). Halotolerant bacteria grow best in the absence of NaCl but are still able to grow in environments where NaCl is present.

1.3 Hypothesis for growth of E.coli in each NaCl environment

NaCl Concentration (%) Growth2.50 Yes5.0 Yes7.50 Yes10.0 No12.50 No

(Todar, 2012).

15.0 No

Table 1.3 shows the predicted results of this experiment,in relation to the growth of the E.coli K-12 strain. As theNaCl percentages 2.5%, 5% and 7.5% all have a water activity of .91Aw or higher, it is expected that in these environments growth will be supported.In comparison, the agar environments containing the NaCl concentrations of 10%, 12.5% and 15% are not expected to show any signs of growth as they have lower water activity compared to the required .91Aw. This experiment will test the effect of varying NaCl concentrated environments on the growth of bacteria to test this hypothesis.

1.4 Variables

1.4.1 Independent Variables

Concentration of Sodium Chloride within the agar(+/- .5%)02.55.07.510.012.515.0

- Note that the 0% plate will be a sterile agar platesinoculated with the same amount of E.coli K-12 strain. They will be treated as the control to

assure that the E.coli K-12 Strain is capable of growth. The results will not be processed.

- These solutions were prepared by a lab technician before the investigation began

1.4.2 Dependent Variables

Description Method of Measuring

Amount of E.coli growth

The number of bacteria colonies will be measured to test the growthof the E.coli after 24 hours.

Using a multifunctional colony counter, the number of bacteria colonies will be counted and marked. Due to the fact that this will rely on a human eye to measure the growth, the uncertainty is (+/-1)

1.4.3 Controlled Variables

Controlled Variable

Purpose of Control Method of Control

Incubator temperature

Bacteria growth can be either discouraged or encouraged by the temperature of thesurrounding environment

The inoculated agar dishes are placed into the incubator, at 37oC, as this is the optimum temperature for E-coli

Time allowed for growth to occur

If more or less time is allowed

All the agar dishes will be

for the growth to occur, the growth of bacteria will be influenced by time, which is notthe purpose of this experiment.

inoculated and sealed at the sametime, then placed into the incubatorat the same time and left for 24 hours.

Controlled Variable

Purpose of Control Method of Control

Number of trials on sample

Because humans arecarrying this out,there is a possibility of human error that would affect the results.

6 trials for each Sodium Chloride concentration (%) solution will be tested to find themean value of the results.

The light available in petridish environment

Due to the possibility of light energy stimulating a series of metabolic reactions, which could either inhibit or encourage the growth of the E.colifurther.

The incubator willsupply a environment that lacks any light.

Amount of Agar in petri dish

The Agar is a source of nutrients for the bacteria, which will provide the energy needed for growth. Therefore,the plates will have the same amount of Agar to provide the same amount of nutrients to all test trials.

The Agar will be prepared followingexact measurementsand the final mixture will be poured to half thesize of the Petri dish.

Size of petri dish The available surface area affects the accessto nutrients, which influences the metabolic reactions.

All petri dishes will all be of thesame size and shape, assured by using the same brand.

Placement of E.coli on petri dish

To assure that colonies are easier to identifyand therefore count, the initialE.coli will be spread across the agar to assure theisolation of colonies.

The ‘zigzag’ method will be used when applyingthe initial E.coli to the agar.

Oxygen availability

Exposure to an environment outside of the agar plate may introduce the E.colito factors that could either inhibit or increase the growth of the bacterium.

Using parafilm, the inoculated agar dishes will be sealed in groups of three tolower the risks ofcontamination and then placed into an isolated incubator.Also, a new sterile inoculation loop was used to assurethat no cross-contamination could occur.

Controlled Variable

Purpose of Control Method of Control

Test Organism Different bacteriastrands will reactdifferently to different concentrations of Sodium Chloride.

All dishes were inoculated from the same pure broth of E.coli K-12strain, which was located from a

Also, the purpose of this experimentit to test E.coli.

reliable source and used to inoculate all the agar dishes.

Initial amount of E.coli placed on Petri dish

The starting amount of E.coli will influence thefinal number of colonies.

6 microliters (ul)of E.coli will be attained using a Micropipette.

Human error involved in data collection

As the counting method is measuredwith a naked, human eye, human errors may occur where colonies areskipped or added.

A multifunction colony counter will be used, which both keeps count of the colonies electronically butalso marks the petri dish once a colony has been counted. This cannot completely eliminate human error, but will minimize it. An uncertainty has been set at (+/- 1) colonies.

2.0 Materials and Methods

2.1 Apparatus 37 standard sized petri

dishes 5 thermometers 5 500mL beakers Microwave 5 stirring rods Heat proof gloves Permanent marker Sterile disposable gloves Fume hood

2.2 Materials 35g Agar powder E.Coli K-12 strain

broth culture 200mL 2.5% NaCl

concentration solution

200mL 5% NaCl concentration solution

200mL 7.5% NaCl concentration solution

200mL 10% NaCl concentration solution

Electronic scales 36 sterile Inoculation loops Parafilm Multifunction colony counter Incubator at 37oC Micropipette 36 Micropipette tips Safety goggles Lab coat

2.3 Method

Day One- preparation of agar dishes 1. A bench top was wiped clean with disinfectant and

sterile disposable gloves were put on. 2. All materials and apparatus were collected. 3. The 500ml beaker is labelled with the concentration

of NaCl solution it will contain (e.g. 2.5% for the appropriate solution)

4. 5.6g agar powder was measured using electronic scales. Once weighed correctly, the powder was placed into a 500mL beaker labelled 2.5%.

5. The prepared 2.5% NaCl solution was measured with a 200mL measuring cylinder, and then 200mL of the solution was poured into the 500mL beaker with the 2.5% label.

6. A stirring rod was used to stir the solution until the agar powder was not visible.

7. The 500mL beaker was placed into the microwave for two minutes. It was then taken out and stirred for 30 seconds. It was placed back in the microwave for 1 minute and 30 seconds. Agar solution had boiled.

8. The solution was taken out of the microwave. Using heatproof gloves, the 500mL beaker was removed and stirred again until the solution was clear.

9. Using a thermometer, the temperature of the solutionwas monitored until it had cooled to 55oC.

10. Whilst cooling occurred, 6 sterile petri disheswere labelled appropriately. On the bottom of the petri dish the date of preparation, concentration ofNaCl solution and trial letter were used for identification. (e.g. 11/3/14, 2.5%, A)(e.g. 11/3/14, 2.5%, B)

2.2 Materials 35g Agar powder E.Coli K-12 strain

broth culture 200mL 2.5% NaCl

concentration solution

200mL 5% NaCl concentration solution

200mL 7.5% NaCl concentration solution

200mL 10% NaCl concentration solution

11. Once the agar solution had cooled to 55oC, the solution was poured into the petri dishes under the fume hood. The solution was poured to fill half of the petri dish. Lids were placed onto the petri dishes, half off to allow cooling.

12. Lids were placed completely onto each petri dish. The petri dishes were left to cool and set until the solution had a jelly-like consistency and they were flipped and placed into the fridge to store.

13. These steps were repeated for each different solution until all 37 plates (6 for each NaCl concentration (%) solution were completed)

14. To assure that the bacteria was active, a control plate was made. The 500ml beaker is labelled(11/3/14, CONTROL).

15. 5.6g agar powder was measured using electronic scales. Once weighed correctly, the powder was placed into a 500mL beaker labelled CONTROL

16. 200mL of distilled water was mixed with the agar.

17. Steps 6-9 were repeated for the control. 18. Whilst cooling occurred, a sterile petri dish

was labelled as follows: 11/3/14, CONTROL, A 19. Steps 11-12 were repeated for the control.

Day Two- Inoculating Plates

1. A bench top was wiped clean with disinfectant and sterile disposable gloves were put on.

2. Remove prepared agar dishes from the fridge and allow time to rest on a sterile bench top.

3. Using an electronic pipette, attach a pipette tip. Set the electronic pipette to 6 microliters.

4. The container of K-12 strain E.coli was opened and the pipette was used to obtain the required (6 microliters) sample.

5. The sample was placed in the centre of the agar dish.

6. Using an inoculation loop, the E.coli sample was then spread in a zigzag motion.

7. The agar dish was rotated 90o and the zigzag patternwas repeated, as demonstrated in figure 2.3.1.

8. Using a new inoculation loop each time, steps 4-6 were repeated for all agar dishes.

9. Using parafilm, the inoculated agar dishes were grouped in groups of three, to seal the plates, therefore preventing the risk of contamination.

10. The grouped inoculated agar dishes were than placed in an incubator at 37oC for 24 hours.

Figure 2.3.1 Diagram of the pattern used to inoculate theplates

Step 6 Step 7

Day Three- Data collection

1. Sterile gloves were put on and the inoculated plateswere removed from the incubator

2. Using a multifunctional colony counter (see appendixfor image), trial 1 for the 2.5% NaCl concentration agar plate was flipped and the visible colonies werecounted and marked to eliminate the possibility of counting a colony twice.

3. The results, displayed on the multifunctional colonycounter, were reported in the raw data table, aligning to trial 1 for 2.5% NaCl.

4. Steps 2-3 were repeated for all inoculated plates. Data was recorded in the table with each plate results aligning with the label.

Figure 2.3.2 Picture of the data collection method, wherethe multifunctional colony counter was used

3.0 Results

3.1 Raw Data

Table 3.1.1: Raw data values collected after 24 hours in incubator showing the number of E.Coli colonies present on the 36 platesSodium Chloride (NaCl) concentration ofAgar solution(%)

Trial Number of E.Coli Colonies present (+/- 1)

2.5 1 6572 7623 7054 5305 6486 505

5.0 1 4982 4753 4324 397

5 4156 387

7.5 1 3502 3283 3694 3945 2976 263

10.0 1 0022 0093 0034 0155 0246 007

12.5 1 02 03 04 05 06 0

Table 3.1.1- Continued: Raw data values collected after 24 hours in incubator showing the number of E.Coli colonies present

Sodium Chloride (NaCl) concentration ofAgar solution (%)

Trial Number of Bacteria Coloniespresent (+/- 1 colony)

15.0 1 02 03 04 05 06 0

- Note: The control test showed a full plate of growth, therefore indicating that E.coli growth was able and little to no contamination effected the results

Uncertainties:Although a multifunction colony counter was used, thedata was still measured based on the judgement of naked human eyes. Therefore it is prone to human errors, hence the +/-1 colony error range.

3.2 Quantitative Results

Table 3.2.1 Recording the physical characteristics of each varying NaCl concentration, to describe the growth of bacteria over the plates Sodium Chloride (NaCl) concentration ofAgar solution(%)

Characteristics of inoculated agar plates after incubation time.

2.5 Bacteria growth not evident in justone area, but rather the whole plate is covered in colonies. E.coli colonies are largest in size on these plates, approximately 2mm in width.

5.0 Bacteria growth present on whole plate, however existing more independently in comparison to colonies on the 2.5% NaCl plate.

7.5 Bacteria growth most evident in thestreaking pattern, with vague colonies forming towards the outer edges of the plate.

10.0 Dispersed colonies small in size found towards the outer edges on the left side of the plate. This suggests that there may have been some contamination or that the E.coli

is able to grow during a short period of time in these conditions but not sustain life.

12.5 No Growth present15.0 No Growth present3.3 Calculations Table

Table 3.3.1 Calculations used to process raw data (presented on table 3.3.1)Calculation

Formula Example

Mean ∑ ofallvaluesNumberofvalues

Raw values for NaCl solution 2.5%

(657+762+705+530+648+505)6

= 635StandardDeviation

This formula was used in Excel and further checked on a GDC calculator

Percentage Difference

x−y(x+y) /2

X100 Mean values of NaCl solutions- 10% and 2.5%

635−10(635+10)/2

X100

¿625322.5

X100

= 193.80%

3.4 Mean and Standard Deviation

The mean for a data set shows the average result and is used to find the average number of bacteria colonies found on each set of agar plates. The standard deviation measures the dispersion of values from the mean of any given data set.

Table 3.4.1 The mean and Standard deviation of the growth of bacteria from each varied NaCl concentration using results found on table 3.3.1Sodium Chloride (NaCl) concentration

Mean (+/- 1 colony) Standard Deviation(colonies)

2.50 635 1005.00 434 447.50 334 4810.0 10 812.5 0 015.0 0 0

Both the results for 12.5% and 15% show no difference, indicating that at 12.5% no E.coli could survive in the agar environment.

Figure 3.4.2 Graph showing the average number of E.Coli colonies present due to inhibition of growth, controlled by the difference NaCl concentration environments the E.Coli grew in, using results found on table 3.4.1

0 2 4 6 8 10 12 14 160

100

200

300

400

500

600

700

The mean number of E.Coli colonies present in varying

NaCl concentration environments after 24 hours

Sodium Chloride Concentration of agar plates (%) Number of E.Coli colonies present (+/- 1

colony

DiscussionThe error bars are representative of the standard deviation of each set of data. The error bars vary in size. The error bars for both 5%, 7.5% and 10% NaCl concentrations are relatively small, indicating that the range of values is small and the means are an accurate representation of the data, as not many of the results varied for the mean. Visually and numerically, the resultfor 10% is the smallest at 8.29, indicating that the meanis a very good representation of the raw values. This could indicate that all the plates were treated the exactsame way without any accidental interference from the outside environment. As seen on figure 3.4.2, 5% and 7.5%show very similar standard deviations at 44.09 colonies and 48.01 colonies respectively. These values are relatively low in light of the experiment, and indicate that the results found were mostly accurate and show a true representation of the effect of NaCl on E.coli. However, the error bars for the 2.5% mean is relatively large (seen in figure 3.4.2), indicating that the mean

may not be the best representative of the data, as the raw values found vary to a larger extent from the mean. This could indicate that the treated plates were not handled exactly the same, resulting in contamination of different initial amounts of E.coli, therefore yielding a wide range of results.

3.5 Percentage Difference

Percentage difference was calculated to show numerically the average difference of growth found between plates. Average results from the NaCl concentrations of 5%, 7.5% and 10% were tested against the average result from 2.5%,to further examine the optimum conditions for growth and where the E.coli strand started to face some resistance from the NaCl environment.

Table 3.5.1 The Percentage difference between 2.5% and three different NaCl concentrations using results found on table 3.4.1NaCl concentration average results tested (%)

Percentage Difference (%)

2.5 and 5.0 372.5 and 7.0 622.5 and 10 1935.0 and 7.5 26

Figure 3.5.2 Graph showing the percentage difference between average number of colonies found in 2.5% NaCl environment and three different NaCl environments (5%, 7.5% and 10& respectively) using the results found in Table 3.4.1

2.5 and 5 2.5 and 7 2.5 and 10050100150200250

The Percentage Difference when comparing the 5%, 7.5% and 10% NaCl solutions to the 2.5% NaCl Concentration, using the means

as a point of comparison

NaCl concentrations tested (%)Percentage Difference (%)

Table 3.5.3 Comparison table of hypothesise and experiment results showing growth of E.coli in varying NaCl concentration environmentsNaCl concentration(%) results

Hypothesized Result

2.5 Yes Yes5.0 Yes Yes7.5 Yes Yes10.0 No Yes12.5 No No15.0 No No

4.0 Conclusion

The results of this experiment clearly show that the amount of Sodium Chloride present in an environment does affect the growth of E.coli K-12 strain. This is supported by the results represented on table 3.4.1, where the varying NaCl concentrations show a difference of mean growth colonies for each concentration, with the exception of 12.5% and 15%, which both showed the presence of 0 colonies. As evident in the data, the 2.5%

concentration showed a mean of 635 colonies, whereas the 5% concentration showed a mean of 434 colonies, both which vary from 7.5% and 10%. However, the results of this experiment are only partially corresponding to the hypothesis, where the 10% solution differed from the hypothesis, as seen in table 3.5.3, as it did show signs of growth. Therefore, it can be stated the results of this investigation did not support my initial hypothesis.

The E.coli K-12 strain showed the most signs of growth in the environment of 2.5% NaCl, where a mean of 635 colonies where counted. This supports the initial research, which can reason this growth, as the environment of this agar was the least hypertonic, and therefore the bacteria would have suffered the least dehydration, due to less movement of water out of the cell. The same can be said for both the 5% and the 7% NaCl environments, which showed little difference in comparison to the other results. The percentage difference between the 5% mean and the 7.5% mean was the smallest, at 26%, which is 11% lower than the difference between 2.5% and 5% percentage difference, at 37.6%. Thissuggests that it is between the 5% and 7.5% concentrationthat the NaCl starts to affect the water activity slightly, but is an environment that still supports the E.coli growth. This can be reasoned by Todar (2012), who stated that the minimum required water activity needed tosupport E.coli in any environment was .91, and therefore the NaCl concentrations between 0.1%-9% should support the E.coli growth, as there is not enough NaCl to truly trigger osmosis to the point of dehydration. However, theresults of this experiment do not completely support Todar’s theory, as the 10% NaCl concentration showed minimal signs of growth in the 24-hour period.

It could be suggested that the amount of time allowed forthe growth, or inhibition of growth, was not enough for all dehydration to occur in the E.coli K-12 strain cells. Whilst on the 12.5% and 15% NaCl environments, where the E.coli initial sample may not have even survived for the first 30 minutes due to the low level of water activity, and therefore high rates or osmosis to find the equilibrium. On the 10% environment, the water activity

may have been just high enough to support the limited number of E.coli colonies present, which was a mean of 10 colonies. However, another theory that could give reason to this growth was if a human error had occurred within the experiment by mistake. For example, the exact amount of agar solution, which contained the NaCl solutions, wasnot measured specifically into each agar dish and insteadwas distributed using human eyes to estimate what was half the size of the Petri dish. This may have been an issue, but the results of the standard deviation, which was only 8.29 colonies for the 10% environment (the smallest standard deviation), suggest that it may have been another human error, as this standard deviation is very low in comparison to the other standard deviations, where the second smallest is 44.09 colonies for the 5% solution.

The error bars seen on figure 3.4.2 show two overlapping error bars for the 5% and 7.5% results of the average number of E.coli colonies present. This overlap indicates that there is no significant difference between these twosets of data. To further test this theory, a T-test couldhave been conducted, however not enough data was initially collected and therefore no further statistical tests could be conducted.

To finally conclude, it is clear that any environment, which has a sodium chloride concentration above 12.5%, will not support the growth of E.coli K-12 strain, only as evident in this investigation. Furthermore, anything below or equal to a 10% environment should be treated as though the water activity will be strong enough against the presence of NaCl to prevent dehydration and thereforesupport the growth of E.coli K-12 strain. In order to find the optimum NaCl concentration that inhibits E.coli growth,the investigation could be performed again measuring a tighter range of NaCl concentrations between 10% and 12.5%, as this investigation identified that growth was inhibited somewhere between these concentrations.

5.0 Evaluation

Strengths of Investigation Evaluation and JustificationStreaking Method By separating the initial

E.coli sample to isolate the colonies, therefore making them easier to identify. This method also allowed theinitial E.coli to form separate colonies, rather than just a few large colonies.

Limitations of Investigation Evaluation and ImprovementsMethod of collecting raw data- using the naked human eye to obtain all raw data risks a high possibility of either missing colonies or counting extra colonies.

Using a microscope to clearly identify all the true colonies, which would therefore lessen the chance of human error impacting theresults, could be used as analternative collection of data.

Amount of agar solution poured into plates- as this would slightly vary the amount of nutrients available.

Further measuring of the agar solutions could have been utilised to fill the Petri dishes with the same exact amount of agar solution.

Limited amount of data- due to the limited amount of data collected, only simple statistical tests could be conducted and therefore the final conclusions are slightly limited due to the lack of diverse statistical justification.

In future experiments, more NaCl solutions could be tested, specifically lookingat the range between 5% NaCland 12.5% NaCl, to see exactly where the NaCl starts to impact growth, andwhich concentration is optimum for inhibiting growth. Also, with more data, a wider range of statistical tests could be carried out to form new conclusions.

Human error- through the In future experiments the

investigation, human error may have played a role in skewing the data without notice and therefore lead toinvalid conclusions.

uncertainties could be managed by using more advanced and specific equipment, especially when measuring out solutions, such as the initial pouring of the NaCl solutions to mixwith the agar, as these would have definitely variedbetween at least +/-2mL.

Bibliography

Todar, K. (2012). Nutrition and Growth of Bacteria. Available: Last accessed 4th February 2014.

Parish, M.. (2006). How do salt and sugar prevent microbial spoilage?.Available: http://www.scientificamerican.com/article/how-do-salt-and-sugar-pre/. Last accessed 4th February 2014.

National Academy of Sciences. (2010). Preservation and Physical Property Roles of Sodium in Foods. Available: http://www.ncbi.nlm.nih.gov/books/NBK50952/. Last accessed 4th February 2014.