When one hears the term "water pollution", one may think of devastating disasters such as the Exxon Valdez oil spill. But in reality, water pollution is much more common and takes place in our everyday life. When fertilizer or soap gets washed off of our yards or driveways into the storm sewers, they go into the river, causing eutrophication, the excessive accumulation of nutrients, leading to oxygen depletion, in turn leading to lower dissolved oxygen levels, and ultimately a reduction in biological diversity. Organic wastes present in farm runoff and sewage have the same effect. Motor oil or antifreeze can leak on the roads or parking lots; they, too, would be harmful and toxic to the organisms living in the river; this would be a more direct way that pollutants reduce biological diversity compared to eutrophication, which reduces biological diversity indirectly. In addition, industrial discharge in the form of wastewater can include many pollutants such as heavy metals, organic toxins, oils, nutrients, and solids. These lead to depletion of oxygen, and are directly harmful and even lethal to many organisms in the river.

In this experiment, we are focusing on one specific aspect of river pollution: the direct effect of river pollutants on river organisms—that is, how toxic or lethal the river pollutants are to the organisms. To simulate this, we are going to use 3 types of bacteria that are readily found in rivers; bacteria is ideal in this situation because they reproduce quickly and takes a relatively short time to grow. We are going to test the effect of 5 different substances that would commonly be discharged into the river We will also investigate the effect of UV radiation of the chemicals on the bacteria’s survival. This can simulate the actual situation, as chemicals that are in the river are exposed to the UV radiation from the Sun. Also, changes in UV can represent the increasing ultraviolet radiation due to ozone depletion.

This experiment will consist of three small investigations:

Question 1. What effect does the concentration of river pollutants have on the zone of inhibition of river bacteria growing in the presence of the pollutants?

Question 2. What effect does UV radiation of river pollutants have on the zone of inhibition of river bacteria growing in the presence of the pollutants?

Question 3. What effect do different types of toxins have on the zone of inhibition of river bacteria growing in the presence of the pollutants?

What are Bacteria?

Bacteria are prokaryotic, unicellular micro-organisms. Like all prokaryotic organisms, they do not have a nucleus within their cell to direct their functions. Bacterial cells are enclosed by a cell membrane, also known as a lipid membrane, this organelle acts as a regulator of substances going in and out of the cell, and plays an essential role in communication with other bacterial cells. Bacteria do not have membrane-bound organelles such as mitochondria, Golgi complex, endoplasmic reticulum, chloroplasts, etc.

All bacteria have basic needs for survival and growth; one of the most important is the process of harvesting energy. Bacteria need a source of carbon, either from photosynthesis and carbon fixation, or from other carbon compounds. Then the carbon is used in cellular respiration, the process by which the compound can be turned into chemical energy (ATP) for the cell to use. In cellular respiration, there are generally three steps—glyclolysis, the krebs cycle, and the electron transport chain. Of these, the electron transport chain produces the most ATP. This step works by passing electrons through an electron transport chain, generating energy in the form of proton-motive force, which can then be used to drive an enzyme called ATP synthase to make ATP. In aerobic bacteria, the final electron receptor required for this process is oxygen. In anaerobic bacteria, an inorganic compound is used as the final electron receptor, such as nitrate, sulfate, or carbon dioxide. Many bacteria are facultative anaerobes, which means they can grow in both aerobic and anaerobic conditions.

In addition to this basic requirement, there are also conditions under which bacteria grow best. For example, each species of bacteria have an optimum temperature and optimum pH in which they experience the fastest growth.

In this experiment, we will be using three different types of bacteria:

1.Escherichia coli is a unicellular, rod shaped, enteric (found in intestines of warm-blooded animals), fecal bacteria. It is 1? µm in length and 0.1?.5 µm in diameter. It is a facultative anaerobe and grows best at warm temperatures. It is present in animal feces (your poo!), and is excreted from the body in large numbers. It can be released into rivers in sewage and effluent, as well as run-off from farms. It is often used as indicators of fecal pollution in rivers. E.coli grows optimally at a pH of 6-7.

2.Pseudomonas florescens is a unicellular, rod shaped obligate aerobe (although some strains are capable of using nitrate instead of oxygen as final electron accepter during cellular respiration). It is found in soil and water, and grows optimally at 25-30°C and at a pH of approximately 6.5. Pseudomonas fluorescence produce lipases and proteases, enzymes that cause milk to spoil. They are capable of using organic compounds as a source of carbon.

3. Pseudomonas Pseudoalcoligenes are unicellular, rod-shaped motile bacteria that utilize aerobic respiration for its energy source. The optimum temperature for pseudoalcoligenes to survive is 28-30°C. These bacteria are capable of using nitrobenzene for a source of nitrogen. They are predominantly found in soils and water under aerobic conditions.

Our Chemicals - all of which are commonly discharged into river.

Toxin A- Industrial Solvents 1:1:1 solution of benzene(C6H6), toluene(C7H8), and xylene(C8H10)

Benzene is a colorless and flammable liquid with a characteristic sweet smell of aromatic hydrocarbon. It is carcinogenic and its use as additive in gasoline is now limited, but it is an important industrial solvent and precursor in the production of drugs, plastics, synthetic rubber, and dyes. Benzene is a naturally present in crude oil, but is usually synthesized from other petroleum components. Benzene exposure has serious health effects. Breathing high levels of benzene can result in death, while low levels can cause dizziness, rapid heart rate, headaches, and unconsciousness. Chronic exposure to benzene primarily affects the blood. Benzene damages the bone marrow and can cause a decrease in red blood cells, leading to anemia. It can also cause excessive bleeding and depress the immune system, increasing the chance of infection. Water and soil contamination are important pathways for transmission of benzene to humans.

Xylene refers to a group of 3 isomers of dimethyl benzene, an aromatic hydrocarbon. Xylenes, like benzene have a planar structure which makes them very stable.It is a colorless, sweet-smelling, and highly flammable liquid, which occurs naturally and industrially in petroleum combustion. The melting point is between −47.87 °C (m-xylene) and 13.26 °C (p-xylene) and the boiling point is for each isomer at around 140 °C. The density is at around 0.87 g/cm? less dense than water. It is soluble in non-polar solvents such as alcohol.

Toluene, or methylbenzene is a clear, sweet-smelling water-insoluble liquid, which naturally at low levels in crude oil and is usually produced in the processes of making gasoline via a catalytic reformer, in an ethylene cracker or making coke from coal. Toluene undergoes substitution, oxidation, and hydrogenation reactions under pressure. Its density is 0.8669 g/cm? Toluene melts at −93 °C and boils at 110.6 °C. Toluene, Xylene are widely used as an industrial feedstock and as a solvent in the printing, rubber, and leather industries. They are also used as a cleaning agent for steel, a pesticide, and a paint thinner. These aromatics are found in small amounts in airplane fuel and gasoline. Toluene and xylene severely affect the brain, lungs, stomach, and reproductive system. Exposure causes headaches, irritation of the skin and eyes, difficulty in breathing, delayed reaction time, and memory difficulties. unconsciousness and death can result from very high levels of inhalation or injestion. Besides occupational exposure, human come in contact with them via soil and groundwater contamination from leaking underground petroleum storage tanks. Another common form of human exposure to xylene is the use of Sharpies.

Toxin B ?Antifreeze 1:1 solution of ethylene and propylene glycol

Antifreeze is a water-based liquid coolant used in gasoline and diesel engines. It is generally is a mixture of ethylene glycol and propylene glycol. This coolant has a high boiling point of 370 °F and is not corrosive, solving many of water's problems including freezing. Ethylene glycol, or ethane-1,2-diol is an odorless, colorless, toxic liquid with a sweet taste. It melts at −12.9 °C and boils at197.3 °C. They form calcium oxalate crystals in the kidneys and can cause acute renal failure and death. Propylene glycol, or propane-1,2-diol, is an tasteless, odorless, and colorless clear oily liquid that is miscible with water, acetone, and chloroform. It melts at -59 °C and boils at188.2 °C Propylene glycol is considerably less toxic and may be labelled as "non-toxic antifreeze".

Toxin C ?20:20:20 Fertilizer

Fertilizers are chemical compounds that promote plant growth, and in this experiment provide the three major plant nutrients (nitrogen, phosphorus, and potassium) in equal ratio, the secondary plant nutrients (calcium, sulfur, magnesium), and trace elements (or micronutrients) with a role in plant nutrition: boron, chlorine, manganese, iron, zinc, copper and molybdenum.

Toxin D - 10W30 Motor oil

Motor oil is a type of liquid oil used for lubrication by various kinds internal combustion engines. The major fraction of the majority of motor oils are derived from heavier petroleum with lower volatility and higher flash point. The Viscosity Index of sample used is 10-W30, which is relatively low.

Toxin E - Laundry detergent

Laundry detergent/soap is a substance that help get the laundry cleaner. A key ingredient in detergents is a surfactant, a substance which, when added to water, significantly reduces the surface tension of the water. This effect allows water to wash surfaces better. There are many different types of organic compounds which can function as surfactants. Most surfactants are thick, viscous liquids, but some are soft, waxy or greasy solids.

Detergent Molecules consist of: CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CHCOONa

Surfactants typically have somewhat longer molecules which are called non-ionic, cationic or anionic, depending on charge. The liquid detergents may also have other solvent liquids, such as alcohol or a hydrotrope, to help blend all the additives together. Laundry detergents are usually basic, and have compounds such as sodium carbonate or sodium bicarbonate to help to control the pH of the water by neutralizing acidic substances in the water.

How do chemicals kill bacteria?

Industrial solvents, motor oil, antifreeze, and laundry detergent all disrupt the cell membrane of bacteria. They solubilize the cell membrane, and extracts lipids out of the phospholipids bilayer. This damages the cell membrane, which also disrupts the cell receptor events. For bacteria, the cell membrane is crucial to their survival. The cell membrane has many necessary functions, including: -Protecting the bacteria -Regulating intake and output of substances from the cell -Intake of nutrients -Interacting with other cells If the cell membrane is damaged, the bacteria would not be able to carry out these basic processes; thus, it would die.

In addition, antifreeze is also known to interfere with electron transport processes in cellular respiration. The electron transport chain represents the step in this process that produces the most energy. Because a cell is constantly using energy to carry out its processes, an interference with its ability to harvest energy means that the organism cannot survive.

The pH of the environment in which a bacteria grows has a significant effect on its growth rate; different bacteria react differently, for example, acidiphiles are bacteria that are likely to grow the best under conditions with a low pH, Neutophiles find neutral environments better for growth, and akaliphiles like basic environments. Most fertilizers contain ammonium; overtime, this ammonium in the soil is converted to nitrates by soil bacteria, through the process of nitrification of first converting the ammonium to nitrite, then to nitrate. When the three hydrogen ions of ammonium are lost, and two oxygen ions are added, the acidity of the soil rises. The acidity of soil is tested by measuring the ratio of H⁺ particles to the basic particles in the soil such as OH^-, if the ratio is higher, the soil is acidic.

The fertilizer also contains copper in the form of copper sulphate; this metal causes metal ion poisoning. At low concentrations, it is required for survival; at high concentrations, it kills bacteria quite effectively.

What is ultraviolet radiation?

Ultraviolet light is electromagnetic radiation with a wavelength ranging from 400?00 nm, which is shorter than that of visible light, but longer than far UV and X-rays.

There are several important characteristics of ultraviolet waves:
- Frequency is the number of complete cycles per second for a vibrating body. For wave motion, it corresponds to the number of complete waves per second.
- The amplitude is of a wave is the distance between the peak of oscillation and the equilibrium during one wave cycle.
- The wavelength is the distance between repeating units of a wave pattern. It is represented by lambda(λ).

For this experiment, the wavelength we used to radiate the chemicals is 365nm

What is the effect of UV on chemicals?

Based on current scientific evidence, UV radiation considerably alters the properties of alkene, alkyne, and aromatics by forming radicals at the double or triple bond. When electrons on a carbon-carbon pi bond absorb UV rays, they gain substantial energy and jump to higher energy levels. Eventually, electrons gain enough energy to break the bond and form free radicals, molecules with an unpaired electron. This single electron makes the radicals highly reactive with each other, oxygen, and trace gases in the air. Reactions involving radical carbons may result in toxic products. Furthermore, radical carbon with the free electron may damage cell function via whatever mechanisms.

Our hypotheses - to correspond with our three small investigations

Hypothesis 1. If the concentration of river pollutants increases, the zone of inhibition of river bacteria growing in the presence of the pollutants will also increase. This is because an increase in the concentration of river pollutants causes a steeper concentration gradient across the agar medium; as this concentration gradient increases, the rate of diffusion would speed up. Therefore, a higher concentration of the chemical used will spread faster through the agar than a lower concentration of the same chemical; this should create a larger zone of inhibition when compared to a lower concentration.

Hypothesis 2. Due to our research, UV treatment affects those with carbon to carbon double/triple bonds, these pi bonds produces then produces radicals, which are highly reactive with other chemicals and are very toxic. Of our toxins, industrial solvents and motor oil are the ones that are under the condition for radicals to form. Chemicals containing radicals, being toxic to humans, is likely to be harmful to bacteria as well; under these circumstances, we predict that Toxins A and D will cause the bacteria to react most differently when UV radiated. If these toxins are affected, it is likely that, when calculating the average diameter of the zone of clearance the overall bacteria reacting to UV treated length will be higher than that of the bacteria reacting to the non-UV treated toxins.

Hypothesis 3. Laundry detergent is likely to have bacteria reacting with the largest diameter of the zone of clearance due to the fact that it is antibacterial, although it may only be able to act upon common bacteria found on our clothes, etc, it may also likely act upon fecal and soil bacteria. Fertilizer is the commonly spread on our lawns, it also contains nutrients that are necessary to bacteria’s survival such as nitrogen, etc; although fertilizer contains copper, which is harmful to most bacteria, its other components may aid towards the bacteria’s survival.

Variables

Variables for Investigation #1

Manipulated: - Concentration of toxins
Responding: - Diameter of zone of clearance
Controlled: - Bacteria
                ?/span>- Temperature for the incubation of bacteria
?/font>                - Size of filter paper
          ??  ?/span>- Extent/time filter paper saturated in toxins
  ?       ?    ?/span>- Toxins ?/span>
?         ?    ?/span>- Time allowed for diffusion of toxins into agar

Variables for Investigation #2

Manipulated: - UV treated/non-UV treated
Responding: - Diameter of zone of clearance
Controlled: - Bacteria
  ?       ?    ?/span>- Temperature for the incubation of bacteria
                ?/span>- Size of filter paper
  ?       ?    ?/span>- Extent/time filter paper saturated in toxins
                ?/span>- Toxins
                 ?/font>- Concentration of toxins
  ?              - Time allowed for diffusion of toxins into agar

Variables for Investigation #3

Manipulated: - Types of toxins
Responding: - Diameter of the zone of clearance
Controlled:?- Bacteria
  ?       ?      - ?/span>Temperature for the incubation of bacteria
?         ?      - Size of Filter Paper
                  - Extent/time filter paper saturated in toxins
                  - Concentration of toxins
                  - Time allowed for diffusion of toxins into agar

Assumptions

1. E.coli, P.florescens, and P.pseudoalcoligenes are present in the Bow River. This is a reasonable assumption due to the fact that E.coli is a fecal bacteria, which is found in the feces of humans and other mammals, in Calgary, through the sewage treatment plant, our feces are disgarded in the river. P.florescens, and P.pseudoalcoligenes are both soil bacteria, in which case they are likely to be present in the river.

2. The conditions under which the bacteria are cultivated is representative of the conditions of the Bow River. This is a reasonable assumption because the LB agar medium has nutrients, which are similar to that of the river, such as salts and tryptone.

3. Only introduced factors such as UV treated and non-UV treated toxins and concentration of the toxins will have an affect on the wellness of the bacteria, no other factors interfere with the growth of the bacteria. This is a necessary assumption in order to analyze the results to be caused not because of un-intended factors.

4. Volatile toxins do not evaporate during the heated incubation in order for bacteria to grow. This assumption is necessary due to the fact that if the toxins evaporated off of the filter paper disk during incubation, the quantity of each of the toxins placed on growing bacteria will be different, therefore, results may be altered.

Procedural and Analytical Methods of Our Experiment

(To go directly to the detailed procedure below, please scroll down.)

In this experiment, the three bacteria which we chose to act as our indicators are listed below. Escherichia coli Pseudomonas florescens Pseudomonas Pseudocoligene These bacteria are inoculated from available stock and diluted with a liquid LB medium, the inoculated bacteria is then spread evenly over a pre-prepared LB agar growth plate.

Everyday house hold contaminants that are known to enter the Bow river regularly are used as toxins to examine the reaction of the bacteria, these contaminants are listed below. Industrial solvents solution [benzene (C6H6), xylene (C8H10), etc.] Motor oil (10-W30) Fertilizer (20-20-20) Antifreeze [ethylene oxide derivatives (C2H4O)] Washing machine detergent [fatty acid salt - sodium palmitate (C15H31COONa)] These toxins are each diluted, through regular and serial dilution, to 50%,10%, 0.1%, and 0.001%, 100% is also used in the experiment. Filter paper is punched into circles that are 7.2 mm in diameter, and placed in each contaminant to soak. The toxin containing filter paper dots are then placed upon the agar medium already containing inoculated bacteria. The plates of growing bacteria are placed in a rotating disk incubator in a room of 28?C.

To detect a change in the growth of a particular bacteria caused by a contaminant, we will use the agar diffusion method. In this method, one species of bacteria is evenly spread onto an agar plate with the nutrients that allow the bacteria to grow. Then, the filter paper discs saturated in the contaminant are placed on the surface of the agar. The plate is then incubated, during which time the chemical diffuses from the disk into the surrounding agar. If the chemical inhibits bacterial growth, it will kill the bacteria already present in the area containing the chemical, and will also stop further bacteria from growing in the area where the chemical is present. After a few days of letting the bacteria grow in the presence of the chemical-saturated discs, we should see a zone around the paper disk where no bacteria are growing. This is called a zone of clearance, or a zone of inhibition. The zone of inhibition should be relatively clearly defined; areas where bacteria continue to grow will appear opaque, and those where bacteria are not present will appear translucent. Measurements can then be made of the diameter of the zone of clearance to compare the effects of different toxins, concentrations, bacteria, etc. A diagram of an expected zone of clearance is presented in the figure below.

The Rate of Diffusion

The process of diffusion is a major component in this experiment, since it is the process that serves as the basis of the agar diffusion method. In this lab, diffusion refers to the net action of matter, specifically the chemical toxins, from an area of high concentration to low concentration. This process does not require energy input, but occurs only if there is a concentration gradient, the difference between the high concentration and the low concentration. The second law of thermodynamics states that in a spontaneous process, the entropy of the universe increases. Change in entropy of the universe is equal to the sum of the change in entropy of a system and the change in entropy of the surroundings. A system refers to the part of the universe being studied; the surroundings are everything else in the universe. Spontaneous change results in dispersal of energy. Spontaneous processes are not reversible and only occur in one direction. No work is required for diffusion in a closed system. Reversibility is associated with equilibrium. Work can be done on the system to change equilibrium. Energy from the surroundings decrease by the amount of work expended from surroundings. Ultimately, there will be a greater increase in entropy in the surroundings than the decrease of entropy in the system.

The rate of diffusion, or how fast a substance diffuses into other substances, is affected by several factors, they are listed below.

- The concentration gradient controls the direction of diffusion and the rate of diffusion. The steeper the concentration gradient (that is, the greater the difference in concentration between two regions), the faster the rate of diffusion.

- The temperature manipulates the rate of diffusion. The higher the temperature when diffusion occurs, the faster the rate of diffusion.

- The surface area across which diffusion occurs is another factor that affects the rate of diffusion; the larger the surface area, the faster the rate of diffusion will be. For example, a cell with more surface area will have a faster diffusion rate than one with less surface area.

- The size and type of molecule is very important in diffusion. Generally, the smaller the molecules, the faster they diffuse. Also, if the substance is soluble in the substances that make up the membrane or barrier, it will diffuse faster. For example, lipid-soluble substances diffuse faster across the cell membrane than those that are not.

The detailed procedure is as follows.

Preparation of Toxins

Serial Dilution
1. Pipette 10.0ml of toxin A from stock solution with a large pipette in a 10ml plastic test tube, label it A-100.

2. Pipette 2.0ml of toxin A into a second test tube, label it A-50
3. Add 2.0ml of ethanol to it, and combine the solution evenly using the vortex mixer.

4. Pipette 1.0ml of toxin A from A-100 to a third test tube, label it A-10.
5. Add 9.0ml of ethanol to it, and combine the solution evenly using the vortex mixer.
6. Pipette 0.1ml content from A-10 to a fourth test tube, label it A-0.1.
7. Add ethanol to the 10.0ml mark in test tube and mix solution using vortex.
8. Repeat steps 1-7 for toxins B, D, E, and Ampicillin (negative control), but use water as the solvent instead of ethanol.
9. For toxin C, make 100% stock solution by weighing out 1.0g of fertilizer and combining with 1.0ml of water.
10. Follow the same steps for serial dilution as the other toxins.

UV Treatment
11. Pour half of the content in each plastic test tube into Petri dishes.
12. Place each sample under a UV lamp (wavelength 365nm) for 60 minutes. Set this up under a fume hood, because the industrial waste and motor oil will produce toxic fumes.

Preparation of Filter Paper
13. Pipette 3.0ml of each toxin solution into Eppindorf tubes.
14. Hole punch about 60 filter paper dots.
15. Place 4 dots into each tube and saturate for 5 minutes.

Preparation of Bacteria

Inoculation of Bacteria
16. Turn on the gas to the Bunsen burner while holding a sparker on top of the burner to light the flame.
17. Label two test tubes with “E.coli? two with “P.F.?and the other two with “P.P.?
18. Using a 1000uL pipetman, carefully rotate the volume adjustment knob until it reaches 1000uL
19. Attach a sterile disposable tip onto the shaft of the pipetman by firmly inserting the shaft into the opening of the tip.
20. Press down the plunger button on the pipetman until it stops.
21. Hold the pipetman vertically and immerse the tip into the LB medium.

22. Allow the plunger button to return slowly to its original position, being careful not to let the button snap up.
23. Withdraw the pipetman from the LB medium.
24. Take the cap off of one of the test tubes with your index and middle finger. Hold the cap in that position so as to not contaminate the cap or the test tube.
25. Wave the opening of the test tube in the flame to sterilize it.
26. Place the tip against the side wall of one of the six test tubes and push the plunger button down slowly.
27. Discard the disposable tip by pushing the ejector button.
28. Repeat steps 19-27 for the other 5 test tubes.
29. Remove frozen stocks of Escherichia coli, Pseudomonas fluorescens, and Pseudomonas pseudoalcaligenes from the cold room.
30. Attach a sterile disposable tip onto the shaft of the pipetman by firmly inserting the shaft into the opening of the tip.
31. Press down the plunger button on the pipetman until it stops.
32. Insert the plunger into the stock container of E.coli.

33. Take a few jabs at the frozen stock. Stop when a visible accumulation of frozen stock has accumulated on the disposable tip.
34. Take the cap off of one of the test tubes marked “E.coli?with your index and middle finger. Hold the cap in that position so as to not contaminate the cap or the test tube.
35. Wave the opening of the test tube in the flame to sterilize it.
36. Immerse the tip with the frozen E. coli stock into the LB medium in the test tube.
37. Press the plunger button down several times, making sure that all of the frozen stock is now in the LB medium.

38. Discard the disposable tip by pushing the ejector button.
39. Repeat steps 30-38for the other 5 test tubes, with bacteria in their corresponding tubes.

Bacteria Plating
40. Sterilize pipette tip using Bunsen Burner; pipette 0.2ml of bacteria E. coli onto plate with agar medium.
41. Sterilize spreader by dipping in 70% ethanol and placing above Bunsen burner.

42. Spread E. coli evenly onto plate.

43. Using sterilized tweezers, place the appropriate filter paper dot on the plate according to concentration, and type of toxin.

44. Repeat steps 40-44 for bacteria P. fluorescence and P. pseudoalcaligenes.
45. Put all plates on rotary disk incubator at 32°C.

46. After 72 hours, take the plates out of the incubator.
47. Hold a plate to a dark surface where the zone of inhibition can be clearly seen.
48. Use a straight plastic ruler to measure the diameter of the zone of inhibition.
49. Repeat steps 47-49 for all agar plates.
50. Repeat steps 1-49 two more times, for a total of three replicates.
51. Take the average of the diameter of zone of clearance for each concentration without UV treatment (using values from all of the bacteria, toxins, and replicates). Use the following formula: average=sum of values/ number of values
52. Repeat step 51 with UV treated concentrations.
53. Calculate the standard deviation using the average obtained in step 51 and all of the values used in step 51. Use the following formula:
SD=sqrt((x1-x)2/n-1)
where SD=standard deviation
sqrt( )= square root of
x1= any value
x= average
n=total number of values (x1) used
54. Repeat step 53 using the average obtained in step 52 and all of the values used in step 51.
55. Repeat steps 51-54 for each toxin, using values from all bacteria, concentrations, and replicates.
56. Make a graph of average diameter of zone of clearance vs. concentration of toxins
57. Make a graph of Average diameter of zone of clearance vs. toxin used.

Observations

To view the complete observations table, please click here.

The largest diameter measured for the zone of inhibition was 15.1mm; the smallest diameter measured for the zone of inhibition was 7.2mm*. The highest concentration of toxins used was 100%; the lowest concentration of toxins used was 0.001%. A general trend in the concentration of toxins can be readily observed; as the concentration of the toxins increase, the zone of inhibiting generally also increases. There are a few exceptions to this trend, such as the trend in the UV-treated toxin D on Pseudomonas fluorescens.

*note - the diameter of the filter paper disk was 7.2 mm, therefore, if there was no zone of clearance visible, the diameter of the zone of clearance would be 7.2 mm.

The following are examples of our observational graphs, the trends in these graphs are state below.

(To view the complete set of observational graphs, please click here.)

This graph shows all three bacteria being non-reactive with the toxins.

This graph shows each of the different bacteria reacting differently to the toxins as their concentration increased.

This graph contains an apparent outlier, in that the 50% concentrated toxin caused a larger zone of clearance than that of the 100%.

This graph shows all three bacteria reacting the similarily to the toxins of different concentrations.

The following are our analysis graphs. (click on the graphs to enlarge) (please click here for a copy of the analysis tables)
The relationship between the concentration of toxins (all toxins) and the average zone of clearance shown by all the bacteria.

The relationship between the UV treated and non-UV treated toxins with their effect on the average zone of clearance of all the bacteria.

Our Findings

In the analysis of our experiment, we used three main graphs to conclude our findings, and to answer our four questions. From figure 2.1, we are able to see the relationship between the average diameter of the zone of clearance, calculated from measurement of all three bacteria’s inhibition zones around the all the toxins, and the concentration of the toxins used (including both the UV treated and non-UV treated contaminants). From the line of best fit, it is apparent that as the concentration of the toxins, in both UV radiated cases and non-UV radiated cases increase from 0.001% to 100%, the average diameter of the zone of clearance steadily increases as well. The average zone of inhibition for toxins which have been affected by UV radiation has both a higher average zone of clearance in the three highest concentrations of toxins, and also a larger slope for the line of best fit, indicating that as the concentration of the toxins become higher, the UV treated half of the toxins display more rapid increases of the average zone of clearance.

In correlation to the background information of this lab report, the rate of diffusion of a substance through a medium always travels down the concentration gradient, this could mean that the higher the concentration of the toxins, the higher the concentration gradient from the toxin to the agar medium, and therefore, the faster the chemicals will diffuse into the LB agar medium which holds the bacteria. When the toxins are well diffused into the agar surrounding the filter paper, the bacteria is able to react to the environment to which it is growing upon.

In figure 2.2, the concept of the relationship between the average diameter of the zone of clearance exhibited by all 3 types of bacteria and the five types of toxins (UV treated and non-UV treated) used is shown. Overall, the UV radiated toxins appear to have caused a larger average zone of inhibition than the non-UV treated contaminants. Individually, each contaminant reacted relative differently; Toxin C was found to be the contaminant that causes the least average diameters of the zone of clearance in the bacteria when used under both UV treated and non-UV treated conditions. The UV treated toxin A caused the three types of bacteria to exhibit the largest average zone of clearance out of all the toxins (both UV treated and non-UV treated). Toxin A, out of all the toxins, created the most dramatic change in the average zone of clearance in the three types of bacteria from non-UV treated to UV treated, this difference ranges from 7.33mm to 7.97mm in the diameter of the zone, with the UV treated toxin instigating the larger clearance length; toxin D was also found to have a significant difference in the diameter of the zone of clearing in response to the UV and non-UV treated contaminants. The three types of bacteria acted upon by the UV treated and non-UV treated toxin B show nearly no difference in their average zone of inhibition, with the difference in diameter being 0.02mm. Our hypothesis regarding the reaction of the bacteria to the different types of toxins, displayed by the zone of inhibition was partly incorrect, we predicted that laundry detergent would be the toxin that caused the bacteria to have the largest diameter for the zone of clearance, but instead, it was toxin A, industrial solvents; the fertilizer however, did result to be the toxin causing the bacteria to have the smallest zone of inhibition.

In toxins A and D, Industrial solvents of benzene, xylene, toluene and motor oil, both caused the bacteria to react dramatically different with ranging average diameters of the zone of clearance between the UV treated and non-UV treated versions of the contaminants. This supports the background information, UV radiation considerably alters the properties of alkene, alkyne, and aromatics by forming radicals at the double or triple bond, and both industrial solvents and motor oil contains carbon to carbon double bonds. These radicals would cause the bacteria to react differently. The hypothesis, which predicted both an overall increase in zone of inhibition for all toxins, and more significant increase for toxins A and D.

There were many sources of error in this experiment that would have caused our results to be altered; these sources of error are listed below.

1. The zone of inhibition of some test plates of bacteria were not perfectly circular, in which case, judgment was used to determine the average diameter of the zone at which to measure. This means that human bias is introduced into the measurements, and how one person determines where to measure is different than how another person would determine this. This would have caused a problem in the results?accuracy, and is a major error in this lab. This is a random error, for it is impossible to determine the exact effect this had on our results (ie, whether it resulted in a larger or smaller measurement). This error may be reduced in the future by having one person complete all the measurements of the diameter for the zone of clearance, or specifying a way of measuring the zone of inhibition—for example, always taking the maximum diameter.

2. The ruler that was used to measure the zone of inhibition may have been lacking in precision; the size of the measurements was relatively small, therefore, precision has a bigger impact on the results. Also, the accuracy of the ruler can only be controlled by the manufacturer, therefore, we have no control over the accuracy of the tool. The error of the ruler used was +/-0.5 mm. This error is a random error, because we do not know how it affected our measurements. A way of reducing this error is by using more precise rulers with smaller increments.

3. Some of the toxins used to produce a response by the growth of the bacteria were extremely volatile, during heated incubation, the toxins may have evaporated. This is a random error because not all of the toxins were volatile and we cannot be certain of which ones did evaporate and which ones did not. This error may have caused the results of our experiment to be altered for if the toxin was not soaked into the agar, it would not be in the presence of the bacteria, and therefore, the bacteria would not react. This error can be reduced in the future by doing a trial to see how much toxin is lost through evaporation, and an extra volume of toxin can be added.

4. In this experiment, we had to assume that no other factors interfered with the growth of the bacteria except for our intended manipulated variables, but it is possible that through unknown sources, the bacteria were contaminated and showed reactions to unintended factors. This is a random error, for we do not know whether or not a plate containing bacteria has been contaminated. This error currently has no ways to reduce its impacts.

5. Lastly, a major assumption that we made previous to the start of this experiment was necessary in order for us to relate our experiment to the environment. If E.coli, P.florescens, and P.pseudoalcoligenes are present in the Bow River and the conditions under which the bacteria were cultivated is representative of the conditions of the Bow River, then we can make the conclusion that the results our experiment may also be symbolic of how river bacteria reacts to water pollution.

Conclusion

From the observations and analysis of our experiment, we are able to make three conclusions. These are stated below.

1. The relationship between the concentration of the river pollutants and the zone of inhibition of river bacteria growing in the presence of these contaminants is that the higher the concentration of the toxins, the larger the zone of inhibition within river bacteria.

2. The relationship between the effect of UV radiation on river pollutants and the zone of inhibition of river bacteria growing in the presence of these contaminants is that when UV treated, toxins cause the river bacteria to have a larger zone of clearance than when the toxins are not radiated with ultraviolet light.

3. The relationship between the different types of toxins and the zone of inhibition exhibited by river bacteria growing in the presence of the contaminants is that industrial solvents and motor oil causes the river bacteria to respond with larger zones of inhibition as opposed to antifreeze, fertilizer, and laundry detergent, which does not cause the river bacteria to exhibit a large zone of inhibition.

Our hypotheses one and two were correct, but hypothesis number three was incorrect. The data gathered for this experiment is very reliable, for our lines of best fit were with in the error range for all graphs, and these results can be used for future references.

So what do our results mean?

Well, we found out how different toxins, different concentrations, and UV radiation affects chemicals?ability to kill bacteria; and here are some implications our experiments can present:

First, there is something very important to clear up. Because of how it is used in everyday contexts, killing bacteria probably sounds like a good thing. But in this context, it really isn’t.

We chose the bacteria as our organisms to test on because they were convenient to grow and measure, and they were readily available at the time. But the three types of bacteria we used are all relatively strong; they can survive in polluted environments, and are found pretty much everywhere. They are more tolerant to pollution than many other organisms that live in or by the river, including:
-Mayfly nymphs
-Stonefly larvae
-Caddisfly nymphs
-Crayfish
-Gilled snails
-Bull trout
just to name a few examples that you could find in the Bow River.

Knowing that the bacteria can tolerate these pollutants better than the all the species just listed, when we find that a particular chemical or a concentration kills a large area of the bacteria we experimented on, it is reasonable to infer that under the same conditions, all of the species that are less tolerant to the pollutants would die too. This would devastate a river ecosystem; it would disrupt the food chains, leading to rapid depletion of dissolved oxygen, accumulation of toxic substances and organic matter, causing further pollution to the river, and ultimately resulting in a significant reduction in biological diversity in the ecosystem. If this condition persists, the river ecosystem would end up with a very small variety of species, consisting of only those that are extremely tolerant to pollution.

The investigation into UV radiation can also have a useful real-life application. From our experiments, we found that chemicals that are exposed to UV radiation generally caused a greater zone of clearance, basically meaning they killed more bacteria. This could apply to one of the major environmental issues we currently have—ozone depletion. Many chemicals used in industry and households, such as CFC’s, react with ozone molecules, causing rapid depletion of the ozone layer, which filters out harmful UV radiation from the Sun. Because of the depletion of the ozone layer however, the Earth and everything on it has more exposure to these Ultraviolet rays. Currently, the ozone hole is starting to increase again; this issue is a continuing environmental problem, and the effect of UV on chemicals could become even more of an environmental issue.

Materials

o 1 bunsen burner

o 6 large test tubes

o    15 small test tubes

o    1 paper holepuncher

o    2 sheets of Whatman’s #1 filter paper

o    42 LB-agar plates

o    stocks of 5 types of toxins:

o    10mL of industrial waste mixture (xylene, benzene, toluene)

o    10g of powdered fertilizer

o    10mL of antifreeze

o    10mL of 10W30motor oil

o    10mL of Tide laundry detergent

o    frozen stocks of bacteria:

o    1mL of Escherichia coli

o    1mL of Pseudomonas fluorescens

o    1mL of Pseudomonas pseudoalcaligenes

o    1 glass spreader

o    1 jar of ethanol

o    1 Gilson pipetman

o    1 box of disposable pipetman tips

o    50 Eppindorf tubes

o    1 vortex

o    1 straight plastic ruler

o    1 rotary disk incubator

o    1 sparker

o    5 petri dishes

We would like to thank many important people who helped us through the long process of designing and carrying out our experiment, a big "thank you" to:

Dr. T. Pike, Mr. D. Nickell, Mrs. D. Miller.

We would also like to thank Miss E. Mah, for helping us with the design and layout of our page.

And most importantly, we would like to thank Dr. R.J. Turner of the University of Calgary, who contributed the most to our learning through this incredible experience.

References

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