A2 Biology Coursework -Investigation into the effect of different concentrations of antibiotics on the growth of bacteria
Biology Coursework
Aim:
My aim is to investigate the effect of different concentrations of antibiotics on the growth of bacteria.
Hypothesis:
I predict that as the concentration of the antibiotics increases the bacteria growth decreases
Null Hypothesis
There is no link between the concentration of antibiotics and the effects it has on the growth of the bacteria
Theory
Antibiotics work in 4 ways, which are listed below:
. Cell Membrane Disruption - This involves making the cell fully permeable which results in substances moving into it causing it to burst and so kills the bacteria
2. Inhibiting Nucleic Acid Synthesis - This method doesn't kill the bacteria off but keeps the growth level static. The bacteria isn't able to replicate its DNA and so no binary fission occurs, causing the growth level to become static
3. Inhibiting Cell Wall Synthesis - This is where an antibiotic inhibits the enzyme required to form cross links within the cell wall and as a result the bacteria looses its structure and is unable to function properly .
4. Inhibiting mRNA Translation - This is where the Translation part of protein synthesis is inhibited by binding across the bacterial ribosome meaning proteins and enzymes the bacteria it requires isn't made and so dies.
Having said how antibiotics work above, it is logical to presume that the higher the concentration of antibiotics the more effective it will be in wiping out, and killing the bacteria. Because the more antibiotic molecules there is in the solution means there is a higher chance that these antibiotic molecules will come upon a bacterial cell and disrupt the cell and kill it .
So in conclusion it is logical to state that the higher the concentration of antibiotic the more effective the antibiotic will be at killing of the bacteria.
Dependant Variable
There are a number of different ways of measuring the effectiveness of the antibiotics but I will be using the technique of bioassays. This method provides a clear output which can easily be measured. The other 2 techniques, Dilution Plating and Heamocytometry could have been used but weren't favorable because they require human assessment as a person is required to count how much bacteria there is, making it prone to human error. Turbidimetery is another alternative but like Dilution plating and Heamocytometry it may produce errors as there are a lot of steps involved in this method.
In conclusion I will be using the method of bioassay to enable me to determine the effectiveness of the antibiotic and the dependant variable will be the diameter of the clear zone.
Independent Variable
For me to fully determine the effectiveness of the antibiotic there will have to be an independent variable. So I have decided that I am going to use a wide degree of concentrations of antibiotic and the gap between the concentrations needs to be kept to a minimum cause the optimum concentration may be left out if the gaps are too big. Also because the results will be analyzed by the spearman's rank correlation I will use 7 data values.
The concentrations I have chosen are listed in the table below:
Test tube
Volume of water (cm3)
Volume of antibiotic (cm3)
Percentage Concentration
(%)
0.0
5.0
00
2
0.5
4.5
90
3
.0
4.0
80
4
.5
3.5
70
5
2.0
3.0
60
6
2.5
2.5
50
7
3.0
2.0
40
8
3.5
.5
30
9
4.0
.0
20
0
4.5
0.5
0
1
5.0
0.0
0
Standardization
To be sure that only the antibiotic is responsible for the clear zone that will be present in the Petri dish all other variables must be kept constant. I will do this by:
Volume - The total volume of the bacteria, buffer and water solution will be 6cm3 .This will eliminate the volume becoming a variable which may give erroneous results.
Surface area of Agar - I will keep this constant by using Petri dishes all the same sizes. This will prevent surface area becoming a variable cause having a higher surface area may affect the rate at which the bacteria are killed.
Concentration - This is my dependant variable.
Surface area of Wells - I will keep this at 5mm, to prevent this becoming a variable as like with the surface area of agar, this may affect the rate at which the bacteria are killed
Temperature - This will be set to 25 degrees. This is to prevent temp affecting the rate at which the bacteria are grown.
PH Level - I will add buffer solution to prevent this factor becoming a variable.
Time - I will use a timer to time the incubation period. This is done to prevent time becoming a variable because more time may result in more killing or more growth of bacterial cells.
Size of equipment - I will try to keep the size of all the equipments at a same size.
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Temperature - This will be set to 25 degrees. This is to prevent temp affecting the rate at which the bacteria are grown.
PH Level - I will add buffer solution to prevent this factor becoming a variable.
Time - I will use a timer to time the incubation period. This is done to prevent time becoming a variable because more time may result in more killing or more growth of bacterial cells.
Size of equipment - I will try to keep the size of all the equipments at a same size.
Nutrients - I will try to keep the amount of nutrients the bacteria get the same so because this can become a variable as different amount of nutrients may result in different growth rates for the bacteria.
Control
For my control I will repeat the whole experiment but this time I will use distilled water instead of the antibiotic to prove whether or not the antibiotic is killing the bacteria and not any other factor.
Equipment
The equipment I will require is outlined below.
Equipment
Quantity
Size
Explanation
Test tube rack
To hold test tubes safely
Test tubes
1
2cmJ
Use to dilute the antibiotic
Petri Dish
1
Separate dish for each experiment to prevent contamination
Agar solution
1
To make agar for bacterial growth
Bunsen burner
Flame bottle necks to minimize contamination risks
Buffer solution
To maintain pH level
Syringe
20
5ml
For accurate measurement of solutions
Glass rod
To spread bacteria equally, to reduce any confounding variable.
Ethanol solution
5cm3
To burn glass rod to kill any unwanted bacteria
Incubator
To incubate bacterial solution at optimum temp
Permanent marker
To label test tubes and Petri-dish
Corks
1
To seal top of test tubes to prevent any airborne contamination
Cork borer
5mmJ
To dig wells
Timers
1
Min
To time incubation period
Ruler
mm
To measure clear zone diameter
Safety goggles
for safety
Safety
Safety is really important when carrying out experiments and so all the necessary precautions must be taken to increase safety. I will try and do this by:
* Wearing lab coats to prevent contamination from person to bacteria.
* Weaning plastic gloves to prevent contamination and stop harmful substances entering the skin surface.
* Wearing safety goggles to protect the eyes and to stop certain chemicals from entering the eyes.
* Keeping test tubes in a rack to prevent them from accidental breakage and to stop them from falling and spilling
* Bunsen burners will be kept in yellow flame when not in use.
Method
. Collect all the necessary equipment that is required
(Agar solution, Petri dishes, Bunsen burner, Scissors)
2. Turn the Bunsen burner to blue flame and cut the cello tape of a Petri dish so that the lid can be opened.
3. Pick up an agar solution, open the lid and flame the top of the agar
4. Open the lid of the Petri dish and pour in the agar solution into it rapidly.
5. The flame the top of the agar solution jar and replace lid.
6. Repeat the procedures from 1 to 5 for al the other Petri dishes.
7. Once this has been done, wait for the agar to cool down and set in the Petri dish.
8. Then collect the following equipment - Syringes, Petri dishes, bacterial solution, cello tape, ethanol, glass rod.
9. Get a syringe and transfer 1cm3 of bacterial solution to the first Petri dish and replace the lid
0. Get a glass rod and dip it in ethanol and then flame it and wait for it to cool down
1. Using the rod, spread the bacterial solution across the dish equally
2. Put the lid back on the Petri dish and fasten it with cello tape.
3. Repeat procedures 9 to 12 for the rest of the Petri dishes.
4. Then put these Petri dish in an incubator at a temp of 25 degrees
5. Collect the following - Test tubes, water, buffer, antibiotic solution and cork)
6. Label the test tubes from 1 to 9 and for each test tube, with a syringe transfer the required amount of water to the test tube and replace cork.
7. Then use a clean sterile syringe and transfer 1cm3 of buffer solution into these test tubes
8. And then use a separate syringe to transfer the stated amount of antibiotic volume in the table above into these test tubes.
9. Replace the cork and shake.
20. Label the Petri dishes with percentage concentration starting from highest to lowest.
21. Get the previous Petri dishes and cut off the cello tape used to fasten them together.
22. Remove the lid (not fully) and using a cork borer dig a 5mm well in the middle of the dish and replace the lid
23. Repeat procedures 21 to 22 for the other Petri dishes
24. Collect the following - diluted antibiotic solution, timers, Petri dishes
25. Use a clean syringe to transfer 5mm of the first dilution into the 5mm well of the Petri dish and get a timer and start it.
26. Replace the lid and refasten the lid with cello tape
27. Place this dish in an incubator at 25 degrees for 15 minutes.
28. Repeat procedures 25 to 27 for the other dishes.
29. After 15 minutes is over get a Petri dish and open the lid by cutting off the cello tape and measure the clear zone using a ruler.
30. Record the size in a table
31. Do this for all the rest of the Petri dishes.
32. Repeat this whole experiment twice
33. Do the experiment again but this time substitute the antibiotic with distilled water.
Precautions
The precautions I will be taking are:
* Clean the work surfaces with disinfectant to prevent contamination
* Do not eat or chew in the lab
* Wear the necessary safety gear
* Turn the Bunsen burner flame to yellow when not in use
* Use scissors to cut the cell o tape rather than mouth to prevent contamination
* When opening the lids of the gar solution, Petri dishes etc I will try to keep the opening to minimum to prevent airborne contamination
* Flame the bottle necks to prevent contamination by killing the bacteria
* Use a separate syringe for each use- prevent cross contamination
* I am going to dip the glass rod in ethanol to kill bacteria
* I am going to place corks on test-tubes to prevent airborne contamination
* Use buffer solution to keep pH level constant to prevent ph becoming a
variable
* Label all test tubes and Petri dishes- to prevent mixtures
* Cello tape the Petri dishes so that the lid is secure but to allow oxygen to enter
Table
Test tube
Percentage Concentration
(%)
Width of clear zone (mm)
Average Diameter (mm)
EX1
EX2
EX3
00
2
90
3
80
4
70
5
60
6
50
7
40
8
30
9
20
0
0
1
0
Graph
Implementation
Apparatus
. Agar Solution
2. Bacteria Culture
3. Antibiotic Solution
4. Distilled sterile Water
5. Petri Dish
6. Bunsen Burner
7. Syringe
8. Scissors
9. Glass Rod
Method
. Firstly I got a lab coat and wore it as a safety measure and also used disinfectant to disinfectant the table I was carrying my experiment out on to rid the area of any bacteria to limit the chances of any contamination
2. I then used hand wash to wash my hands
3. I obtained a heat proof mat and placed this on the table and then got a Bunsen burner and lit it and put it on a yellow flame
4. I got a test tube rack and gathered all the test tubes I required and with a marker pen I ladled these.
5. I then collected the bacterial solution and the nutrient solution and mixed these 2 together but after opening the lids I waved the necks of the bottles near the Bunsen burner to prevent any contamination
6. I then got 2 beakers and filled 1 beaker with antibiotic solution and the other with sterile water, I also gathered some syringes
7. I then used 2 syringes, one for the water and the other for the antibiotic solution to suck up the stated amount of antibiotic solution and then poured them into their labelled test tubes accordingly and also I used a cotton wool to stop anything from entering the test tubes.
(The volumes of water and antibiotics used to make up the different concentrations were as follows :)
Antibiotic Concentration
(%)
Volume of sterile water
(cm3)
Volume of antibiotic solution
(cm3)
0
5.0
0
0
4.5
0.5
20
4.0
.0
30
3.5
.5
40
3.0
2.0
50
2.5
2.5
60
2.0
3.0
70
.5
3.5
80
.0
4.0
90
0.5
4.5
00
0
5.0
8. I then got the bacterial solution and placed it in my hand and opened the lid and then flamed the neck off the bottle and then using a syringe I sucked up 5ml and took out the wool which was in place of all the test tubes and poured it into each test tube. As quickly as I could I put the cotton wool back in the test tube to avoid any contamination and also flamed the bottlenecks of the bacterial solution.
9. I then placed all the test tubes in a beaker and placed it into an incubator at a temperature of around 30 degrees and left it there for 24 hours.
0. After 24 hours had passed I collected the beaker containing the test tubes
1. I then obtained a colorimeter and 2 cuvettes to measure the light absorbency of each of the solution in all the test tubes.
2. For the blank solution I used the antibiotic solution and mixed 2ml of antibiotic solution together with the bacteria culture solution poured this into a cuvette
3. I placed this cuvette in the colorimeter with the clear side of the cuvette facing towards the light and calibrated it. I did this calibrating so that I only gained the value for the light absorbency and nothing else and then I took it out of the colorimeter.
4. I then gathered the first test tube which was the one which contained 100% antibiotic solution and poured a bit of this solution into a different cuvette and placed the lid back on it and took the value down and recorded it in a table.
5. I repeated this step for all the rest of the test tubes using 2 different cuvettes, one for the blank solution and the other for the solution which was in the test tubes (I washed the cuvette after each test tube to reduce contamination and distortion of light absorbency values) and recorded the values in a table.
6. I carried out this experiment 2 more times and then used the results to obtain an average set of results.
Safety and Precautions
The safety and precautions I took while I carried out this experiment were that:
* I washed my hands with a hand wash
* I wore a Lab coat
* I wiped the table with a disinfectant to reduce contamination by killing any bacteria on the surface
* I made sure that the apparatus I used was capable of carrying a certain amount of solution to reduce the risk of overflowing
* I flamed the necks of the bottles and test tubes to kill any bacteria
* I washed the cuvettes after I used a different test tubes solution to make sure the readings on the colorimeter were accurate
* I used separate syringes while doing the experiment to make sure that cross contamination was reduced.
* Cotton wools were placed inside the test tubes to prevent contamination
Control
For my control I used no antibiotic and just 5ml of sterile water together with 5ml of bacterial solution in the buffer and nutrients solution to prove that only the antibiotic had an affect on the bacteria and nothing else.
Analysis
Results Table
Concentration of solution
Colorimeter Readings Absorbance mode (Arbitrary Units)
Experiment 1
Experiment 2
Experiment 3
Average
00.00%
0.02
0
0
0.01
90.00%
0.02
0
0
0.01
80.00%
0.02
0.02
0.02
0.02
70.00%
0.05
0.06
0.06
0.06
60.00%
0.05
0.05
0.07
0.06
50.00%
0.1
0.08
0.05
0.05
40.00%
0.15
0.1
0.1
0.06
30.00%
0.1
0.15
0.1
0.06
20.00%
0.2
0.27
0.1
0.1
0.00%
0.35
0.25
0.23
0.3
0.00%
0.42
0.27
0.25
0.3
Spearman's Rank Calculations: Averages
% Concentration of Antibiotic
Light Absorbency
Rank % Concentration of Antibiotic
Rank Light Absorbency
Difference (D)
Difference2 (D2)
00.00%
0.01
1
.5
9.5
90.25
90.00%
0.01
0
.5
8.5
72.25
80.00%
0.02
9
3
6
36
70.00%
0.06
8
6.5
.5
2.25
60.00%
0.06
7
6.5
0.5
0.25
50.00%
0.05
6
4
2
4
40.00%
0.06
5
6.5
-1.5
2.25
30.00%
0.06
4
6.5
-2.5
6.25
20.00%
0.1
3
9
-6
36
0.00%
0.3
2
0.5
-8.5
72.25
0.00%
0.3
0.5
-9.5
90.25
412
Rs = ((6*412)/(11*(121-1)) = - 0.8727
This value suggests a negative correlation meaning that as the antibiotic concentrations increase the more bacteria that are killed.
Table
My control which didn't contain any antibiotic solution what so ever had the highest absorbance value in all 3 experiments suggesting and proving the fact those bacteria has grown without the presence of antibiotic.
Graph
(On a separate Paper)
Graph Analysis
In graph 1, which contains the graphs for experiments 1, 2 and 3 I can see that the light absorbency tends to decrease as the concentrations increase from 0% to 100% with a few anomalies in concentrations 20% in graph 1 and 90% in graph 3. This means that more bacteria are being killed off as the antibiotic concentration increases. This also suggests that I there may have been some contamination due to the fact that there is a few anomalies in the graphs. This may be because I wasn't carefully rinsing out the cuvettes after the solution from the next test tubes was to be poured into it and also by not performing aseptically.
From Graph 2 which contains the average of all experiments I can see that there again is trend, because as the concentration of the antibiotic increases the lower the light absorbency reading is getting suggesting the bacteria are being killed and that the antibiotic is effective at that. However there are a few anomalies which are at the concentration levels of 50% 60% and 70%, again meaning that I may not have been performing in an aseptic manour which may have brought about these anomalous results.
Evaluation
Overall the experiment went well apart from the few anomalies which were found that may have been due to poor aseptic techniques, it does prove my hypothesis correct that as the concentration of antibiotics increases the bacteria level falls.
During this experiment there many limitations, which are:
* When I was using the cuvettes to measure the light absorbency using the colorimeter I had to use the same cuvettes after a solution from each test tube was measured. The only possible solution to this was to wash the cuvette after each sample had been measured but still this could have lead to contamination which may have given those anomalous results present in my graph.
* Also the limitations with the syringes was that they had air bubbles within them and I tried to get rid of them by flicking them but even then some still remained. If these bubbles were in the bacterial solution, it would have decreased the amount of bacteria present in it resulting in a lower light absorbency reading and vice versa with the antibiotics. (This would result in a lower amount of antibiotic meaning a reduced amount of bacteria may have been killed because of this resulting in a higher absorbency value.)
* Also the cotton wools placed in the test tubes to prevent oxygen from entering was a slightly different size and may not have totally prevented the air entering into the tube meaning that if it was bigger the would have been a higher chance that less oxygen may have entered, decreasing the bacteria's ability to grow much faster. De to the wool being smaller in size, more oxygen may have entered meaning that more bacteria were growing faster which means that the absorbency reading will be higher, this may be the cause of my anomalous results.
* The amount of each solution had to be the same cause if they weren't then this would have affected the growth and death of bacteria because if more antibiotic solution was present then more bacteria would have died resulting in a decrease in light absorbency and vice versa with the bacterial solution.
* Also the light absorbency of all the test tubes differed as they could not all be measured in one go meaning that in some test tubes some bacteria had more time to grow, giving an invalid absorbency reading.
The method I used could have been improved since I used turbidamitry which gives total cell count, both living and dead isn't really accurate or representative. I could have improved this by using bioassays which is a method that will only count the living cells which may have given a better representation. I could also improve the experiment next time by making sure that the size of the cotton wool was decent sized so that it has a higher chance of preventing oxygen entering into the test tube allowing the bacteria to grow more faster. Another way to improve the experiment is to make sure to properly rinse out the cuvettes after each sample from each test tube was measured in the colorimeter to prevent contamination and an invalid light absorbency reading.
Kamrul Hassan
