There are two types of antibiotics: bacteriacidal antibiotics which destroy bacteria and bacteriostatic antibiotics which has an indirect effect. A bacteriostatic antibiotic prevents the multiplication of pathogens. It then allows host immune system to destroy the pathogen.
Antibiotics work in several ways to disrupt the growth of bacteria. These includes: Inhibiting of cell wall synthesis in bacteria, bacteria cell lyses caused by disruption of cell membrane and Inhibition of nucleic acid synthesis, replication and translation
Hypothesis
The antibiotic will inhibit the growth of bacteria, as the concentration of sodium chloride increases, the inhibiting ability of the antibiotics decreases as the growth of bacteria around the disc will increase. At low concentration of sodium chloride the inhibiting ability of the antibiotics will increase. As the inhibiting ability of the antibiotics increase, there will be a decrease in bacteria growth around the disc.
Variables
Dependent variable
This is the variable being measured. I.e. the diameter of area of inhibition which is the measure of the effect of the action of antibiotics on the growth of bacteria.
Independent variables
Thus is the variable that will be changing during the course of this investigation. To maintain accuracy it is important to change only one variable at a time. The independent variable in this investigation will be sodium chloride.
Controlled variables
These variables will be kept constant through out the investigation. These are Temperature, incubation time and volume of broth. Temperature will affect the rate of growth the bacteria. Warm and humid condition favours increase in bacteria growth. Hence it is important to keep the incubation temperature constant to prevent any inaccuracy which might arise from temperature differences.
Incubation time: Time will affect the extent of bacteria growth. This is why it is important to keep incubation time constant. The diameter of area of inhibition will be measured at the same time interval for each agar plate after incubation.
Volume of bacteria broth: varying volumes of nutrient will affect the rate of growth of bacteria. The volume of broth will be kept constant.
Preliminary
I carried out a preliminary trial to find the most effective antibiotics and compare effectiveness of selected antibiotics which included. A clear zone around the disc impregnated with various antibiotics provides a method of comparing the effectiveness of different antibiotics. The wider the zone that remains clear due to no bacterial growth, the more effective the antibiotics is at preventing growth.
The antibiotics used for the trial were streptomycin, tetracycline and penicillin. They were placed on inoculated medium of agar and the plates were incubated at 25˚C.
Evaluation of preliminary
Penicillin proved to be the most effective in preventing bacterial growth. There were no growth inhibition in zones surrounded by streptomycin and tetracycline in all the 5 agar plates tested. This showed that out of the three antibiotics tested, penicillin was the most effective. Therefore I will be using penicillin for my investigation.
Risk assessment
Table 1
Table 2
Penicillin antibiotic disc
Tripod
Maker pen for labelling dishes.
10 Sterilised Mc Cartney bottles containing 15cm3 of nutrient agar in an agar bottle
Masking tape.
Access to non-pathogenic culture of Escherichia coli
Access to autoclave and autoclave bags or pressure cooker
250cm3 beaker
Sterile Petri dish
Method
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Make standard solution of varying concentration, see appendix A.
- Prepare agar plates with the sodium chloride solution and nutrient broth seeded with bacteria
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follow all safety precautions, see risk assessment
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Collect the bottle with the 15cm3 of sterile agar nutrients. Unscrew the bottle, and then place it in a hot water bath of about 50ºC to melt.
- Leave the agar in the water bath until you are ready to use them.
-
Dissolve 28.86g of NaCl in 250cm3 of distilled water in a volumetric flask. Make solution of concentration as shown in table 1, see appendix 1
- Place each of the standard dilution formed into separate sterile McCartney bottles.
- Place the NaCl McCartney bottles and its content in the 50ºC water until you are ready to use them.
- with your working area with ethanol, then set up a yellow Bunsen flame to keep contaminants out
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Place sodium chloride solution from each McCartney bottle into a designated Petri dish using aseptic technique. Lift lid only enough to allow entry of pipette. Pour in the 15cm3 of the agar nutrients into the plate using a sterilised volumetric cylinder. Use an inoculating loop to evenly spread the e.coli over the plate Allow the agar to slightly set before placing the penicillin antibiotic disk.
- Using sterile forceps, place a penicillin antibacterial disc onto the surface of the inoculated nutrient agar. Ensure that the antibiotic discs are in contact with the agar surface before closing and sealing the dish.
- Close the Petri dish and tape it. It is important that the Petri dish is not taped all round to prevent anaerobic growth of bacteria.
- Using maker pen, label the underside of the plates with their concentration, control, test and date to distinguish between the different concentrations.
- Incubate the plates for 24 hours at 25ºC
- Ensure that all controlled variables are kept constant throughout this investigation
Repeat step 8 -14 for all the concentrations in the table 3 below
For each concentration of sodium chloride solution make a control plate and three test plates.
NB: A control plate in this investigation is an agar plate with bacteria, sodium chloride solution and no penicillin disc. For each agar plate of sodium chloride concentration, prepare 1 control plate and 3 test plates.
Concentration of sodium chloride in agar solution is calculated as follows:
Concentration of sodium chloride in mixture = concentration of NaCl × volume of KI
Total volume
Stock solution of NaCl = 3.00 moldm-3
Total volume =10cm3
For each experimental plate A
Concentration = 3 × (10/10)= 3.00moldm-3
3×(9/10)= 2.70 moldm-3
Table 3
Reference:
[1]
[2][Salters Nuffield advanced biology]
[3]Essential A2 biology for OCR, nelson thornes; Glenn, Susan toole
[Preliminary Method] http://secure.sciencecompany.com/Bacteria-Growing-Experiments-in-Petri-Dishes-W54C659.aspx
[4]http://www.microbiologyonline.org.uk/ecoli.htm
Fig 1:
Bibliography: salter-nufield advanced biology A2, topic 6 infection, immunity and forensics
Implementation
Table 4
Analysis
Graph showing the relationship between sodium chloride concentration in agar and the diameter of zone of growth inhibition
The graph of the diameter of zone of growth inhibition against sodium chloride concentration shows a slightly strong negative correlation. This relationship can be interpreted as: an increase in sodium chloride concentration decreases the effectiveness of the antibiotics penicillin at preventing the growth of E.coli. This means that salt concentration has a negative effect on the action of antibiotics. Take agar plate A with concentration of 3moldm-3 had an average diameter of 1.96cm which is relatively smaller than an average diameter of 2.70cm with sodium concentration of 0.90moldm-3 in agar plate H. control agar plate which had no antibiotics did not exhibit any signs of growth inhibition.
Spearman’s ranks correlation coefficient is a good method used to identify the relationship between to variables. I have decided to use it to analyse my data to see if the relationship between sodium chloride concentration and area of growth inhibition shown on the graph above will be supported with the results from the calculation of spearman’s rank correlation coefficient.
Spearman’s rank correlation coefficient
Rs = 1 - 6(∑D2 )
(n2 – 1)
∑D2 = 322
n =10
rs = 1 - 6(322 )
10(102 – 1)
rs = 1 – 1.95151515152
= - 0.95151515152
= -0.952 (3s.f)
From the calculation rs value is negative and close to -1 . This suggests that the concentration of sodium chloride in agar and diameter of zone of growth inhibition are negatively correlated.
It is possible to get nonsense correlations, correlation between sodium chloride concentration and diameter of zone of growth inhibition does not necessarily mean that the two variables are linked. To close an existing gap between cause and causation I have decided to put the results of my investigation to a null hypothesis test.
Null hypothesis
Increasing NaCl concentration will not have an effect on rate of growth of bacteria at 5% significance level. There is no significant difference between the diameter of zone of growth inhibition and the concentration of sodium chloride in agar.
To accept or reject the null hypothesis I have decided to compare the calculated rs value with the critical values of spearman’s rank correlation coefficient at 5%(p<0.05) significance level .From the table the critical value at 5% significance with 10 data pairs is 0.648. The calculated value exceeds this critical value. Therefore I reject the null hypothesis.
Systematic error - these are errors associated with equipment, to achieve maintain systematic error in this investigation I used the same weighing balance, burette, thermometer, volumetric flask and venier callipers for measurements.
To increase reliability of data for each concentration, there were three agar plates with the same conditions.
Percentage errors
graph of percentage error
Analysis and conclusion
The trend line passes through most data points with one exception. The agar plate C with sodium concentration of 2.10moldm-3 appears to be an anomalous data. This anomalous result has occurred possibly due to lack of homogeneity of agar solution. It could also be that the technicians who were incubating the agar plated had tampered with the particular set of plates which gave rise to the anomalous data. Therefore I’ve decided not include it in the graph of error bars and any further calculation. The graph of percentage errors shows a strong positive correlation with the trend line passing through every single error bar. This shows that the results of this investigation are accurate and any inaccuracies which have occurred are as a result of the difficulty in controlling controlled variables. To analyse the relationship between sodium concentration and zone of growth inhibition of e.coli I have decided to borrow the knowledge in the journal, American journal of respiratory cell and molecular biology, for a detailed analysis. In conclusion there is a relationship between the sodium chloride concentration in agar and the diameter of Zone of bacteria growth inhibition.
Fig2 cell wall of bacteria and formation of antimicrobial factor
Growth inhibition does not require specific ions but its worth mentioning that sodium ion a divalent (+2 charge) cation is a greater inhibitor than monovalent (+1 charge) cations. The interaction of sodium cations and the electronegative chlorine anions with polar groups in penicillin possibly inhibits the antimicrobial activity in the antibiotic. Penicillin works by [6]“destroying the cell wall of the micro organism. It does this by inactivating an enzyme necessary for the cross linking of bacterial cell walls. The enzyme accepts the penicillin as a substrate” The interaction of sodium cations and chlorine anions with polar groups possibly disrupts the shape of the penicillin molecule affecting the shape of the substrate preventing the binding of penicillin-binding-proteins(antimicrobial transcription factor in fig 2) onto the active site of the enzyme necessary for the cross-liking of bacterial cell way. If the synthesis of cell wall of e.coli is not inhibited then growth of e.coli will not be inhibited too. This makes penicillin ineffective.
When This principle is applied to cystic fibrosis, I can infer that the reduction in antimicrobial activity in cystic fibrosis patients is due to interaction of sodium cations and the electronegative chlorine anions with polar groups in lyzomes and other natural antimicrobial proteins in cystic fibrosis patients makes them prone to infection.
Overall this investigation have been successful at proving that as sodium chloride concentration increases the diameter of zone of growth inhibition decreases at 5% significance level. From the compelling results of this investigation and with reference to the research studies published in the American journal of respiratory and molecular biology I conclude that large salt concentration weakens antimicrobial activity in the airways of CF patients.
I believe there are limitations in method and the variety of antibiotics available of the investigation. I suggest that further experiments should be carried to with varied range of concentration to see if the trend shown in my graph is continuous. I suggest that other antibiotics which have similar properties to penicillin should be investigated and other salts should be investigated. Further research in this area could result in the possibility of treating cystic fibrosis with salt-insensitive antimicrobial factors and can be extended to other areas to treat other upper respiratory tract infections.
Reference:
[5] American journal of respiratory and molecular biology, authors of article: M. Travis, Barbara-Ann D. Conway, Joseph Zabner, Jeffrey J. Smith, Norma N. Anderson, Pradeep K. Singh, E. Peter Greenberg, and Michael J. Welsh
Activity of Abundant Antimicrobials of the Human Airway
Am. J. Respir. Cell Mol. Biol. 1999; 20: 872-879 : I believe this is a reliable source of information as it a reputable journal which shows off the research studies carried out by scientists in the field of respiratory and molecular biology.
[6]
[fig2]http://www.3dscience.com/img/Products/Images/clip_art/Inhibition%20of%20Penicillin-Binding%20Proteins_web.jpg
Penicillin molecule: http://www.bio.miami.edu/~cmallery/255/255enz/penicillin.gif