The Antibiotics:
The Penicillin that we will be using is Penicillin G, which only affects Gram-positive bacteria. Penicillin refers to a group of antibiotics called β lactam. These are used in the treatment of bacterial infections usually caused by Gram-positive bacteria, although, it is possible for penicillin antibiotics to treat Gram-negative bacteria when developed, such as ampicillin which can treat both Gram-positive bacteria and Gram-negative. Penicillin works by forming peptidoglycan cross links in the bacterial wall, which causes the cell wall to weaken when it tries to divide, causing cytolysis.
Streptomycin is an antibiotic that works well on both Gram-negative bacteria and Gram-positive, and was the first remedy discovered to cure tuberculosis. It works by stopping bacterial growth by damaging the cell membrane and inhibiting protein synthesis(C).
Prediction:
As Penicillin G is only effective against Gram-positive bacteria(D), and E. coli is Gram-negative, due to this I believe that it will have very little, if no effect at all on the E. coli. However, the streptomycin is effective against both Gram-positive and Gram-negative bacteria, which is why I predict that the streptomycin will have an affect on the bacteria, by killing off the bacteria in close proximity to the streptomycin.
Apparatus:
- Bunsen burner
- Heat mat
- Sterile Petri dishes
- Wire loop
- Bottle containing E. coli on the surface of agar slope
- Bottle containing molten agar
- Filter paper
- Penicillin G
- Streptomycin
Bunsen burner
This will be used to flame the rims of the bottles containing the E. coli culture, this will kill anything that happens to be on the rim of the bottle, preventing anything from accidentally falling into the culture when the lid is off. It will also be used to flame the wire loop so that it is sterile and does not let any unwanted pathogens into the culture.
Heat mat
This will prevent the work-surface from being burnt by the Bunsen burner.
Sterile Petri dishes
These will be used to grow and incubate the cultures within, and must be sterile to ensure that the experiment is fair, and also to prevent unwanted pathogens from growing inside the cultures
Wire loop
This will be heated until it is red, to sterilise, and then used to withdraw the bacteria in the agar, from the original culture.
Bottle containing E. coli on the surface of agar slope
From this I will scrape a sample of the bacteria to be placed in a new bottle containing molten agar, producing a new culture of E. coli.
Method:
There are a two main methods used to investigate the effects of antibiotics on bacterial growth, these are the pour plate method and the streak plate method. The pour plate method is more precise than the streak plate method and is done by placing a couple samples of an antibiotic in spots on the agar. The streak plate method would be done by spreading the antibiotic back and forth over the agar, using a wire loop. However, this method is almost only used for the isolation of particular strains of bacteria, and would therefore be unadvisable to use the streak plate method in an experiment of this nature. It would also be harder to compare and measure the outcome of the streak plate method due to the setup being less precise, and the bacteria not being evenly distributed over the agar.
Pour plates are usually the method of choice for counting the number of colony-forming bacteria in fluids, as they give a very even distribution of the bacteria being used; this allows the measurement of the area killed off at the end of the incubation to be measured more easily and accurately. Their preparation is, however, time consuming. Other disadvantages are the reduced growth rate of obligate aerobes in the depth of the agar and the danger of killing heat sensitive organisms with the hot agar. When preparing a pour plate, you have to mix all the bacteria into molten agar, which causes there to be bacteria at all depths of the agar, when it would be more desirable for the bacteria to be solely on the surface. This could be sorted out by pouring the molten agar containing the bacteria to a depth of just 1 or 2mm in the Petri dish. These disadvantages cannot be raised against the streak plate method, as it is easier to perform; only it gives less reliable results. But due to the nature of the preparation for it, it would be a much harder method to measure the final outcome.
For the above reasons I have decided to perform the pour plate method in my investigation, as I want to obtain the most accurate results possible.
Key factors to take into account
- Temperature to incubate at.
- Period of time left to leave for and frequency of times to view it.
- Number of times to repeat.
- Control disc.
- Number of impregnated discs to put in each Petri dish.
- Concentration of antibiotic.
- Method of measurement.
- Sterile techniques
Temperature to incubate at:
It is important to incubate all the Petri dishes at the same temperature, so that the experiment is a fair one, and also to choose an appropriate temperature to use. Although temperatures around body levels seem to give the maximum possible growth, you should never incubate microbiological cultures above 30°C as this would be approaching the temperature that is ideal for human pathogens to grow, which is 37°C. This can be avoided if the temperature is lowered so that it is less likely for this to occur. I will therefore be incubating the cultures at a temperature of 30°C. This is therefore warm enough to ensure that the antibiotic in the culture continues to have an affect on the bacteria, and not too warm that unwanted pathogens start to grow. (E)
Period of time left to leave for and frequency of times to view it:
To determine the length of time that I will leave the cultures to incubate, I will perform a preliminary experiment. I will observe the culture each day, and from that be able to decide the optimum length of time to leave it for. From this preliminary I will also be able to tell if I need to look at the cultures more or less often than once a day, however, I presume that once a day will be sufficient, and until the preliminary is performed, will also presume that 5 days is a sufficient time period. I will also ensure that I view the culture at the same time each day, so that an equal period of time is left in each gap of 24 hours.
Number of times to repeat the experiment:
It will not be necessary to repeat the experiment more than twice. This is because I will be able to determine the effects after just one experiment, as I am only trying to find out which is more effective at killing E. coli, and the second is merely to check that nothing went wrong.
Control Disc:
The control disc is a disc of filter paper that has been soaked in sterile water, and then allowed to dry. The control disc will have no affect on the bacteria, and will therefore prove that it is not just the filter paper that is killing the bacteria, but the actual antibiotics which it is soaked in.
Number of impregnated discs to put in each Petri dish:
I will put one disc of filter paper soaked in Penicillin G, and one soaked in Streptomycin in each of the Petri dishes. They will be spaced at equal distances, not only from each other, but also from a control disc, to ensure that there is plenty of space around each disc of filter paper, preventing the antibiotics from interfering with each other.
Concentration of antibiotic:
I have decided that I will make up penicillin G and streptomycin to equal concentrations of 50μg/ml. I will do this by obtaining the antibiotics with an equal or higher concentration to 50μg/ml. If I manage to obtain the antibiotic at the concentration required, I will not need to do anything. However, if it is stronger I will have to perform methods of dilution using distilled water.
Method of measurement:
I will be measuring the outcome of the experiment using a ruler. I will be measuring the diameter of the space killed off by the antibiotic; this is presuming that the area killed is a perfect circle. If this is not the case, and the shape killed by the antibiotic is a more oval shape, I will measure the longest and shortest diameters of the area, and then find the average of the two.
Lab safety(F)
I must make sure that all equipment and techniques are sterile throughout the procedure to reduce the chance of any unwanted micro-organisms from spreading into the work.
- This can be done by ensuring that I am well on the day of the experiment, and covering any cuts or grazes with a clean and waterproof plaster.
- It is important to use a lab coat when dealing with bacteria, as they can be hazardous to your health, and it is important not to get them on yourself or your clothing, to reduce the possibility of contaminating the cultures. The coats must also be cleaned after they have been used.
- I will wash my hands before and after handling any of the bacteria with anti-bacterial soap.
- I will be taping the Petri dishes closed after I have placed the antibiotic tabs inside, and then label what they are. This will prevent the dish from bursting open if it is dropped, and enable it to be identified quicker if it happens to break, so that it can be cleaned up effectively and quickly.
- It is essential that goggles are worn throughout the duration of the experiment to protect eyes from the bacteria, and when handling the Bunsen burner.
- The worktop must be cleaned with disinfectant before and after working, with 10% sodium chlorate (I) or 1% virkon cleaners.
Procedure:
- Get a closed bottle containing 15cm³ of sterile nutrient agar, then melt it by placing the bottle in a bath of water at 100°C
- Remover the bottle and leave to cool until it is between 42°C and 50°C, remaining in liquid form.
- The following step should be done under sterile conditions to avoid contamination, it should also be done as fast as possible to prevent any unwanted pathogens from entering the cultures.
- Sterilise a wire loop by flaming it until it goes red.
- Remove the lid from the bottle containing the E. coli, then flame the rim of the bottle to sterilise.
- Then remove the lid from the bottle containing the liquid, and flame the mouth. Then with the sterile wire loop, transfer one scoop of the E. coli culture into the liquid agar, dipping the loop in fully, so as to properly mix it in.
- Then immediately pour the liquid agar containing the E. coli into a warm (to prevent water condensing on the lid as I pour), sterile Petri dish by only lifting the lid just enough to pour it in, therefore preventing contamination.
- Then replace the lid, and make sure that the culture is evenly distributed and flat throughout.
- Once the agar has hardened, place 2 discs of filter paper on the surface, one which has been soaked in penicillin G and dried, the other, soaked in streptomycin and allowed to dry. The two discs should then be transferred using sterile forceps for each.
- Place the control disc at equal distances from the other two discs, on the surface of the agar using sterile forceps.
- Then secure the lid of the Petri dish with sellotape, and then turn it upside down to prevent condensation forming on the lid whilst it is being incubated.
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Leave the culture in the incubator for 5 days, at a temperature of 30°C.(G)
Below I have included the table that I will use to show my results:
Bibliography
Image 1 - http://fig.cox.miami.edu/Faculty/Dana/monera.html.
Image 2 - taken from Wikipedia - http://en.wikipedia.org/wiki/Bacterium.
Image 3 – Pg 84 in “Essential A2 Biology” by Glenn and Susan Toole.
Information (A) – Pg 216 in “Biology 1” by Mary Jones, Richard Fosberry and Dennis Taylor
Information (B) – Pg 64 in “Biology, Approved Specifications” by Oxford Cambridge and RSA Examinations
Information (C) and (D) – Pg 116 in “Microbiology and Biotechnology” by Lowrie and Wells
Information (E) and (F) – Pg 24 in “Biology 2” by Mary Jones, Richard Fosberry and Dennis Taylor
Information (G) – Pg 166 in “Biology: A functional approach” by Roberts and King
