Investigating how different concentrations of a antibiotic effects the growth of a bacterium.

Population

Of bacteria

        1   2         3     4

                        Time (hours)

1. The first of these is known as the Lag phase, and at this phase the numbers of bacteria remain fairly constant as the bacteria adjust the new conditions. During this time the bacteria synthesise enzymes needed to use nutrients in the broth available/
2. The second of these phases is known as the Log Phase, in this phase the bacteria reproduce exponentially. I.e. population continually doubles. Nutrients are plentiful and waste products have not built to the harmful levels.
3. Thirdly, titled Stationary Phase, where the nutrients begin to run out, the rate of growth begins to slow down due to ever increasing rate of competition. Accumulation of waste products inhibits the metabolism. Population size remains constant.
4. The fourth and final phase, known as Decline Phase, or “death phase”. In this phase the quantity of the nutrients decrease. The bacteria can longer reproduce and individuals die.   

My quantitative prediction at this point is that as the concentration of the antibiotic increases, for example, from 0 to 1%, the area that the bacteria has not grown in will increase, for example, from 1mm to 5mm.

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                    Area affected    

                     by antibiotic

                                               Concentration of antibiotic

Plan-For the practical there will be a number of aspects needed to be followed so that the experiment can be a fair and safe experiment.

The plan/method of the experiment will be as follows:

1. Collect the apparatus that is needed for the experiment. The apparatus needed is:

• A culture of bacterium, Micrococcus luteus
• Molten agar at about 50oC
• 12 Petri dishes
• A 1% solution of antibiotic
• 3 filter papers
• Disinfectant (Virkon) and paper towels
• Marker pen
• Hole punch
• 6 Test tubes
• Test tube rack
• Distilled water
• 1 mm syringes
• 5 cm3 syringes
• Spreader
• Ethanol
• Callipers

2. Before any of the apparatus is to be used the working surface should be cleaned using the disinfectant provided and paper towels. Along with this hands of those involved should be cleaned to reduce the chances of foreign bacteria entered the agar gel. It would also be important to make sure that petri dishes have been cleaned.
3. Before the actual start of the practical the 6 petri dishes should be collected and with the marker that is available should be labelled. These should have the initials of those carrying out the experiment, and then on 3 of the dishes they should be labelled experiment 1-5 and then the 6th labelled control, in the experiment this will not be used but instead rather as a test to make that the dishes have been cleaned and treated correctly before the experiment.  
4. The next point would be to pour the molten agar into the petri dishes that are available. When using the molten agar, eye protection should be worn at all times and care should be taken, as there could be a risk of scolding. When carrying out this task with the agar it should be made sure that agar is poured on quickly and the lid then placed back on, with the care not to breathe on the agar nutrients. When the agar gel has been poured into the petri dishes it would be safe to move them away from the area so that gel has time to solidify. This action should be repeated for the 5 different agar plates.
5. While waiting for the agar gel to solidify it may then be a good time to collect the filter paper available along with the hole punch that will be used to create small paper discs. Using the filter paper the person should try to “punch” 5 similar circles for each of the five experiments. These should be collected using the method of hole punch but removing the plastic protector. 
6. After this it would then be time to collect the 5 test tubes, with the test tube racks, distilled water and the antibiotic. Making sure to label the test tubes from 1 to 5. Setting up the rack and test tubes correctly. Using the 5cm3 syringe in total 9 cm3 should be taken and then placed into Test Tube 1, next again using the same syringe 1cm3 of the antibiotic should be taken and placed in Test Tube 2, and this needs to be diluted with 9cm3 of water. 1cm3 of the solution should be removed via the 5cm3 syringe and placed in the Test Tube 3 that would also be diluted. This should be repeated onto each of the test tubes.    
7. Once the test tubes and the serial dilution have been completed the paper circles should be collected and then 1 each should be placed in the test tube to absorb the antibiotic solution. This should be done by collecting the remaining petri dishes that are available and placing that on the desk, without the bottoms attached, the different concentrations should then be placed within these and then add the small paper discs.
8. While this is being left the bacterium should then be spread across the five different petri dishes containing the agar gel. This should be done be using the spreader and care should be taken not to breathe on the agar and to spread the bacteria evenly across the whole agar sample. Repeating this on each of the petri dishes. Once this has been done it will then be time to collect the small circles that have been placed within the test tubes and placed onto agar gel within the petri dishes. It is important during the spreading of the bacteria that the ethanol is placed and then ignited before use to ensure sterilisation, the bottle contained the Micrococcus luteus should when opened be run past a flame. Repeating this for each of the different agar plates.
9. On completing this the discs which should have became saturated at this point should then be removed from the different petri dish lids, individually, and then placed in the different agar plates and noted which concentration these are, aim to leave some distance between each. One from each of the antibiotic concentrations should place in each agar plate.
10. Once all the small discs have been placed into the dishes, it would be important to seal the lids together with the use of cellotape to ensure that they are not opened and the results destroyed.
11. After the designated time is over, the different plates should be collected and from these the areas surrounding the different discs should be measured with the use of callipers.
12. Once the results have been collected they should then be placed in a table. Along with the inputting of the results into he table they could also be used to produce a graph. The table to be used can be seen underneath the heading of Presentation of Results.

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Variables-

Risk Assessment/Safety- When implementing the practical there are a number of points or issues that would need to be taken into consideration, which are part of the Risk and Safety Assessment.

In assessing the different risks, I will note them and then begin assessment and the preventions that can be taken. The different risks include:

• Aseptic technique
• Ethanol
• Risk of person
• High temperature

Aseptic technique- Aseptic means "without micro organisms." Aseptic technique refers to practices that help reduce the risk of post procedure infections in clients by decreasing the likelihood that microorganisms will enter the body during clinical procedures. Some of these practices are also designed to reduce service providers' risk of exposure to potentially infectious blood and tissue during clinical procedures. So when assessing the risk of this during the practical it can be seen as a major risk if the right precautions or preventions are taken. Different examples of the techniques that could be use include:

• Remove or kill microorganisms from hands and objects.
• Employ sterile instruments and other items.
• Reduce clients' risk of exposure to microorganisms that cannot be removed.

When looking at the size of the risk it could be quite large. As there are three different groups that could be at risk, these include, students working with the apparatus, staff cleaning or using and community or people that may come into contact with the room or yourself.

Ethanol- Ethanol is a monohybrid primary alcohol. It melts at -117.3°C and boils at 78.5°C. It is miscible (i.e., mixes without separation) with water in all proportions and is separated from water only with difficulty; ethanol that is completely free of water is called absolute ethanol. Ethanol burns in air with a blue flame, forming carbon dioxide and water. It reacts with active metals to form the metal ethoxide and hydrogen, e.g., with sodium it forms sodium ethoxide. It reacts with certain acids to form esters, e.g., with acetic acid it forms ethyl acetate. It can be oxidized to form acetic acid and acetaldehyde. It can be dehydrated to form diethyl ether or, at higher temperatures, ethylene.

When reading the properties of Ethanol it can be seen that it is a very high risk when using as it may react at high temperatures and a number of metals. Therefore there must be a number of precautions taken. These include:

• The precaution of the high temperature from the Bunsen and ethanol, so they always been kept away from each other
• Not spread in excess

Risk of person(s)- as the number of different issues raised so have been concerned with the different apparatus and the techniques being used there are also risks concerning the persons alone. The general precautions that should be taken to reduce the chance of risk are:

• Virkon should be used before and after to reduce the spreading bacteria on surrounding area.
• Ties and hair should be either tied or tucked within shirt to reduce risks such as fire and bacteria.
• Bags should be removed

Accuracy of Results- By referring to the results that have been collected some may same to not be correct, but there are a number of ways during the practical where results may have been damaged. The different errors that may have occurred include:

• Sterilisation of surrounding area and equipment
• Spreading of agar, either the spreading technique was not correct or during this process foreign bodies were introduced
• Not sealed correctly
• Discs unsaturated with antibiotics fairly

Presentation of Results-

After completing the practical the results that were gathered can be seen in the tables below. An important point to consider on the results is that produced on the Control plate. The details of this there were no bacteria grown after leaving these for three days, therefore the sterilisation technique had been correct and so that results gathered can be deemed reliable. 

After the collection of the results and the presentation of these, which can be seen in table above. I will also go through the process of a t-test so that the different results can be tested to make sure that they are correct difference rather then chance or mistake.

In the t-test the two different results that will be gathered to see significance is the two concentrations 0.1 and 0.0001, so that it can be shown that these have no significance.

The formula for the T-test is:

T=(average 0f A-average of B)

SDA/5 + SDB/5

First of all we must work out the standard deviation for this two concentrations.

Concentration 0.1%

The average length at this concentration was 1.718 mm.

Concentration 0.0001%

The average length at this concentration is 0.785mm

With these the Standard Deviations can then be worked out by applying the t-test formula.

T=(average 0f A-average of B)

SDA/5 + SDB/5

T=1.718-0.785

0.841485/5+0.00608125/5

T=0.933

0.168297+0.00121625

T=0.933

0.408755122

=2.382540205

When the figure has been calculated has been entered it to the t-test, under the heading of 8 degrees of freedom it shows that there is a large significance between the results that were gathered.

So by the use of the t-test formula this proves that the results which were gathered had a significant difference compared to each other, therefore the difference that is shown in the results was that of actual difference but not that which could have occurred by error or chance.

Conclusion-In conclusion to this investigation it can be seen by referring to the table of results and the graph produced that the trend that the results have taken, suggests that as the concentration percentage of solution that the discs were saturated in increased, the area that was affected or killed by the bacteria increased. This can be seen with reference to the graph that has been produced and the results, for example, the average area affected by the 0.0001% solution was only 0.785mm whereas the 0.1% was much larger at an area of 1.718mm. Other conclusions that can be made by looking back at the results that were gathered and the chart that has been produced are that as the concentration of the antibiotics increases, the difference of measurements between two of the different concentration increase. E.g. Between 0 and 0.00001 the difference can be seen as 0.050cm whereas between the final two concentrations the difference was a large as 0.5.

The scientific information that will support the different conclusions that have been made, are concerned with the penicillin and antibiotics and how they affect the bacterial growth and binary fission.

As mentioned previously, in the scientific background antibiotics are effective as they affect bacterium in a number of different locations, examples before included cell wall synthesis, metabolic reactions and transcription. More specifically I mentioned how penicillin affects bacterium cells, the details that were mentioned included that Penicillin works by inhibiting the bacteria cell from forming a cell wall. This may not seem important, but the cell wall is critical to the cell. If a cell cannot make a wall it will rupture and die. This means that a single cell divides to produce two cells. During this division the cell wall doubles in size and finally separates forming two cells. How Penicillin works is that it cannot affect an already formed cell wall, but it prevents the cell from making more of that wall and inhibits the cell from multiplying. Penicillin kills bacteria by interfering with the ability to synthesize cell wall.   This plays a very important role, as the method of cell division that is used by bacteria is of Binary Fission. Binary Fission simple means “two division”, and the penicillin mode of attack has the important affect as during the process of binary division the final process id the dividing or separation of the two copies of gametes, but as the wall of the cell is being damaged it cannot synthesize new cell wall, therefore the process of cell division is damaged.  

When the information that has been mentioned above is then applied to the different conclusions that I have produced above, it makes them seem more reliable and correct. So in final conclusion it can be seen that the increase in the concentration mean that the binary fission process is less likely to be complete due to the cell wall synthesis inhibition. Therefore the greater the concentration percentage, the more inhibition is likely to occur and the grater the area surrounding the saturated discs will be.

Evaluation: In the evaluation we will assess the procedure that took place, review the results that were gathered, state were there may be a number of areas of error, the different areas that could be improved.

First of all I will comment on the suitability of the procedure that took place. After completing the practical and the investigation I believe that the suitability of the procedure was correct of fair. The reasons for this opinion are that as it was an investigation concerning the different concentrations of the antibiotics on bacteria. So as this was quite a simple practical relatively compared to the amount of the results that were gathered and the methods in which they could be used. But on the other hand, the method used can be seen as unsuitable as the task of pouring the agar, spreading of the agar, and this could be seen as a lot of work which a great chance of being erroneous.

On commenting on the review of the results, I will comment on the accuracy of the results that were gathered and any anomalous results that may have damaged the graph, t-test etc. In collecting the results that were collected there are a number of areas that may have made the results erroneous and less accurate. These have been mentioned previously but in summary they include:

• Pouring of agar
• Spreading of bacteria
• Preparation of discs
• Serial dilution

When commenting on the different results that were gathered there are some that seem to be out of place or wrong these have been high lightened into the tables under the heading presentation of results.

In more detail, there are a number of areas that error may have occurred. These include:

• Sterilisation of equipment and area-during the experiment there where a number of times where the equipment and areas would need to be cleaned and sterilised. But throughout the experiment this may have been forgotten or not carried this out correctly, affecting different parts of the practical such as the spreading of the agar plates. The effect of this may have been the poor spreading of the bacteria, addition of foreign bodies.  
• Disc diameter- as the production of the discs was simple done using a hole punch there was not much guarantee that these would have been the same in diameter, this in turn may have and an effect on the absorption of antibiotic solution, although small differences some results may have differed.  
• Serial dilution- for the preparation of the different concentrations this involved the addition of water etc. Problems may have occurred in wrong measurement of water and antibiotic solutions, test tubes filled etc, damaged all results gathered
• Pouring of agar- as this was to be a very important but this could go wrong quite simply as during this it may have been breathed, the addition of foreign bodies etc.  
• Spreading of bacteria- this in the practical was the area where most error may have occurred. The different errors that may have occurred may have been the poor spreading of bacteria across the agar nutrients, sterilisation, and cutting of agar.
• Saturation of discs- during the practical when the different solutions had been made, they were then poured into the different solutions, where the discs where shortly added. The problem with this method is that there is no way to ensure that they have been covered with the solution and for the same amount of time.

If there were the chance to repeat to practical there would be certain area that would need to be improved. The different areas include:

• The different concentrations that were selected, it would be better to see a larger range.
• Better method of production for discs
• Spreading technique- longer time may be better so that time was not rushed. Possibly more practice at the technique

If the investigation was to be repeated a new method that could be used may be