1.2.2- Control experiment - A positive control which always inhibits e coli growth will be used (bleach), and a negative of control which never inhibits e coli growth will be used (water)
1.3.0- Experimental method
1.3.1- Materials and apparatus (table 2)
1.3.2- Safety aspects (table 3)
Part 1- Preparation of agar plates
1-Draw numbers on the agar plates (numbers correspond to a substance, eg- bleach)
2-Place e coli sample into an empty Petri dish
3-Use the glass spreader to pick up some e coli and spread it evenly over the surface of the agar
4-Close the agar plate
5-Repeate for 4 more agar plates (Glass spreader decontaminated by soaking it in mentholated spirits and heating under Bunsen burner)
Part 2- Preparation of growth colonies
1-Soak paper disks in a given substance (water, bleach or toilet cleaners)
2-Place the paper disk using tweezers on top of its corresponding number on the agar plate pressing down slightly. (Decontaminate the tweezers by soaking it in mentholated spirits and heating them under a Bunsen burner between uses)
3-Repete this step for all substances (1 to 6)
4-Reprete for remaining agar plates
5-Seal agar plates and incubate for 24 hours
Part 2- Recording results
1- Record which substances have caused zones of inhibition.
2-Using a ruler measure the diameter of the regions of inhibition (from the bottom of the agar plate)
3-Compare control and different toilet cleaning products.
1.3.4- Diagram of experimental setup (diagram 1)
2.0.0 Data collection and processing
2.1.0 Recording raw data
2.1.1- Changes to experimental design
Due to human circumstance the results of the experiment were taken after two days instead of one. However after the samples were placed into a fridge which would have caused bacterial death (cryostasis) largely preserving the result of a 24 hour time period. As good experimental practice an event like this should be avoided in future experiments.
2.1.2- Qualitative data
Different samples of toilet cleaner seemed to have different levels of viscosity. For example samples of duck toilet cleaner formed large droplets upon the paper disks while in other less viscose samples such as bleach the solution flowed spread outwards, to attempt to remedy this situation excess was allowed to drip onto a paper towel. When paper disks where placed upon the agar plates they slipped around slightly before they were placed into the correct positions. This resulted in some of the solution from the given plate being dispersed on the surface of the agar. As mentioned with the viscosity of the different products not all cleaners appeared equal. Samples of bleach and King White both consisted of a strong smelling vapour; additionally both use child proof seals. Harpic and Duck toilet cleaners where pink and blue in colour respectively and smelt quite inviting (like flowers). After the 24 hour period zones of inhibition were examined, the diameters of inhibition where indentified as sections of clear agar in contrast to darker surrounding agar. These zones where circular but many where not perfectly circular. Some had diameters that where larger than others. The measurement of diameter was taken from the largest point for all samples. Some zones of inhibitions crossed with other and some even hit the edges of agar plate, like the other samples the largest complete diameter of these particular samples was found.
2.1.3- Raw data table (table 3) - The diameter in millimetres of the zone of inhibition in a e coli growth colony after 24 hours when tested with different cleaning products.
2.2.0 Processing raw data
2.2.1- Overview- Each different cleaning product showed results that were fairly similar. Hence the independent values can be averaged (including the calculation of standard deviation). This average will represent the average diameter of the zone of inhibition for a given substance. These averages with respective standard deviations will be placed into a bar graph so that the effectiveness of different products can be visually compared.
2.2.2- Calculations used (table 4)
2.2.3- Experimental setup (figure 2)- inserted a bit later
2.3.0 Presenting raw data
2.3.1- Processed data table (table 5) – The average diameter (mm2) of the zone of inhibition of e coli growth colonies after 24 hours for difference different cleaning products.
2.3.1- Processed data graph (figure 3)
The smallest zone of inhibition was the negative control of diluted water, the highest inhibition was observed for the positive control of bleach solution. Closely following bleach was the product King White. The zone of inhibition for Harpic was about half the size of bleach. Home brand and Duck toilet cleaner, take 4th and 5th largest diameter respectively with similar diameters. The standard deviations where relatively large for all samples, the standard deviation if considered could allow the samples to be split in to two groups. The first group is bleach and King White which showed similar diameters, and the second group being Harpic, home brand and Duck toilet cleaner. The standard deviation for these three cleaners is not large enough to challenge the position of King White or bleach even considering the standard deviation. However the positions within the two groups could change if the standard deviation was implicated.
3.0.0 Conclusion and evaluation
The hypothesis “King White toilet cleaner will most adversely affect (kill) the bacteria Escherichia coli (e coli) to the highest degree when compared to a range of toilet cleaning products” was suggested to be valid as it showed the largest zone of inhibition of any of the toilet products at an average of 28.4mm in diameter (figure3). All of the other toilet cleaning products showed at least some adverse affects on the e-coli. Harpic displayed an average of 16.4mm in diameter (figure 3). Harpic and King White where the only toilet cleaning products to reach the “susceptible” level in the Kirby Bauer test (A Olsen, pg 1, 1995). Home brand and duck toilet cleaners both displayed the “intermediate” level with 12.6mm and 10mm in diameter respectively (A Olsen, pg 1, 1995).
As expected the control of water did not show any inhibition (figure 3). Water does not affect bacterial cells because all bacteria posses an external capsule white prevents them from bursting, even in a high pressure environment (Gary K, pg 1, 2006). In contrast all of the cleaning products showed at least some inhibition of e coli, inhibition could have occurred by two processes. The first process is that the reproduction of e coli in the zone of inhibition was being restricted; therefore the bacteria could not multiply. This could occur if one of the enzymes related to binary fission is denatured by the cleaning product. For example in bacteria enzymes are used to generate the kinetic energy required to “separate” daughter cells, if this enzyme is denatured then the bacteria would not divide (William B, pg 1, 1998).
The other possible explanation is that the cleaning products where physically killing the e coli Bactria through processes such as cytotoxic attack. For instance T killer cells employ cytotoxins in order to deactivate other living cells (C. Venkat, pg 1, 2003). By this process all of the e coli bacteria would die in the zone of inhibition therefore no growth would be experienced in that sector, living cells would continue to grow surrounded in areas surrounding the zone of inhibition.
One or both of the processes could have occurred for any of the solutions, and in the end both would end with the same result, the elimination of e coli growth. However it is important in future experiments that the individual process is identified as each has different implications (one is possibly more effective than the other). The second method is likely not as effective as the first and it can even be dangerous. If some bacteria survive the cleaning product then they will still reproduce, these new bacteria will all be resistant to the respective cleaning produce.
In contrast the first method would not allow reproduction and therefore any resistance would not be passed on/developed. The first method would also be superior as it could be safer. For the second method to be effective the substance would need to be cytotoxic, these cytotoxins could be harmful to humans. In contrast a substance that simply inhibits reproduction can be designed to be specific to e-coli’s reproduction enzyme/s rather than the entire cell, hence increasing the safety of the product.
Outside of the biological field implications may exist. In particular would be the commercial implication. For example some companies state that there bacteria will kill 99% of germs. Assuming this to be true the product would have to engage in the second method which would mean that resistant bacteria could be created. This means that the companies could be endangering the public by allowing the possibility of bacterial mutation. It is important that this report is extrapolated upon to give consumers a better understanding of the products they buy. A consumer may prefer to buy a product that reduces growth via the first method rather than the second.
Moving on to consumer preference it was found that some products were (substantially) more effective than others. The most effective cleaner was the positive control of bleach. The products Duck, Harpic and home brand considering the standard deviation would fall largely in to the same range of effectiveness. King White however performed well above other toilet products and even rivalled the performance of bleach, considering the standard deviation it is even possible that King White performed above bleach. It could be appropriate to separate the substances into two grounds. The first group bleach and king white, and the second group Harpic, Home brand and Duck, each group showed substances with similar diameters of inhibition. This may suggest the chemical composition of each group is quite similar. King White also produces bleach products for example so it possible that its bleach products share the same properties as the King White cleaning properties (King white, pg 1, 2010).
If this is the case it may be more appropriate for the consumer to simply purchase bleach in the place of branded toilet cleaning products. Bleach showed performance similar and above many general toilet cleaning products. Unlike toilet cleaning products bleach is also used in other household tasks such as washing clothes. Bleach could be used as an “all” purpose product eliminating the need to purchase toilet cleaners, hence reducing consumer expenditure. With the sacrifice of the aromatic properties of toilet cleaners however.
There are several flaws in the experiment however. For instance some properties were more viscous then others, this means that they less dense substances would diffuse over the surface of the agar more than denser subjects, therefore covering a larger surface diameter and possible increasing the size of the zones of inhibition. The size of the standard deviation was quite large for all substances (except water). This could be attributed to several limitations. For example the exact amount of substance placed on to the paper disks was not controlled. Also some difficulty was experienced when placing the disks themselves, some disks slid around over the agar when trying to position them which would have deposited some of the substance in ulterior sections. The deviation may have been created by the combination of a host of limitations which will be further explored in the limitations to experimental design.
However even if the standard deviation was taken into account the performance of King white and bleach would have been well above that of the other products. All toilet brands will reduce the growth of e coli bacteria, not all products will perform equally however. With further experimentation upon the thesis of this experiment which includes a better separation of the process by which growth is reduced, , better control of random errors and finally a wider range of cleaning products which includes products that are not toilet cleaners. This will allow consumers to better informed decisions on their purchases.
3.2.0- Limitations of experimental design
While not relevant to the hypothesis directly the process by which e coli bacteria are actually removed has important implications in the fields of biology and commerce. This could achieved by using a microscope to examine and physical changes in the bacteria, or by examining the chemical compositions of products to find if their composition can be linked to the method of bacterial removal.
A limitation that may have contributed to the differentiation between difference substances was experienced. This limitation is that different substances possessed differing levels of viscosity. This means that the specific amount of substance on the paper disks may have been greater for denser substances as the substance would form in large droplets rather than simply soaking in the paper. Additionally less dense substances would have diffused more over the surface of the agar plate. These processes in tandem would result in denser samples having a higher concentration then their less dense counterparts, at the same time less dense samples would cover a larger surface diameter on the agar then dense substances resulting in a larger zone of inhibition. Both of these limitations could be remedied by using static volumes of each solution, in regards to the diffusion of less dense substances it may be responsible to spread each substance (of the same volume) over the same diameter rather than simply placing the paper disk and allowing diffusion to occur passively.
Some zones of inhibition crossed with either the walls of the agar plate or with other zones of inhibition. This made it difficult to measure the diameter of the zones as the circles where not complete. In future experiments the number of samples allocated to individual agar plates should be reduced or the size of the agar plates should be increased. These methods shall avoid any cross over between samples.
The agar plates themselves may have been subject to error. For instance when the e coli colonies where prepared they were simply spread over the agar starting in one corner. This is a large chance that this method would have greater colonies of unequal density, The bacteria would be more difficult to remove in diameters of high density vs. diameters of low density, resulting in smaller diameters of inhibition for samples within the e coli dense diameters. In future experiments the grid square crossover method should be used as it creates colonies from the same source in a single continues stroke rather than the random movements seen in the spread method.
Also the agar plates themselves where often moved. Considering that some of the solutions used where highly fluid this may have caused some of the solutions to shift. And hence some of the zones of inhibition may have changed. Some random errors also occurred. For instance the experiment was performed in a relatively public environment with dozens of other students who where all rushing to complete their own experiments. Students even shared the same decontamination stations when decontaminating equipment (Same methylated spirits for example). This would have increased the likelihood of cross contamination between experimenters. This issue could be remedied by simply performing the experiment at different times then other experimenters.
3.3.0- Limitations of experimental design (table 6)
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