E.coli K12: Extended Experimental Investigation

Extended Experimental Investigation
Year 11, Microbiology
BYRNES, Olivia

Extended Experimental Investigation
Year 11, Microbiology
BYRNES, Olivia

Abstract
The following report discusses whether the bacteria E.coli K12 are able to gain resistance to nickel and aluminium over generations. It discusses the importance of bacteria and how it is used throughout everyday life. It discusses that some bacteria is used for human applications and shows how it can be helpful in certain situations. This report will discuss the background information on E.coli K12 and the uses of bacteria. Throughout the investigation a series of experiments will be conducted to determine if metals such as nickel and aluminium have a resistance to E.coli K12 over a sequence of generations. To complete the investigation four Macconkey agar plates were inoculated with E.coli k12 and four paper discs of aluminium and nickel that were soaked in metal salts were placed into the plates to see the resistance that would occur over. Once resistance had occurred, the zone of inhibition was scraped and placed into a new broth to create a bigger resistance over time. In conclusion aluminium had a larger resistance to E.coli K12 although in the final generations both metals decreased.
Introduction
The aim of this experimental report is to discuss whether E.coli K12, over six generations could become resistant to metals and then we will refer it to antibiotic resistance.
Bacteria are single celled prokaryotic organisms in the Kingdom Monera. Bacteria exist as parasites or independent free living organisms on the search for nourishment. Bacteria are very small, too small to be seen by the naked eye therefore is measured in micrometres. Bacteria are present in everyday life but only few are harmful to humans. Bacteria reproduce by a cell division called binary fission. Binary fission also known as prokaryotic fission is the process of where a cell divides asexually to produce the exact same bacteria. Although this is an effective way to reproduce bacteria, it does present difficulties. Due to the cells produced through identical reproduction they become prone to identical antibiotics therefore bacteria can increase variation by integrating genes from bacteria’s (How Do Bacteria Reproduce?. 2012).

Bacteria are present in everyday life and used for many human applications. Bacteria are present in: * Insulin-certain types of bacteria can produce insulin in the human body. Research is now being completed to one day use healthy bacteria for diabetics as it initiates are production for insulin in the body. Recombinant DNA is used in this element. Recombinant DNA is the genetic material from several sources to create structures that cannot be found in other organisms. All organisms share the same chemical structure although differ in the nucleotides within the structure, which is a link to a phosphate group that form the basic structure of DNA. Therefore, when different DNA structures are linked, they are able to be replicated to another (Jae Ireland, 2011). * Pest Controls- Particular bacteria’s cause pests to degenerate and slow down the reproduction system there is a form of natural pest control (Jae Ireland, 2011).. * Immune System- Bacteria is present in the human body immune system to break down food substances and prevent the body from serious illnesses (Jae Ireland, 2011). * Cheese- Bacteria is found in lactic acid that has relation to the aging of cheese. Bacteria are stimulated within the cheese causing the aging of the cheese to move on giving the individual flavours (Jae Ireland, 2011).
Human body illnesses are brought about by infectious bacteria entering the human body either by food or water poisoning or inhaling bacteria. Once the bacteria have entered the body, the bacteria reproduce and duplicates and enters into cells. In the perspective of killing a bacterial infection, antibiotics are a common items used to do so. Antibiotics are known as a selective poison and its especially chosen to kill the infectious bacteria in the body but not the body’s cells. Each antibiotic has a different effect on bacteria. It specifically works to disable the bacteria’s specific ability that affects the body. Although if a particular antibiotic is being used persistently the bacteria becomes resistant to it and the antibiotic will eventually have no effect on the bacteria (Department of health, 2012).
Bacteria have a very basic structure and consist without membrane organelles. The internal structure consists of DNA which is situated in the central area of the cell. It is a visible from the rest of the cell interior. The ribosomes are what give the bacteria an appearance in micrographs. Smaller ribosomes have a similar function in translating genetic RNA into the production of peptide sequences. The storage granules which are the nutrients stored in the cytoplasm become glycogen, lipids, polyphosphate and in other cases sulphur and nitrogen. The surface structure consists of the capsule, which is made up of polysaccharides. This is what protects the bacterial cell and is in relation to pathogenic bacteria because it acts as the barrier again phagocytosis. The outer membrane is the lipid bilayer is the source of lipopolysaccharide (LPS). LPS is toxic and turns on the immune system off. The cell wall is made up of polysaccharides and protein, otherwise known as peptidoglycan. The cell wall is the maintenance of the structure of the cell. The cellular area is found in bacteria that have an outer membrane and plasma membrane. This compartment consists of enzymes that help digest and move nutrients into the cell. The lipid bilayer, similar to the plasma membrane allows ions, nutrients and waste throughout the membrane (Jae Ireland, 2011).
E. coli K12 is the inhabitants of warm blooded mammals. Due to its wide range of use for research in many microbial genetics, physiology, and industrial applications, E.coli K12 is the most widely studied microorganisms. E. coli K12, has a history of safe use and its by-products are often used in industrial applications, human drugs, insulin and somatostain. With the exemption of strains known to be pathogenic, E. coli K12 is known to be Class 1 Agent under NIH guidelines. (EPA, 2006)
Bacteria are present in everyday life. Bacteria can be found in many forms, such as the air, soil, water, plants and animals and even in the human body. Although bacteria can be used for good uses it can also be harmful. There are two ways to exterminate bacteria, physically and chemically.
Disinfectants and antiseptics are used commonly to avoid the spread of bacteria. A variety of substances are used to kill bacteria in household appliances. Bacteria are known to have an ability to grow resistance to household appliances such as bleach. If the bleach concentration is low, bacteria grow a resistance to it and are able to avoid an affect from cleaning. From contact with hypochlorous acid , it works in a way that has an ability to stimulate a gene in bacteria that to fight off infection (Kathryn Senior, 2012).
Bacteria can often become resistant to either antibiotics or heavy metals. The bacteria becomes useful to these materials and becomes resistant to them, therefore they have no effect on the bacteria. Antibiotic resistance is an antibiotics inability to successfully control and kill bacteria. The bacterium becomes resistant therefore will continue to reproduce in the existence of antibiotic. Bacterial resistance can occur from several procedures. Increased reproduction of the bacteria, therefore there will be an increasing amount of the bacteria that the antibiotic will be unable to inactivate the bacteria. Another way bacterial resistance can occur is for the bacteria to gain a gene to transfer genes from one bacterium to another that allows the bacteria to become resistant to the antibiotic (Alliance for the Prudent Use of Antibiotics, 2012).
Heavy metal resistance is similar to antibiotic resistance in the fact the bacteria both becomes resistant to it. Heavy metal is a substance that is able to kill bacteria, although over time bacteria becomes resistant to it. Bacteria become resistant over several procedures spoken about above.
Virulence is the amount of fatality and severity within a group of organisms and its ability to invade particular cells or tissues. Virulence describes a particular organism’s ability to cause infection or disease and is determined by virulency factors that indicate this. Bacteria virulency is determined by the number of bacteria in the area, how common the bacteria is, the path of the bacteria into the body, the characteristics and host of defence mechanism which is the body’s response to the infection and how well its defence is to the bacteria.

Method
Method 1
The area performing the experiment was sterilized thoroughly to ensure a clean area. The aseptic technique was performed to ensure the inoculating loop, tweezers and glass rake was sterilized before using it. Four Macconkey’s agar plates were inoculated with E. coli K12 using a glass rake to ensure the bacteria was evenly spread among the agar plate. The lid of the agar plate was kept on at all times to ensure no bacteria were able to get onto the plate. Once this was completed the glass rake was sterilized and repeated for the other three. The glass rake was again sterilized and placed into the rack to hold the equipment. A set of tweezers were sterilized and let cool down. Eight paper discs of aluminium were removed individually by the tweezers and placed onto a paper towel to let the discs drain off. The lid was removed from the agar plate and four aluminium discs were individually placed into the agar plate. The discs were evenly spread apart to allow for resistance to occur. The other four aluminium discs were placed into the other agar plate and were evenly spread apart. The lid was then placed on quickly after to ensure no bacteria was able to reach the agar plate.
The aseptic technique was used for the tweezers and was left to cool. Eight paper discs of nickel were individually removed and placed onto a paper towel and to allow the discs to drain off. Four metal discs were individually placed onto an agar plate and repeated for the other agar plates. Once all agar plates were completed, the agar plates were marked with permanent marker the date, our groups name, the bacteria E.coli K12, the teachers name and the name of the metal. These eight agar plates were then placed into an incubator with a temperature of 37° and left for 24 hours to allow resistance to occur.

Method 2
Once resistance had occurred, the eight agar plates were removed from the incubator. Measurements were taken if resistance had occurred and noted down. An inoculating loop was sterilized and left to cool. One of the agar plates lid was removed that contained nickel and the zone of inhibition was scraped for all four paper discs and mixed into a new brother of E.coli K12. The inoculating loop was sterilized using the aseptic technique and left to cool. This step was repeated for the other agar plate containing nickel and placed into the same broth.
The lid was removed from an agar plate containing aluminium. The zone of inhibition was scraped from all four metal discs and once again placed into a broth containing E.coli K12. The inoculating loop was sterilized using aseptic technique and left to cool. This step was repeated for the other agar plate containing aluminium and placed into the same broth.
Both methods were completed for all six generations.

Risk Analysis
Whilst conducting this investigation and completing experiments in the lab, the risks that could occur are high so in order to prevent this, particular steps must be put in place to reduce these risks. During this experiment, a Bunsen burner is used to sterilize equipment. Many risks come with this task such as a burn from the Bunsen burner. To prevent this risk from happening, hair must be tired back to minimise risks occurring. The Bunsen burner should be kept on the safety flam until it is in use. If this risk does occur it is important to inform the teacher. The burn must be under a run of constant cold water to reduce the sting. If the burn is extremely bad, medical assistance should be informed.
The use of bacteria plays a major factor in this experiment. It is important that steps must be put in place to ensure safety is put in place whilst experimenting. A risk of growing dangerous bacteria could occur from the E. Coli K 12. This can be prevented by keeping the agar sanitized before ensuring only required items placed on it. All equipment used should be sterilised before and after use. If this risk is to occur, the teacher should be informed as the teacher can decontaminate the agar. Different solutions are used throughout this experiment such as metal solutions and bacteria. A risk of foreign solutions to have contact with eyes could occur. It is important to wear safety glasses at all times while conducting the experiment to reduce this risk. If chemical contact with the eyes was to occur, the eyes must be washed thoroughly with water and the teacher must be informed. If the injury is severe medical attention should be required.
The use of metal solution is another major part of this experiment. Spilling metal solutions causing skin irritation is another risk that could occur. To prevent this from happening, ensure lids are closed and only opened when metal disks are required. Is this risk is to occur, removed contaminated clothing and wash skin with water. The teacher should be alerted and if further medical attention is need, this should be done.

Results and Observation
Graph 1:

Graph 2:

Throughout the experiment it was noticed that the bacterial resistance began to let off an unpleasant smell at the fifth generation. This could have been due to unknown bacteria growing in the broth setting off the smell. This could have been due to not sterilizing the equipment used and incorrect aseptic technique being used. Human error could have been the cause of this. It was noticed that the resistance to the metals aluminium and nickel was changing over the six generations by increasing and decreasing. The metals should have had a constant resistance to the E.coli K12, this error could have possibly been due to human error. Throughout the experiment it was observed that the broth the zone of inhibition had been scraped and placed into would become a reddish colour. This may have been due to scraping the Agar plates instead of the bacteria.

Discussion
The aim of this experimental report is to discuss whether E.coli K12, over six generations could become resistant to metals and relate it to antibiotic resistance.
Whilst conducting this experiment, unusual results occurred. It was hypothesised that aluminium and nickel would show constant and increase in resistance to the bacteria E. coli K12 over the six generations. The results showed otherwise.
Graph 1 showed the aluminium had a constant gradient of 0mm for the first three generations. Aluminium had a constant resistance to E.coli K12. Generation four showed that aluminium had a resistance of 0.5mm in generation four. This could have been due to human error. In generation five nickel had a resistance to E.coli K12 of 0mm and rose again to its highest point of 0.5mm in generation six.
Graph 1 showed that to begin with nickel had a resistance of 1.6mm in generation 1. In generation 2 and 3 no resistance was occurring. In generation 2 nickel reached a resistance of 1.7mm and in generation 3 a resistance of 1.4mm. Resistance to E.coli K12 increased in generation 4 and dropped down to a resistance of 1.9mm. In generation 5 the resistance decreased and reached its climax of 2.7mm and increased substantially to a point of 1.3mm in generation 6. Nickel should have had a constant resistance over the six generations the reasons for this may have been due to human error.
Graph 2 displays the average results of the two metals. To begin with nickel and aluminium began with their most resistance to E.coli K12. In generation 1, nickel had a resistance of 1.6mm and aluminium had an average of 0mm. Both metals lost resistance over the six generations. Nickel averaged at around 2.1mm and aluminium averaged at around 0.3mm. It was hypothesised that both metals would have gained resistance to E.coli K12 over the generations.
Blundell, 1969 commenced a study that shows that a metal salts concentration is increasing and how it effects bacterial growth becomes more evident because fewer proteins were able to be made as the IC ratio decreased. This research is similar to the experiment conducted because the metals used had a higher concentration of metal salts than the ones found in Blundell, 1969. This is reason the bacteria E.coli K12 did not gain resistance to the metals aluminium. This research is comparable to the experiment as the metal salts are increasing, bacterial growth is affected and throughout this experiment metals such as nickel and aluminium are being examined to grow resistance to bacteria such as E.coli K12.
Throughout the experiment, the metals nickel and aluminium should have had no resistance to E.coli K12 to begin with. This means that the metals should have had a constant reaction to E.coli K12 although the graphs recorded show different results. The results could have been altered from many human errors that could have occurred. Throughout conducting the experiment, it was important to scrape the zone of inhibition to create a new broth to become resistant to E.coli K12. Human error could have occurred when we were supposed to be scraping of the zone of inhibition but instead was scraping the bacteria. This error would cause the new broth to become less resistant. When placing the metal discs into the agar plates it was important to allow the discs to dry off from the solution so that the same amount of solution was on each disc initiating the same results to take place. The metal discs may have had more solution than other discs causing the metals to have more of a resistance than others. It was important to place the metal discs carefully onto the agar plates so that only a certain area was infected by the metals. When placing the metal discs onto the agar plates, the metals could have been dragged across the bacteria causing the metal to have a larger effect on the bacteria. Before creating and finishing a new generation, it was important to conduct the Aseptic technique to ensure all equipment was sterilised. Human error could have occurred when conducting the aseptic technique. The equipment used might not have been sterilised properly causing them to be infected. The equipment could have been infected from previous experiments or students breathing on the equipment. From the equipment being infected it could have had an effect on the bacteria. The metal discs are kept in a solution to allow them to remain new. Due to this, the metal solution concentration could have been changing over the three week period. This could have had an effect on the experiment making the resistance become either stronger or weaker. When completing the experiment it was important to sterilize the equipment. After the equipment had been placed above the fire to sterilize not enough time was given for the object to cool down and was the placed into the bacteria. From this, this could have caused an effect on the bacteria.
The method could be improved by ensuring that throughout conducting this experiment, the steps must be completed carefully to avoid altering results. The zone of inhibition must be scraped carefully to ensure that only the zone of inhibition is scraped and not the agar plate. When the metal discs are removed from the metal salts and left to dry a time should be allowed for the metal discs to dry to ensure they are all equally dry to make it a fair experiment. The aseptic technique should be performed twice and correctly to ensure equipment is completely sterilized. A time should be allowed for the equipment to cool to ensure the bacteria and metals are not affected by the heat of equipment. The metal discs that were kept in the metal salts should be renewed over the six generations to ensure there is no change in metals.
In Townsville, there are copper and zinc metal refineries. The environments surrounding these refineries are infected by exposure to these metals. Once bacteria is exposed to this environment the bacteria then gains metal resistance and due to co-selection, bacteria also gains resistance to antibiotics. Once this infection reaches civilization, due to antibiotic resistance gained on this bacteria, antibiotics is not treatable to this as the bacteria.
Conclusion:
The aim of this experimental report was to discuss whether E.coli K12, over six generations could become resistant to metals. It was hypothesized that metals aluminium and nickel would gain a resistance to E.coli K12 over six generations. In conclusion, nickel and aluminium had uncommon results to gaining resistance although the results shown did not support this. Both metals did not gain a resistance to E.coli K12 over the generations. This could have been due to human error. Aluminium had a constant gradient of 0mm to E.coli K12 for the first three generations although in generations 4 and 6 lost resistance. Nickel did not have any resistance to E.coli K12 although in generation 6 had a gained a substantial amount of resistance to E.coli K12. There is a high possibility the results was altered due to human error. In conclusion, no resistance was gained for metals aluminium and nickel.

Annotated Bibliography
Escherichia coli K-12 TSCA Section 5(h)(4) Exemption: Final Decision Document | Biotechnology Program Under Toxic Substances Control Act (TSCA) | US EPA. 2012. Escherichia coli K-12 TSCA Section 5(h)(4) Exemption: Final Decision Document | Biotechnology Program Under Toxic Substances Control Act (TSCA) | US EPA. [ONLINE] Available at: http://www.epa.gov/biotech_rule/pubs/fra/fd004.htm.
The article discusses the Environmental Protection Agency proposing review for certain microorganism’s uses under certain use and conditions. The article discusses the use of E. coli K 12 and of its most extensively studied organism. It discusses the conditions of exemption whilst the particular organism is in use. This article is relevant to my investigation as it discusses the exemption conditions whilst the microorganism E. coli K 12 is in use. The article discusses the history of E. coli K 12 and of its safe use. The article could have been better if it discussed more of E. coli K 12 resistance to metal or antibiotics. The information is reliable as it confers about the uses of E. coli K 12 and the safety of the microorganism. This is useful as we are using E. coli K 12 in our investigation. The article was educational as it gave facts and background knowledge about E. coli K 12. The information was not bias.
Wright, m.s.w, 2006. Co-Selection of antibiotic and metal resistance. Co-selection of antibiotic and metal resistance, 14, 176-182.
The article discusses the co-resistance process and cross-resistance process. Co-resistance is meaning different factors present in the same genetic element and cross-resistance meaning that the same genetic factors responsible for metal and antibiotic resistance. This article relates to the investigation as it discusses a bacteria’s resistance to a metal and antibiotic. It discusses that when a bacteria is resistant to a metal it automatically becomes resistant to an antibiotic. This article could have been more useful if the articles vocabulary was up to a high school student standard. The information was valid and useful as it discussed similar investigations that will be conducted in our investigations. The information was not bias.
David J Groves, d.j.d, 1975. Epidemiology of Antibiotic and Heavy Metal resistance in bacteria: Resistance Patterns in Staphylococci isolated from Populations Not Known to be Exposed to Heavy Metals, 7, 614-621
This article discusses the patterns of resistance to antibiotics and heavy metals in bacteria. It briefly discusses particular bacteria, Staphylococci being isolated from specimens obtained to patients and exposed to heavy metals abnormal to the bacteria. It discusses that genes for antibiotic and heavy metal resistance are both located on the plasmids and chromosomes in the bacteria. This article is in relation to the investigation being conducted this term as we are removing a certain bacteria, E. coli K 12 and exposing it to abnormal metals such as nickel and aluminium. It discusses the genes for heavy metal and antibiotic resistance being located in the chromosomes and plasmids. This article could have been improved by discussing the reactions Staphylococci had to the abnormal heavy metals to give the reader an idea of the reaction that would occur from exposing the bacteria to metals. The information was reliable, valid and well researched. The information was not bias.

Appendix | Gen 1 | Gen 2 | Gen 3 | Gen4 | Gen 5 | Gen 6 | | Ni | Al | Ni | Al | Ni | Al | Ni | Al | Ni | Al | Ni | Al | 1 | 2 | 0 | 1.5 | 0 | 2 | | 1.5 | 0.5 | 3 | 0 | 1 | 0.5 | 2 | 1 | 0 | 1.5 | 0 | 1 | | 4 | 0.5 | 3 | 0 | 1 | 0.5 | 3 | 2 | 0 | 1 | 0 | 1 | | 3 | 0.5 | 3 | 0 | 1 | 0.5 | 4 | 1.5 | 0 | 1 | 0 | 3 | | 3 | 0.5 | 4 | 0 | 1 | 0.5 | 5 | 1.5 | 0 | 2 | 0 | 3 | | 0.5 | 0.5 | 2 | 0 | 1 | 0.5 | 6 | 2 | 0 | 2 | 0 | 3 | | 1 | 0.5 | 3 | 0 | 1.5 | 0.5 | 7 | 1.5 | 0 | 3 | 0 | 3 | | 1.5 | 0.5 | 2 | 0 | 2 | 0.5 | 8 | 1.5 | 0 | 3 | 0 | 3 | | 1 | 0.5 | 2 | 0 | 2 | 0.5 | average | 1.6 | 0 | 1.9 | 0 | 2.4 | 0 | 1.9 | 0.5 | 2.8 | 0 | 1.3 | 0.5 |

Acknowledgments
My appreciation in particular goes to my fellow lab partners, Liyana Hallen and Hannah Coman. I would also like to acknowledge teachers such as Mr Koulakis and Mrs Barton for assistance throughout this experimental report.

Bibliography: Wright, m.s.w, 2006. Co-Selection of antibiotic and metal resistance. Co-selection of antibiotic and metal resistance, 14, 176-182.