Antibiotic Resistant Bacteria Experiment

An experiment was conducted to observe the distinctions in amounts of antibiotic resistant bacterias from 3 East Lansing locations, Burger King, Meijer, and Dejà Vu. A hypothesis was developed which stated that the restrooms could have more bacteria which is repellent to antibiotics, meaning there would be difference between the two areas, with a null of there being no significant difference between the two. This is due to the idea that bath rooms are constantly exposed to antibiotics, whereas the trafficked areas are hardly ever exposed to antibiotics in comparison. An example was extracted from each bathroom, and a sample taken from a highly trafficked area for the reason that location. These locations were swabbed onto growth plates to develop a colony and then transferred onto plates with Ampicillin, Tetracycline, and Kanamycin to check for level of resistance to antibiotics. A gram stain, KOH test, MacConkey, and EMB dish examined gram-positivity or negativity, gel electrophoresis analyzed for plasmids and lower DNA period, PCR examined for what bacteria was present, and restriction digestion tested for addition of antibiotic repellent bacterias. Conclusions show that there was no significant difference in the quantity of antibiotic resistant bacteria grown in bathrooms versus highly trafficked areas, and the types of bacteria have not obtained results at the moment.

Introduction

With antibiotic protected bacteria becoming more and more hazardous, it is important to understand where in fact the bacteria have a tendency to conjugate. The goal of this experiment is to answer this question by identifying whether areas of heavy human traffic at popular locals or the bathrooms at those locals house more antibiotic immune bacteria.

Independent parameters of the test include anything that the researchers have control over. Independent variables of this experiment will be the locations chosen, the heat the samples were incubated at (37 degrees Celsius), volume of plates made, if the bacteria is gram positive or gram negative, the antibiotic that the bacterias were subjected to, and the amount of time they were incubated for. Dependent factors are parameters that change depending on the independent variables. Because of this experiment that would include amount of bacterias produced, the antibiotic amount of resistance, and how the bacteria separates in the gel electrophoresis.

In society, antibiotics are the commodity and are often given out without just cause. These were often thought of as a cure all medicine and provided to anyone who it might help, as well as those who could just not accept being unwell without a treat (Campbell et al. 2008). Additionally, when given these antibiotics for true medical reasons, people might not exactly take the full dosage of the prescription and stop treatment at a stage where the bacterias are not completely wiped out. The bacteria that are still alive tend to be more immune to the antibiotics that they have been exposed to. Furthermore, they can pass on their resistance easily through a process called conjugation. The bacteria that already house the antibiotic resilient DNA can use a structure called the making love pilus to form a bridge with bacteria that will not have the amount of resistance (Campbell et al. 2008). The donor bacteria can give the non-resistant bacterias a level of resistance plasmid, which binds to its DNA, so that it is more protected to antibiotic hazards. This escalates the amount of antibiotic immune bacteria on the planet. Locations constantly exposed to antibiotics are finding that the antibiotics are no more working. The regular exposure to antibiotics has lead to resilient types of the bacteria living on and reproducing (Campbell et al. 2008). Due to this issue, it has come crucial to understand antibiotic protected bacterias and where they are commonly located.

Bathrooms have a better resistance to antibiotics than the highly trafficked areas credited to continual contact with antibiotic cleaning products and human wastes that are laced with antibiotics because of their supply. Since humans are constantly consuming antibiotics, their waste materials is filled with antibiotics. The antibiotic waste is a frequent way to obtain natural selection for bacteria in restrooms, triggering the weak to die away and the strong to go on. Therefore, it can be inferred that restrooms would have more exposure to antibiotics than closely trafficked areas, resulting in increased levels antibiotic protected bacteria.

Finally, aims of his test are to determine the magnitude of bacterias cultivated in each environment, and to determine which types of bacterias can be found.

This experiment is being presented to see between areas of heavy human being traffic and contact, like a club, a shelf, or a play place for children, and bath rooms constantly exposed to cleaning and for that reason antibiotics, which area got more antibiotic resistance.

Independent variables of the experiment include whatever the research workers have control over. Self-employed variables of this experiment will be the locations chosen, the temperatures the examples were incubated at (37 levels Celsius), range of plates made, the antibiotic that the bacteria were exposed to, and the amount of time they were incubated for. Dependent factors are factors that change with regards to the independent variables. Because of this experiment that could include amount of bacterias produced, the antibiotic level of resistance, whether the bacterias is gram positive or gram negative, and the way the bacterias separates in the gel electrophoresis.

In modern society, antibiotics are a commodity and tend to be given out without just cause. These were often thought of as a remedy all medicine and provided to anyone who it might help, as well as those who could just not accept being unwell without a stop. Moreover, when given these antibiotics for true medical reasons, people may well not take the full dosage of the prescription and stop treatment at a stage where the bacteria are not completely killed. The bacterias that remain alive will be more immune to the antibiotics they have been exposed to. They can propagate their resistance easily, and when the same medical concern that they triggered arises again, it might be much harder to treat.

This increases the amount of antibiotic resilient bacteria on earth. Locations constantly exposed to antibiotics have found themselves facing the challenge of the antibiotics no longer working. Because of this issue, it includes come essential to understand antibiotic resilient bacterias and where they are generally located.

Bathrooms have an improved amount of resistance to antibiotics than the highly trafficked areas scheduled to continual contact with antibiotic cleaning products. Also, since humans are regularly taking in antibiotics and selecting for antibiotic level of resistance in their immune system systems, the bathrooms would have more exposure to antibiotic resistant bacterias excreted in real human waste. Therefore, this might create a null of no difference between degrees of bacteria.

Finally, goals of his test are to determine the magnitude of bacterias grown in each environment, and also to determine which types of bacterias are present.

Methods

First, environmental examples of bacterias were obtained. This was carried out by taking a sterile swab with a sterile solution of phosphate buffered saline (PBS), and transferring it over the surface where the bacteria were wishing to be obtained. The swabs were then taken into laboratory and swabbed onto agar only plates. Lawns of bacteria were cultivated after being incubated every day and night, and from these plates, more agar only plates were made from these and used as get better at plates. Once these were expanded, colonies were used and put onto patch plates, each made up of an antibiotic including Kanamycin, Ampicillin and Tetracycline, and also a plate containing only LB basic, which was used for a control. Specific colonies were cultivated in each one of the 16 sections that the plates have been split into, and these colonies were resilient to the antibiotic that was at the plate that they were grown on. To make sure that these plates remained healthy, these were redone every 2 weeks. These were incubated every day and night, and then counted to investigate the antibiotic level of resistance of the bacteria from each environment.

Next, Gram discolorations were preformed. A colony of bacteria was diluted in drinking water and located onto a slide. To adhere the bacterias onto the glide, it was handed over a slip 2 to 3 3 times. The slide was then flooded with crystal violet for 60 a few moments and rinsed with drinking water. It had been then flooded with iodine for 60 mere seconds and rinsed with ethanol. After, it was flooded with safranin as a counter-top stain and rinsed with normal water. The glide was air-dried. Immersion petrol was placed on the slide and the stain was looked at at 100x magnification.

The KOH test was also conducted to determine if the bacteria was Gram positive or Gram negative. Bacterias was placed on the microscope glide and subjected to KOH. A steel hoop was handled to the combination of bacteria and KOH. In the event the mixture jammed to the hoop, then it is Gram negative, if it did not adhere to the hoop, it is gram positive.

Gel electrophoresis was used to split up DNA nucleotides into bands. The gel was made out MacConkey Agar, which when cooled, varieties pores in the matrix to let the DNA run through when exposed to a power current. To make the gel, either 40mL or 60mL of the TBE is blended with either. 4g or. 6g of agarose respectively. We were holding heated, mixed jointly, and remaining to cool. Following, 2ïl of ethidium bromide was added. The DNA ran from the negative end of the cell to the positive end and made columns of bands with specific lengths.

Plasmids were isolated from the environmental samples utilizing a miniprep process. After antibiotic resistance was developed, the colonies were converted into liquid civilizations. These civilizations were incubated for 24 hours. After incubation, 10 ml of the sample were placed in a centrifuge, spun, and the bacteria were separated from the perfect solution is. It had been then re-suspended and lysed to eliminate genetic materials and proteins. The sample was spun again, and the plasmids were drained and put into a spin column. Using nuclease free normal water, plasmids were taken off the spin column into a centrifuge tube. The isolated plasmids are then tell you a gel electrophoresis, and using the NEB cutter V2. 0 program from New England Biolabs, a plasmid map can be produced. Following the plasmid was produced, it was tell you gel electrophoresis and shown under UV light to verify the procedure functioned.

To inoculate liquid press, the following method was followed. 5mL tubes with liquid medium were obtained. Next, 5 ïL of appropriate antibiotic were added. With a sterilized loop, an individual colony from an antibiotic streak plate was taken and added to the mix. Finally, the pipe was capped and placed in the shaker for 24 hours.

After plasmids were isolated, in cases like this only the blue control, the control plasmids and plasmids from proficient E. coli cells were considered and a heat shock procedure was used to drive the bacteria to obtain the plasmid. Three examples, two formulated with plasmids from blue control and from E. coli, and one with P. Litmus and LB only plates as control buttons, were heat stunned for ninety a few moments, then blended with warm LB broth and incubated. The examples were then propagate onto an LB and antibiotic plate to determine resistance transferred in one bacterium to some other.

A limitation digest was completed as follows. 10 ïl of the restriction enzyme, in this case Ava 1 was chosen, and 20ïl of the DNA from the miniprep were mixed. Next, 3ïl of the buffer #4 4 were added to the mixture. 100ïl of BSA was added previous. This focus was then incubated for one hour. 3. 3ïl of gel launching buffer was added following the incubation directly into the effect. The effect was then stored in the freezer until we were prepared to run the gel. A gel electrophoresis was run to determine the results of the digest.

Results

Written by Stacy Tipton

The experiment began with gathering two bacteria samples from three different locations. All bacterias were then grown up on LB only plates, which offered as professional plates for the bacterias in the test (Shape 1). All bacteria for the experiment were taken ultimately from these plates. Bacteria from these plates were then grown up onto patch plates that comprised Ampicillin, Tetracycline, and Kanamycin in the LB, so that only bacteria resistant to that antibiotic would grow (Physique 2).

Gram Negative bacterium expands lawns of bacterias on MacConkey plates. Making use of this test, the Dejà vu bathroom AMP immune bacterias, and Burger King Play area TET repellent bacteria came out to be Gram negative. The Meijer bathroom AMP resistant bacteria and the Dejà vu bar AMP resistant bacterias arrived Gram positive (Body 3). Gram stain checks for both gram negativity and positivity. Gram-positive spots pink during a Gram stain, while Gram negative stains purple (Body 4 ). The Dejà vu bathroom arrived up gram negative, while Meijer bathroom, Dejà vu pub, and Burger Ruler play structure emerged up gram-positive (Stand 1). The KOH test is sticky if it's gram-negative rather than sticky for gram-positive bacterias. Dejà vu bathroom arrived up gram-negative, as the Meijer bathroom, Dejà vu pub, and Burger Ruler play structure came back gram-positive (Stand 2).

Mini-preps on cultured bacteria were preformed. The mini-prep isolates plasmids from a bacterium. The mini-preps were then run through a gel electrophoresis. This was to test for plasmids in the bacteria. A ladder was run with the bacterias to identify plasmids, and a control was set you back ensure the test did the trick properly. No plasmids were within the cultured bacterias (Table 3), another gel was run to ensure the effect was accurate (Amount 9).

EMB plates were run. Gram-negative bacterium develops on EMB agar. A bacterial colony from each cultured antibiotic resilient plate was considered and subjected to the EMB agar. The results were that colonies subjected to the EMB were gram positive.

A chi square test was conducted. It included 1 degree of freedom, and possessed a value value of 0. 05. This intended that the Chi square syndication was at 3. 84 (Stand 4). Results said there is no definite difference in the quantity of antibiotic resistant bacterias found in bath rooms than in highly trafficked areas.

A PCR was set you back identify the bacterias by reproducing the DNA inside of it and comparing known examples of DNA to those being tested. These results never have been completed yet.

A restriction digest was also run, but right results have never been yielded out of this experiment yet. Bacteria has not yet been properly cut and observed on a gel electrophoresis test.

Discussion

Written and Revised by Stacy Tipton

There is little difference in the amount of antibiotic resistant bacterias found in bathing rooms versus found in highly trafficked areas. It was hypothesized that when the experiment was done to see which environment, the bathing rooms or the highly trafficked areas, would have the best antibiotic resistance, that the bathroom would have greater resistance. This is because of the theory that the bathrooms are constantly exposed to antibiotics, where as locations like a store shelf, stripper club, or play area are almost never if ever washed with antibiotics. It had been thought that a lot more the bacterias were exposed to antibiotic resistant bacterias, the more likely it would be that antibiotic level of resistance was naturally preferred for. The results contradict the hypothesis. There was found to be little difference in the quantity of antibiotic resistant bacterias in each location. According to the Chi square test, no factor between the two locations.

As we realized from previous research, exposure to antibiotics selects for antibiotic amount of resistance. When the antibiotics that the bacterias is subjected to does not kill all bacteria, the strongest bacteria that remain alive reproduce and pass on their resistance both through duplication and the passage of plasmids. Our studies suggest that when subjected to the same bacterias, it doesn't matter just how many times the bacteria face the antibiotics, once it is picked for, antibiotic amount of resistance stays in the populace.

There were specific problems in the research that could have afflicted the results. First, no plasmids were found in the bacteria. Which means that the passage of the resistance via plasmid spreading could not be examined. Also, several tests needed to be done regularly to get results. This means that the very same samples weren't used, but reproduced as just as possible. However, since you can find variation in which colonies were tested in each experiment, it's possible that the email address details are skewed somewhat.

There are several options for even more studies in this experiment. Further experiments could be used to find plasmids in the bacterias. One could go farther to see if the antibiotics that were used were supposed to kill the bacterias found. Also, one could experiment to discover if the bacterias were repellent to other antibiotics that use mechanisms similar to the ones tested.

References

Written and Revised by Stacy Tipton

Campbell, N. A. , J. B. Reece, L. A. Urry, M. L. Cain, S. A. Wasserman, P. V. Minorsky, and R. B. Jackson. 2008. Biology 8th ed. Pearson Education, Top Saddle River, NJ

Cognato, A. 2010. Recitatation_031710 PowerPoint. Webpages 1-10.

Lu, H. , X. Wang, X. Lang, Y. Wang, Y. Dang, F. Zhang, J. Tang, X. Li, X. Feng. 2009. Preparation and application of microarrays for the detection of antibiotic level of resistance genes in examples isolated from Changchun, China. Molecular Biology Records. Volume level 37. 1857-1865.

Rusin, P. , P. Orosz-Coughlin, C. Gerba. 1998. Reduced amount of faecal coliform, coliform and heterotrophic dish count bacteria in family members kitchen and bathroom by disinfection with hypochlorite cleaners. Journal of Applied Microbiology. Volume level 85. 819-828.

Figures

Written and Modified by Stacy Tipton

Figure 1: Swab Plates. Each different environment was swabbed and two different locations were extracted from each. The bacterias were grown on an LB only grasp plate before subjected to any antibiotics. Each small dot on the dish represents a new colony of bacteria. These plates served as the get better at plate for all those bacteria in the experiment and all examined bacteria ultimately originated from this plate. The small dots throughout the dish in the shape each represent an alternative colony of bacteria. These may be the same species of bacterias, or there may be several species on one plate due to the fact that each environment the samples were taken from was exposed to all varieties of bacterias. Afterward, these bacteria will be studied and subjected to antibiotics to check for amount of resistance. These bacteria were swabbed from a host using a swab coated with PBS. A shows Burger Ruler play framework, with specific colonies of bacterias. B shows Burger Ruler bathroom with individual colonies of bacterias. C shows Dejà vu club with a yard of bacteria. D shows Dejà vu bathroom with a yard of bacterias. E shows Meijer bathroom with a garden of bacteria. F shows Meijer shelf with a garden of bacterias.

Figure 2: Patch Plates. Inside the above amount A represents Meijer Bathroom Ampicillin, B signifies Meijer Shelf LB only patch dish, C signifies Burger Ruler Play Composition, D represents Tetracycline Dejà Vu Bathroom, and E presents Dejà Vu Pub Ampicillin patch plates. Each cultured bacterias was taken from the original professional plate and exposed to some other antibiotic dish. Each dish was divided into sixteen sections, and a new colony of bacteria from the same location were put on each section. This means several different species of bacteria could be on one plate, but didn't necessarily need to be. Bacteria grown on these plates are immune to the antibiotic that is in the LB that they grew on. A, B, C, and F all acquired 16 colonies of expansion, D possessed 8 colonies of growth. They are important in determining the amount of bacterias that was resilient in each environment so that statistical lab tests could be set you back determine if there was factor in the quantity of antibiotic amount of resistance in each area. Ampicillin plates comprised 100ïg of antibiotic permL, and Tetracycline and Kanamycin both comprised 50ïg of antibiotic per mL.

Figure 3: MacConkey Plates. MacConkey plates test for gram-positive or gram-negative bacterias. Gram-negative bacteria increase on MacConkey plates, gram-positive bacterias does not. Clockwise from top departed: Dejà Vu bathroom, Burger King Play Framework, Meijer Bathroom, and Dejà Vu bar. Only one colony of bacterias was picked to develop onto each dish, and therefore it is only one species of bacteria displayed per dish. B and C have development on the plates, and are therefore gram-negative. The A and D show no growth and are therefore gram-positive.

Figure 4: Gram Stain. B: Dejà Vu Pub AMP Gram Stain, B and C: Burger Ruler Play Framework TET Gram Stain D: Dejà Vu Bathroom AMP Gram Stain, E: Meijer Bathroom AMP Gram Stain. The dark color crimson color shows gram-negativity. Gram-negative bacteria lack a thicker cell wall membrane, and also have their part of peptidoglycan more uncovered than gram-positive bacteria. Gram stain runs about identifying gram negativity or positivity by coloring the part under the wall membrane. Since the wall structure isn't as thick, the dye is able to better stain that layer of peptidoglycan. They are rod-shaped bacteria, otherwise known as bacillus molded. The green color illustrated gram positivity. Gram-positive bacteria have a thicker cell wall membrane, this means their level of peptidoglycan is less subjected to the environment. A gram stain runs about figuring out gram negativity or positivity by coloring the level under the wall structure. Since the wall structure is thicker, the dye is not able to stain that part of peptidoglycan as well, and it turns up as a red color. This is a lawn of bacteria, usually coccus (circular) shaped, but some are bacilli formed as well.

Figure 5: Gel Electrophoresis Plasmid Isolation. The gel separates nucleic acids and protein. Lane 1 shows the 1 KB ladder, which is employed to help identify undiscovered plasmids by demonstrating lanes of known platform pairs. Lane 2 illustrates the blue control plasmid. This Plasmid ensures that the gel performed properly. A control of E. Coli bacteria was used. Lanes 3 through 6 show where a gel was run with lanes 1 through 6 filled utilizing a micropipetter, and where no plasmids were found. These lanes were examples form each one of the environmental plates that experienced already been subjected to antibiotics. Meijer bathroom, Dejà vu pub, and Dejà vu bathroom in lanes 3 through 5 were all from Ampicillin plates. Street 6 presents the sample from the Burger Ruler play structure that had been subjected to tetracycline. It really is obvious no plasmids were found, because there are no glowing bands that appear in the gel in lanes 3 through 6. Nothing of the surroundings acquired plasmids isolated from them. This was significant because level of resistance is moved through plasmids. To make this gel, . 4 g of TBE were blended with 40 mL of agarose. After these cooled, . 2ïL of ethidium bromide was added.

Tables

Written and Revised by Stacy Tipton

Table 1: Gram Positive/Negative Results

Gram Stain

KOH Test

Miejer Bathroom

Pos

Pos

dejà vu bathroom

Neg

Neg

Dejà vu bar

Pos

Neg

BK play structure

Neg

Pos

Table 3: Gel Electrophoresis Results

Positive*

Negative*

Meijer bathroom

X

Dejà Vu bathroom

X

Dejà Vu bar

X

BK play structure

X

*Indicates positive or negative for plasmids

Table 4: Chi Square Test

Antibiotic resist. colonies

Non-antibiotic resist. colonies

Total

Bathroom

167

25

192

Highly Trafficked Area

153

39

192

Total

320

64

384

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