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Antibiotic Resistant Bacteria Experiment

An experiment was conducted to observe the differences in amounts of antibiotic resistant bacteria obtained from 3 East Lansing locations, Burger King, Meijer, and Déjà Vu. A hypothesis was developed which stated that the bathrooms would have more bacteria which is resistant to antibiotics, meaning there would be difference between the two areas, with a null of there being no significant difference between the two. This was due to the idea that bathrooms are constantly exposed to antibiotics, whereas the trafficked areas are rarely exposed to antibiotics in comparison. A sample was taken from each bathroom, as well as a sample taken from a highly trafficked area in that location. These locations were swabbed onto growth plates to grow a colony and then transferred onto plates with Ampicillin, Tetracycline, and Kanamycin to test for resistance to antibiotics. A gram stain, KOH test, MacConkey, and EMB plate tested gram-positivity or negativity, gel electrophoresis tested for plasmids and cut DNA length, PCR tested for what bacteria was present, and restriction digestion tested for addition of antibiotic resistant bacteria. Conclusions show that there was no significant difference in the amount of antibiotic resistant bacteria grown in bathrooms versus highly trafficked areas, and the types of bacteria have not gotten results at this time.
Introduction With antibiotic resistant bacteria becoming increasingly hazardous, it is important to understand where the bacteria tend to conjugate. The purpose of this experiment is to answer this question by determining whether areas of heavy human traffic at popular locals or the bathrooms at those locals house more antibiotic resistant bacteria.
Independent variables of the experiment include anything that the researchers have control over. Independent variables of this experiment are the locations chosen, the temperature the samples were incubated at (37 degrees Celsius), number of plates made, whether the bacteria is gram positive or gram negative, the antibiotic that the bacteria were exposed to, and the amount of time they were incubated for. Dependent variables are variables that change depending on the independent variables. For this experiment that would include amount of bacteria grown, the antibiotic resistance, and how the bacteria separates in the gel electrophoresis.
In modern society, antibiotics are a common commodity and are often given out without just cause. They were often thought of as a cure all drug and given out to anyone who it could help, as well as those who could just not accept being sick without a cure (Campbell et al. 2008). Moreover, when given these antibiotics for true medical reasons, people may not take the full dose of the prescription and stop treatment at a stage where the bacteria are not completely killed. The bacteria that are still alive are more resistant to the antibiotics that they have been exposed to. Moreover, they can spread their resistance easily through a process called conjugation. The bacteria that already house the antibiotic resistant DNA can use a structure called the sex pilus to form a bridge with bacteria that does not have the resistance (Campbell et al. 2008). The donor bacteria can give the non-resistant bacteria a resistance plasmid, which binds to its DNA, making it more resistant to antibiotic threats. This increases the amount of antibiotic resistant bacteria in the world. Locations constantly exposed to antibiotics are finding that the antibiotics are no longer working. The constant exposure to antibiotics has lead to resistant forms of the bacteria living on and reproducing (Campbell et al. 2008). Due to this issue, it has come crucial to understand antibiotic resistant bacteria and where they are commonly located.
Bathrooms have a better resistance to antibiotics than the highly trafficked areas due to continual exposure to antibiotic cleaning products and human wastes that are laced with antibiotics due to their availability. Since humans are continually taking in antibiotics, their waste is filled with antibiotics. The antibiotic waste is a constant source of natural selection for bacteria in restrooms, causing the weak to die out and the strong to live on. Therefore, it can be inferred that bathrooms would have more exposure to antibiotics than heavily trafficked areas, leading to increased levels antibiotic resistant bacteria.
Finally, aims of his experiment are to determine the magnitude of bacteria grown in each environment, and to determine which types of bacteria are present.
This experiment is being held to see between areas of heavy human traffic and contact, such as a bar, a shelf, or a play place for children, and bathrooms constantly exposed to cleaning and therefore antibiotics, which area had more antibiotic resistance.
Independent variables of the experiment include anything that the researchers have control over. Independent variables of this experiment are the locations chosen, the temperature the samples were incubated at (37 degrees Celsius), number of plates made, the antibiotic that the bacteria were exposed to, and the amount of time they were incubated for. Dependent variables are variables that change depending on the independent variables. For this experiment that would include amount of bacteria grown, the antibiotic resistance, whether the bacteria is gram positive or gram negative, and how the bacteria separates in the gel electrophoresis.
In modern society, antibiotics are a common commodity and are often given out without just cause. They were often thought of as a cure all drug and given out to anyone who it could help, as well as those who could just not accept being sick without a cure. Moreover, when given these antibiotics for true medical reasons, people may not take the full dose of the prescription and stop treatment at a stage where the bacteria are not completely killed. The bacteria that are still alive are more resistant to the antibiotics that they have been exposed to. They can spread their resistance easily, and if the same medical issue that they caused arises again, it may be much harder to cure.
This increases the amount of antibiotic resistant bacteria in the world. Locations constantly exposed to antibiotics are finding themselves facing the problem of the antibiotics no longer working. Due to this issue, it has come crucial to understand antibiotic resistant bacteria and where they are commonly located.
Bathrooms have a better resistance to antibiotics than the highly trafficked areas due to continual exposure to antibiotic cleaning products. Also, since humans are continually taking in antibiotics and selecting for antibiotic resistance in their immune systems, the bathrooms would have more exposure to antibiotic resistant bacteria excreted in human waste. Therefore, this would produce a null of no difference between levels of bacteria.
Finally, aims of his experiment are to determine the magnitude of bacteria grown in each environment, and to determine which types of bacteria are present.
Methods First, environmental samples of bacteria were obtained. This was carried out by taking a sterile swab with a sterile solution of phosphate buffered saline (PBS), and passing it over the surface where the bacteria were hoping to be obtained. The swabs were then taken into lab and swabbed onto agar only plates. Lawns of bacteria were grown after being incubated for 24 hours, and from these plates, more agar only plates were made from these and used as master plates. Once these were grown, colonies were taken and put onto patch plates, each containing an antibiotic including Kanamycin, Ampicillin and Tetracycline, and also a plate containing only LB base, which was used for a control. Individual colonies were grown in each of the 16 sections that the plates had been divided into, and these colonies were resistant to the antibiotic that was in the plate that they were grown on. To ensure that these plates stayed healthy, they were redone every 2 weeks. These were incubated for 24 hours, and then counted to analyze the antibiotic resistance of the bacteria from each environment.
Next, Gram stains were preformed. A colony of bacteria was diluted in water and placed onto a slide. To adhere the bacteria onto the slide, it was passed over a slide 2 to 3 times. The slide was then flooded with crystal violet for 60 seconds and rinsed with water. It was then flooded with iodine for 60 seconds and rinsed with ethanol. After, it was flooded with safranin as a counter stain and rinsed with water. The slide was air-dried. Immersion oil was placed on the slide and the stain was viewed at 100x magnification.
The KOH test was also conducted to determine if the bacteria was Gram positive or Gram negative. Bacteria was placed on a microscope slide and exposed to KOH. A metal hoop was touched to the mixture of bacteria and KOH. If the mixture stuck to the hoop, then it is Gram negative, if it did not stick to the hoop, it is gram positive.
Gel electrophoresis was used to separate DNA nucleotides into bands. The gel was made out MacConkey Agar, which when cooled, forms pores in the matrix to let the DNA run through when exposed to an electric current. To make the gel, either 40mL or 60mL of the TBE is mixed with either .4g or .6g of agarose respectively. These were heated, mixed together, and left to cool. Following, 2l of ethidium bromide was added. The DNA ran from the negative end of the cell to the positive end and formed columns of bands with specific lengths.
Plasmids were isolated from the environmental samples using a miniprep protocol. After antibiotic resistance was developed, the colonies were converted into liquid cultures. These cultures 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 solution. It was then re-suspended and lysed to remove genetic material and proteins. The sample was spun again, and the plasmids were drained and put into a spin column. Using nuclease free water, plasmids were removed from the spin column into a centrifuge tube. The isolated plasmids are then run through a gel electrophoresis, and using the NEB cutter V2.0 program from New England Biolabs, a plasmid map can be produced. After the plasmid was produced, it was run through gel electrophoresis and shown under UV light to confirm the procedure worked.
To inoculate liquid media, the following procedure was followed. 5mL tubes with liquid medium were obtained. Next, 5 L of appropriate antibiotic were added. With a sterilized loop, a single colony from an antibiotic streak plate was taken and added to the mixture. Finally, the tube was capped and placed in the shaker for 24 hours.
After plasmids were isolated, in this case only the blue control, the control plasmids and plasmids from competent E. coli cells were taken and a heat shock procedure was used to force the bacteria to obtain the plasmid. Three samples, two containing plasmids from blue control and from E. coli, and one with P. Litmus and LB only plates as controls, were heat shocked for ninety seconds, then mixed with warm LB broth and incubated. The samples were then spread onto an LB and antibiotic plate to determine resistance transferred from one bacterium to another.
A restriction digest was completed as follows. 10 l of the restriction enzyme, in this case Ava 1 was chosen, and 20l of the DNA from the miniprep were mixed. Next, 3l of the buffer number 4 were added to the mixture. 100l of BSA was added last. This concentration was then incubated for an hour. 3.3l of gel loading buffer was added after the incubation right into the reaction. The reaction was then stored in the freezer until we were ready 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 bacteria were then grown on LB only plates, which served as master plates for the bacteria in the experiment (Figure 1). All bacteria for the experiment were taken ultimately from these plates. Bacteria from these plates were then grown onto patch plates that contained Ampicillin, Tetracycline, and Kanamycin in the LB, so that only bacteria resistant to that antibiotic would grow (Figure 2).
Gram Negative bacterium grows lawns of bacteria on MacConkey plates. Using this test, the Déjà vu bathroom AMP resistant bacteria, and Burger King Play area TET resistant bacteria came out to be Gram negative. The Meijer bathroom AMP resistant bacteria and the Déjà vu bar AMP resistant bacteria came out Gram positive (Figure 3). Gram stain tests for both gram negativity and positivity. Gram-positive stains pink during a Gram stain, while Gram negative stains purple (Figure 4 ). The Déjà vu bathroom came up gram negative, while Meijer bathroom, Déjà vu bar, and Burger King play structure came up gram-positive (Table 1). The KOH test is sticky if it is gram-negative and not sticky for gram-positive bacteria. Déjà vu bathroom came up gram-negative, while the Meijer bathroom, Déjà vu bar, and Burger King play structure came back gram-positive (Table 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 bacteria to identify plasmids, and a control was run to ensure the test worked properly. No plasmids were found in the cultured bacteria (Table 3), and a second gel was run to ensure the result was correct (Figure 9).
EMB plates were run. Gram-negative bacterium grows on EMB agar. A bacterial colony from each cultured antibiotic resistant plate was taken and exposed to the EMB agar. The results were that all colonies exposed to the EMB were gram positive.
A chi square test was conducted. It included 1 degree of freedom, and had a significance value of 0.05. This meant that the Chi square distribution was at 3.84 (Table 4). Results said there was no definite difference in the amount of antibiotic resistant bacteria found in bathrooms than in highly trafficked areas.
A PCR was run to identify the bacteria by reproducing the DNA inside of it and comparing known samples of DNA to the ones being tested. These results have not been completed yet.
A restriction digest was also run, but correct results have not been yielded from this experiment yet. Bacteria has not yet been properly cut and observed on a gel electrophoresis experiment.
Discussion Written and Revised by Stacy Tipton
There is little difference in the amount of antibiotic resistant bacteria found in bathrooms versus found in highly trafficked areas. It was hypothesized that when the experiment was done to see which environment, the bathrooms or the highly trafficked areas, would have the greatest antibiotic resistance, that the bathroom would have greater resistance. This was due to the theory that the bathrooms are constantly exposed to antibiotics, where as locations such as a store shelf, stripper bar, or play area are rarely if ever cleaned with antibiotics. It was thought that the more the bacteria were exposed to antibiotic resistant bacteria, the more likely it would be that antibiotic resistance was naturally selected for. The results contradict the hypothesis. There was found to be little difference in the amount of antibiotic resistant bacteria in each location. According to the Chi square test, no significant difference between the two locations.
As we knew from previous research, exposure to antibiotics selects for antibiotic resistance. If the antibiotics that the bacteria is exposed to does not kill all bacteria, the strongest bacteria that are still alive reproduce and pass on their resistance both through reproduction and the passing of plasmids. Our findings suggest that when exposed to the same bacteria, it doesn’t matter how many times the bacteria are exposed to the antibiotics, once it is selected for, antibiotic resistance stays in the population.
There were specific problems in the research that could have affected the results. First, no plasmids were found in the bacteria. This means that the passing of the resistance via plasmid spreading could not be tested. Also, several experiments had to be done repeatedly to get results. This means that the exact same samples were not used, but reproduced as exactly as possible. However, since there is variation in which colonies were tested in each experiment, it is possible that the results are skewed somewhat.
There are several options for further studies in this experiment. Further experiments could be used to find plasmids in the bacteria. One could go farther to see if the antibiotics that were used were supposed to kill the bacteria found. Also, one could experiment to find out if the bacteria were resistant 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, Upper Saddle River, NJ
Cognato, A. 2010. Recitatation_031710 PowerPoint. Pages 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 resistance genes in samples isolated from Changchun, China. Molecular Biology Reports. Volume 37. 1857-1865.
Rusin, P., P. Orosz-Coughlin, C. Gerba. 1998. Reduction of faecal coliform, coliform and heterotrophic plate count bacteria in the household kitchen and bathroom by disinfection with hypochlorite cleaners. Journal of Applied Microbiology. Volume 85. 819-828.
Figures Written and Revised by Stacy Tipton
Figure 1: Swab Plates. Each different environment was swabbed and two different locations were taken from each. The bacteria were grown on an LB only master plate before exposed to any antibiotics. Each small dot on the plate represents a different colony of bacteria. These plates served as the master plate for all bacteria in the experiment and all tested bacteria ultimately came from this plate. The small dots throughout the plate in the figure each represent a different colony of bacteria. These could be the same species of bacteria, or there could be several species on one plate due to the fact that each environment the samples were taken from was exposed to all kinds of bacteria. Afterward, these bacteria will be taken and exposed to antibiotics to test for resistance. These bacteria were swabbed from an environment using a swab coated with PBS. A shows Burger King play structure, with individual colonies of bacteria. B shows Burger King bathroom with individual colonies of bacteria. C shows Déjà vu bar with a lawn of bacteria. D shows Déjà vu bathroom with a lawn of bacteria. E shows Meijer bathroom with a lawn of bacteria. F shows Meijer shelf with a lawn of bacteria.
Figure 2: Patch Plates. In the above figure A represents Meijer Bathroom Ampicillin, B represents Meijer Shelf LB only patch plate, C represents Burger King Play Structure, D represents Tetracycline Déjà Vu Bathroom, and E represents Déjà Vu Bar Ampicillin patch plates. Each cultured bacteria was taken from the original master plate and exposed to a different antibiotic plate. Each plate was divided into sixteen sections, and a different colony of bacteria from the same location were placed on each section. This means several different species of bacteria could be on one plate, but did not necessarily have to be. Bacteria grown on these plates are resistant to the antibiotic that is in the LB that they grew on. A, B, C, and F all had 16 colonies of growth, D had 8 colonies of growth. These are important in determining the amount of bacteria that was resistant in each environment so that statistical tests could be run to determine if there was significant difference in the amount of antibiotic resistance in each area. Ampicillin plates contained 100g of antibiotic permL, and Tetracycline and Kanamycin both contained 50g of antibiotic per mL.
Figure 3: MacConkey Plates. MacConkey plates test for gram-positive or gram-negative bacteria. Gram-negative bacteria grow on MacConkey plates, gram-positive bacteria does not. Clockwise from top left: Déjà Vu bathroom, Burger King Play Structure, Meijer Bathroom, and Déjà Vu bar. Only one colony of bacteria was selected to grow onto each plate, and therefore it is only one species of bacteria represented per plate. B and C have growth 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: Déjà Vu Bar AMP Gram Stain, B and C: Burger King Play Structure TET Gram Stain D: Déjà Vu Bathroom AMP Gram Stain, E: Meijer Bathroom AMP Gram Stain. The dark color purple color shows gram-negativity. Gram-negative bacteria lack a thicker cell wall, and have their layer of peptidoglycan more exposed than gram-positive bacteria. Gram stain goes about identifying gram negativity or positivity by coloring the layer under the wall. Since the wall isn’t as thick, the dye is able to better stain that layer of peptidoglycan. These are rod-shaped bacteria, otherwise known as bacillus shaped.The pink color illustrated gram positivity. Gram-positive bacteria have a thicker cell wall, which means their layer of peptidoglycan is less exposed to the environment. A gram stain goes about identifying gram negativity or positivity by coloring the layer under the wall. Since the wall is thicker, the dye is not able to stain that layer of peptidoglycan as well, and it shows up as a pink color. This is a lawn of bacteria, mostly coccus (round) shaped, but some are bacilli shaped as well.
Figure 5: Gel Electrophoresis Plasmid Isolation. The gel separates nucleic acids and proteins. Lane 1 shows the 1 KB ladder, which is used to help identify unknown plasmids by showing lanes of known base pairs. Lane 2 illustrates the blue control plasmid. This Plasmid ensures that the gel worked properly. A control of E. Coli bacteria was used. Lanes 3 through 6 show where a gel was run with lanes 1 through 6 loaded using a micropipetter, and where no plasmids were found. These lanes were samples form each of the environmental plates that had already been exposed to antibiotics. Meijer bathroom, Déjà vu bar, and Déjà vu bathroom in lanes 3 through 5 were all from Ampicillin plates. Lane 6 represents the sample from the Burger King play structure that had been exposed to tetracycline. It is obvious no plasmids were found, because there are no bright bands that appear in the gel in lanes 3 through 6. None of the environments had plasmids isolated from them. This was significant because resistance is transferred through plasmids. To make this gel, .4 g of TBE were mixed with 40 mL of agarose. After these cooled, .2L 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
déjà vu bathroom
Neg
Neg
Déjà vu bar
Pos
Neg
BK play structure
Neg
Pos
Table 3: Gel Electrophoresis Results Positive*
Negative*
Meijer bathroom
X
Déjà Vu bathroom
X
Déjà 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

Spatial Learning Ability of Male and Female Mice in Mazes

PREPARED BY:
ELANCHELIAN A/ L THEVER
FARAH HANI BINTI MUNJIAT
NURUL FATINIZZATI BINTI ABD HALIM
LUCIA EMPINA HARRY
NURFAZRINA BINTI JAINULABADEEN
INTRODUCTION:
The house mouse (Mus musculus) is a small mammal of the order Rodentia, characteristically having a pointed snout, small rounded ears, and a long naked or almost hairless tail. House mouse can live together with humans. The house mouse has been domesticated as the pet and the laboratory mouse, which is one of the most important model organisms in biology and medicine.
House mice have an adult body length (nose to base of tail) of 7.5–10 cm and a tail length of 5–10 cm. The weight is typically 10–25 g. In the wild they vary in colour from light to dark agouti (light to dark brown) but domesticated mice and laboratory mice are produced in many colours ranging from white to champagne to black. They have short hair, ears and tail have little hair. The hind feet are short, only 15–19 mm long; the walking way is a run with a stride of about 4.5 cm, though they can jump vertically up to 45 cm. The voice is a high-pitched squeak. House mice can live under various conditions: they are found in and around homes commercial structures, open fields and agricultural lands.
New-born males and females can be easily distinguished. From the age of about 10 days females have five pairs of mammary glands and nipples; males have no nipples. When sexually mature the most striking and obvious difference is the presence of testicles on the males. These are large compared to the rest of the body and can be retracted into the body. Tail has only a thin covering of hair as it is the main peripheral organ of heat loss and used for balancing.
House mice usually run, walk, or stand on all fours, but when eating, fighting, or orienting themselves, they rear up on their hind legs with additional support from the tail. This behaviour known as tripoding. Mice are good jumpers, climbers, and swimmers, and are generally considered to be thigmotactic, means maintain contact with vertical surfaces.
Mice are mostly nocturnal. The average sleep time is 12.5 hours per day. Mice are territorial, and one dominant male usually lives together with several females and young. Dominant males respect each other’s territory and normally enter another’s territory only if it is vacant. If two or more males are housed together in a cage, they will often become aggressive unless they have been raised together from birth.
House mice primarily feed on plant matter, but are omnivorous. They will eat their own faeces to acquire nutrients produced by bacteria in their intestines. House mice, like most other rodents, do not vomit. Mice predators are rats which often kill and eat mice, a behaviour known as muricide.
Humans and rodents share some similar characteristics and genetic similarities since both are mammals. This makes rodents, especially mice, very suitable lab animals to conduct experiments that cannot be done on human beings. There are many types of maze that have been created for different type of studies, from classic maze, T-maze, radial arm maze, Barnes maze and even water mazes. These maze studies are used to study spatial learning and memory in mice. Maze studies have given tremendous help to study the general principles about learning for a better understanding not just in mice, but also other species including humans. There are a lot of maze studies conducted by various researchers who are curious to know whether different treatments or conditions affect learning and memory in mice. Our experiment is mainly focused on spatial learning ability between male and female mice in finishing the mazes.
OBJECTIVES:
I. To test the memory power of mice from both sexes
II. To find out the intelligence mice from both sexes to find out way to get the food
III. To find out which mice (male or female) learn faster
HYPOTHESIS:
Female mice will learn faster than the male mice.
MATERIALS:
MAZE:
Glue
Scissors/ Penknife
Cellophane tape
Boxes
Transparent wrapping paper
USED IN THE EXPERIMENT:
Biscuits (to lure the mice)
Stopwatch (to record the time)
Video camera/DSLR
PROCEDURE:
3 mazes with different patterns were made. There were simple, moderate and complicated pattern.
The simple maze was labelled as A, moderate as B and the complicated one was labelled as C.
Male and female mice were prepared.
The foods were placed at the end of each maze. The foods used in this experiment were biscuits.
The male mouse was released first into maze A.
The time until the mouse reaches the food was recorded.
3 readings were recorded with 5 minutes interval for each reading.
Then, the male mouse was released into the maze B followed by maze C and the readings were recorded for each maze.
Step 5, 6, 7 and 8 were repeated by using the female mouse.
The average time taken for each mouse to reach the food in different mazes was calculated.
RESULT:
1st Maze
2nd Maze
3rd Maze
1st Trial
1min 59s
2min 30s
12min 41s
2nd Trial
1min 8s
1min 28s
3min
3rd Trial
44s
2min 13s
3min 8s
Average
1 min 17 s
2 mi 4 s
6 min 27s
Table 1 shows time taken for a male mouse to complete 3 mazes with 3 trials.
The mazes consist of vertical walls and a transparent ceiling. The rat starts in one location, runs through the maze, and finishes in another location where they were rewarded with biscuits as treat. In the first maze, we can see that the time taken for the male to complete the maze decreases as the male attempted three trials. During the first trial, it took 1 minutes and 59 seconds for the male to reach the end for the first time. After a 5 minutes interval, we proceed with the second trial and found that the male agility improved as he took a shorter period to complete the maze, that is 1 minutes 8 seconds. The last trial for the first maze only took 44 seconds to be solved by the male mouse. Less time is needed to finish the maze as the male mouse learned better, and perhaps remember better from solving the first and second maze.
In the second maze, different trend can be observed. The time taken for the male mouse to complete the maze fluctuated between the first and second trial by 1 minute 2 seconds as what we assumed them to be. However, the male mouse took a longer time to finish the third trial. Instead of taking a shorter period to solve the maze, he took 45 seconds more of his time to reach the end. This is due to the mouse behaviour, where during the experiment was carried out in the maze, eventually he will stop to clean himself for a few times, which extended the time taken to complete the maze.
When we first introduced the third maze to the male, he took the longest time to finish the maze by taking up to 12 minutes 41 seconds before he finally reached the end. When the experiment was conducted, we observed that the male mouse took a longer time to explore the maze rather than trying to complete the maze to earn his treat as he was already satiated. There’s also up to a point when the male would not move at a certain place, as if he was resting or getting bored with the maze. There’s a huge different between the first and second trial of the third maze where the male only took about 3 minutes to complete the maze, with a slight increase when he finished the third trial by an increase of only 8 seconds.
1st Maze
2nd Maze
3rd Maze
1st Trial
43 s
33 s
32 s
2nd Trial
42 s
39 s
30 s
3rd Trial
50 s
37 s
35 s
Average
45 s
36 s
32 s
Table 2 shows time taken for a female mouse to complete 3 mazes with 3 trials

Figure 1 shows the average time taken by the male and female mouse to complete all three mazes
From Table 2, it is clear that the time taken for the female mouse to complete the maze of all three mazes did not decrease consecutively as what we have hypothesized. The first maze shows a slight decrease by merely one second between the first and the second trial, but increased to 50 seconds in the third trial. The same pattern can be observed in the third maze. The second maze shows a different pattern where the female mouse took a longer time to complete the second trial than the first trial. Eventually, the female mouse managed to solve the maze in a shorter period during the third trial compared to the second trial.
Comparatively, the female is a faster learner compared to the male mouse. From figure 1, we can analyse that in average, the female took a shorter time to solve all three mazes compared to the male. However, the extreme data obtained when the male completed the third maze could have affected the average time for the male mouse. Astonishingly, the more difficult the level of the maze, the shorter time was needed by the female mouse to complete the maze. The first maze is the easiest level, followed by the second and the third. This is different from the male, where the more difficult it gets, the longer time is taken to complete the maze, just like what we hypothesized for this experiment.
DISCUSSION:
Shorter time in maze completion by the female mouse than the male mouse shows that the female is a better learner. Sex differences in spatial learning ability and memory performance is proven in this experiment, with the female mouse as the better performer. This is supported by a research where mating system type predicts the presence of a sex difference in maze performance (Gaulin et al., 1989) since mice practices polygamy. According to Gaulin, sex differences in completing the maze are generally present in polygamous species but not in monogamous ones. Other factor that might influence the time taken to complete the maze could be due to the gender morphological difference. The male mice are usually bigger and heavier on their feet. Comparatively, the females are light bodied, which makes them to have better agility to solve the maze puzzle.
The level of difficulties also affects the mice performance to complete the maze. The number of junctions increases from maze A, followed by B and C. The more junctions in the maze, the more decision the mice have to make to reach the end of the maze, causing more errors especially during the first trial. Over multiple trials, mice tend to run the maze with fewer errors and quicker, hence a shorter time. The possible proximal causation is it could be because of the hippocampus is involved in the mice explicit memories (Edward D.L. ,Jerry J.B., 2006) which helps them to memorize the correct turns over multiple trials, enabling them to learn better by reducing the errors. Looking into their evolutionary history, mice, like other small burrowing rodents, have been digging and navigating their way around underground tunnels which are similar to mazes. Solving maze could be their natural ability since mice can navigate their ways through the maze.
Olfactory system functions as the sensory system which is to be used for sense of smell. Smell is important sense in many species as it is also for life quality. Throughout this experiment, the mice used their olfactory system to find their way out of the maze. Mice is a special animal whereby they only uses the sense of smell and touch in order to survive well in captivity as they do not need sight or hearing. A mouse actually does not see colour. In fact, black-and-white sight only enables the animals to easily spot movement. Even in the total darkness, the mouse relies on other senses, touch, hearing and smell. Besides of using its sensory smell, the mouse uses its whiskers to feel the way around. Food has been placed at the end of each different maze which each type of maze represents different level of difficulties. One of the ways that the mice used in order to escape the enclosed maze is to follow the scents of the food (biscuits). The food releases its scent which also called odorants (chemical molecules that stimulates the olfactory system) in the maze. Therefore the mouse follows the trail by using its olfactory senses where the odorants are detected by receptors which is located in the olfactory epithelium which then signalling to the hypothalamus and back to the muscles, which then allowing them to find the food in the end of the maze.
It is an ethologically relevant procedure that takes advantage of the mouse’s tendency to use olfactory cue to forage for food. Mice learnt the odour discrimination for 5 minutes. After 5 minutes being isolate from the maze, the mouse seemed to know where it has been throughout the maze which that explains the reason why the mouse got so fast in ending the maze. One of the ways that we observed throughout the experiment is the mouse also urinates in some of the mazes. This is because besides using the scent that the food has released, it follows the faint scent trail of urine to find its way to the finish line.
However, the result obtained could be bias since there are several factors that might have caused the mice to take long time to reach the end of the maze.
Mice is satiated:
Since mice already satiated, they does not attracted or interested to the food. In some observation, the mice show behaviour where they only see the food from few centimetres from them. Besides, they didn’t go to get the food until a few second after they show this behaviour. Then, they just go close to the food but did not eat the food.
Mice suddenly sit down to start to clean themselves:
Most of the observation, mice suddenly sit down and start to clean up themselves. This behaviour repeatedly occurred in some observation; maybe they become tired and not interested in running in the maze.
Mice urinate and defecate:
Sometimes, mice urinate and defecate in the maze. This factor will affect the time taken of the mice to reach end of maze. It is either they become disturbed with the scent of their own excretion or use it as an advantage to trail the end of maze.
Error that we encountered during handling this experiment:
The first error that occurred in this experiment was when the mice did not want to approach the food and ended up resting in the maze. This may be because the mice were not hungry. The second error is that the mice tend to give up in finding food. This error might occur because the mice get tired or maybe because the pathway was long.
The third error was accuracy in recording the time. At some time, we recorded the starting time and ending time slightly shorter or longer from the exact time since we got distracted in watching the mice in the maze. The next error was wrong selection of food. At first, we placed grain at the end of maze and we found out that the mice didn’t seem to be attracted to it. Then, we decided to put biscuits instead of the grains. The mice turned out to get attracted to the biscuits.
Other error that occurred was the materials that we used to make the maze. The mazes were made by using the boxes, which absorbs the urine of the mice and release bad odor. This odor caused the mice to get distracted.
The last error that we encountered in this experiment is that the environment during the experiment and the way in handling the mice. This experiment was conducted in a bright room. This may be the reason the mice do not wanted to come out from the box where they were placed since mice are sensitive to light. And also, the way we handled the mice could be one of the reason the mice stayed inside the box. Maybe the way we handled the mice were not that proper, causing them to think that we might harm them.
Precautionary steps and suggestions
Do not give the mice any food one day before experiment start. This is to starve them, making them to be attracted and interested to the food placed at the end of maze. Therefore, mice will take less time to reach end of the maze.
Studies show that playing classical music will increase their learning abilities. Mice exposed to classical music will take shorter time to reach the end of the maze.
Clean up faeces and urine of the mice might help to reduce the time taken of mice to reach end of maze. Besides, mice can detect the scent of the food effectively without being disturbed by the scent of their own excretion. We should have used more suitable materials to make the maze such as plastic-based materials or polystyrene so that the urine of the mice will not be absorbed. We can also clean the maze if these materials were used. No bad odour and no disturbance for the mice in finding the food.
Do the experiment in an environment that is conducive for the mice. Mice always prefer dark environment. We should also practice the correct ways in handling the mice.
Other than that, this experiment should be done by at least 3 people at a time so that the time can be recorded more accurately as there are more people to observe the mice in the maze.
REFERENCES:
Edward D.L. and Jerry J.B. (2006). Animal Models of Cognitive Impairment. Boca Raton, FL: CRC Press. Retrieved from http://books.google.com.my/books?id=VRi-mHJDOVsC

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