Biology 11 Bacteria Inquiry

Background Research

Staphylococcus is a group of bacteria that can cause a big variation of diseases. It is a gram positive bacteria that is also recognized as cocci. Staphylococcus is generally found in the nose and on various areas of the skin. This bacterium can easily and quickly be passed on even between species. It is passed on sometimes just by physical touch. Staphylococcus originally comes from the Greek word staphyle, which means a bunch of grapes. The bacteria came to its name during the research of staph bacteria because under the microscope it had a close resemblance to exactly that, grapes. It was found that 25%-30% of healthy adults carry the staphylococcus bacteria, and that 25% of hospital workers also carry it. It is important to know that staphylococcus infections are not caused just by having the bacteria. Staph infection occurs when there is a direct infection and/or by the toxins that the bacteria produces. There are more than 30 different types of staphylococcus, but the most common form is called staphylococcus aureus. This form of the bacteria has a huge affect on people everyday. It is one of the five most common bacteria that cause infections once an injury or surgery has occurred. On average, staph aureus infections effects 500,000 patients every year in American hospitals. Anyone is susceptible to staph infections, but there are a few groups of people that are much more at risk. These groups include, newborns, breastfeeding women, and people that are suffering from chronic illness. There is a pretty sizeable list of illnesses that can than lead to developing staphylococcus infection; such as skin infections, pneumonia, food poisoning, toxic shock syndrome, and blood poisoning. Another aspect of the staph bacteria is known as MRSA or Methicillin-Resistant Staphylococcus Aureus. This variation on the bacteria is carried by an average of 2% of our world population. It is harder to treat because of the resistance it has built up against methicillin. Antibiotics are usually what is used to treat bacterial infections such as staph infection. Antibiotics can be very dangerous if they are used to fight viruses, if they are used when not necessary, and if the prescription is not taken as by the instructions. All of these are examples of ways that bacteria can grow antibiotic resistance therefore doing more harm than good. When certain bacteria become resistant, it means that they can’t be killed off by that antibiotic and will continue to grow rapidly inside your body. Amoxicillin is a type of antibiotic that is commonly used to fight staph infections. It is a semisynthetic antibiotic that can kill off gram-positive and gram-negative bacteria. It is stable in the presence of gastric acid (which is important depending on where the infection is), and works the fastest after being administered orally. Amoxicillin is very close to other antibiotics like penicillin and ampicillin. Amoxicillin fights infections by the inhibition of cell wall biosynthesis. Research has shown that another way to fight staphylococcus is by using natural oils such as tea tree oil. Tea tree oil, otherwise known as melaleuca alternifolia, was originally used in Australia and is an anti-septic, anti-inflammatory, and anti-microbial. Tea tree oil is made up of terpene hydrocarbons such as monoterpenes and sesquiterpenes in addition to the needed associated alcohols. This natural oil can also be used to treat acne, fungus infections, and athlete’s foot. It should be a pale yellow or colourless and transparent. It has a camphoraceous (strong scented, white crystalline compound) odor. Tea tree oil can only be applied topically. It is poisonous if consumed through or around the mouth.

Big Question

What will be the difference in bacterial growth (staphylococcus) when antibiotics are used (amoxicillin) vs when natural oils (tea tree oil) are used to kill the bacteria?

Hypothesis

If antibiotics are used to kill off the staphylococcus bacteria, the bacterial growth will be much smaller then if tea tree oil was used to kill the bacteria. This is because, antibiotics were designed to have the necessary amount of strength to kill bacteria, but tea tree oil was not originally designed for use against bacteria, therefore it won’t have the same amount of power as antibiotics.

Safety Concerns

In order to stay safe during this inquiry, it was important to wear goggles and an apron at all times. They were needed because there were many elements that were being dealt with such as, fire, oils, antibiotics, agar, and a variety of dyes. Another risk, was broken glass because there were many beakers, stir rods, and petri dishes being moved around that could have easily broken. Luckily nothing did break, but if it would have, the proper thing to do would be to let Mrs.Becker know, and to put broken glass in the broken glass bin. Another thing to be conscious of was the agar and amoxicillin, which were both a fine white powder that could have easily been consumed through the nose or mouth. It was important not to ever smell chemicals or compounds or taste them. If the odor would have been an important observation, the odor would have to be wafted to the nose. Bunsen burners and hot plates were also used in this inquiry, in order to stay safe, it was important never to touch the burners flame or directly touch the hot plate surface. Flammable materials needed to be a safe distance away from the burner and plate and once finished, it was important to shut off the gas and unplug everything. If the hot plate was no longer needed but was still hot, it was necessary to place it on top of the shelf to alert others not to touch it. The last thing that was a big safety concern was the dyes and liquids that were being used in the process of gram staining. It was very important not to spill the liquids and to try not to get them on skin. If they were spilt, they needed to be cleaned up immediately from either the counters, floor, or skin where they were spilt.

Variables

Independent variable: Amoxicillin to kill bacteria vs tea tree oil to kill bacteria

Dependant variable: The surface area of bacterial growth

Control variable: Staphylococcus bacteria

Procedure

1) Make agar plates-sterilize 6 petri dishes-use scale, scoopula, and element boat to measure out 2.8g of agar-mix agar into large beaker filled with 200mL of distilled water using a stir rod-place beaker on hot plate and heat until boiling and the liquid is almost completely transparent-allow to cool -fill each petri dish ¾ of the way full with agar liquid-place in fridge to set overnight

2) Once agar has set, use a marker to label and split the petri dish into 6 different spaces3) Get 3 boys and 3 girls to each place a thumbprint into one of the separated spaces of the set

agar4) Place petri dishes in room temperature incubator overnight5) Once set, examine agar to see which fingerprint has the staphylococcus bacteria- (white,

circular bacteria) (it may be necessary to examine under a microscope) 6) Following step 1, make more agar plates7) Cut out 24 small circular pieces of filter paper (approx. 5mm around) and put aside8) Fill a small beaker with approximately ½ an inch of distilled water, and mix in the amoxicillin

from a 500mg capsule9) Fill a separate small beaker with approximately ½ an inch of tea tree oil10) Place 12 circles of filter paper in each beaker11) Let filter paper soak while labeling the petri dishes:

-using an marker, draw a line down the bottom of the dishes splitting them in half-label each side of each petri dish with the variable that will be held in that side and the trial number

12) Once labeled, use one end of a Q-tip to swab the hand of the person who had the staph bacteria. To swab:-dip Q-tip in distilled water, use Q-tip to rub on hand

AntibioticTrial 1

AntibioticTrial 2

Tea tree oilTrial 1

Tea tree oilTrial 2

-take the Q-tip and rub gently on one side of the agar plate (use new Q-tip for each side)-swab for each side of each agar plate, making sure each swab line is similar size and shape

13) Using tweezers, pick up filter paper circles from the antibiotic beaker. Place 3 pieces randomly spaced along each swab line for the antibiotic trials

14) Do step 13 again, this time using the filter pieces from the tea tree oil beaker and placing only in the tea tree oil trials

15) Do not place anything on the control trials16) Place lids on petri dishes and places the dishes in the body temperature incubator overnight17) Once they have been incubated overnight, take out the petri dishes and observe their

bacteria growth18) Take pictures of each petri dish19) Using 1cm grid paper, trace each petri dish and make an approximate drawing of the spaces

that were without growth20) Using the drawing for each petri dish, calculate the surface area of bacteria growth to later

use for a data table21) Gram stain the bacteria

-get out a gram staining bin (containing the necessary supplies)-place a drop of distilled water on a clean glass slide-sterilize a bacterial loop using a Bunsen burner and pick up a small amount of the bacteria-rub the bacteria into the distilled water on the slide-allow the smear to air dry-heat fix the slide by passing it through the Bunsen burner 3-4 times, allow slide to cool-flood the prepared slide with crystal violet stain and let stand for 1 minute-rinse slide gently with distilled water-flood the slide with gram iodine solution, let stand for 1 minute-rinse slide gently with distilled water-rinse slide with alcohol for 5-10 seconds (complete when solution runs clear)-rinse with distilled water-flood slide with safranin stain, let stand 1 minute-rinse slide with distilled water-blot slide dry with a kimwipe and observe under the oil immersion lens of the microscope-you will know if bacteria is gram positive or negative based on the colour it is stained (gram positive=purple stain and gram negative=pink stain)

22) When observing under the microscope, examine for the morphology of the bacteria23) Using a camera phone, take pictures of the bacteria through the lens 24) Use collected data to make inquiry write-up

Observations

In observing the growth of the staph bacteria, I observed that I only grew one type of bacteria. There was such a high level of growth in the control trials, that the entire petri dish was covered. In the antibiotic trials, the petri dishes were almost completely covered, and there was absolutely no

Tea tree oil trial Antibiotic trial Control trial

Gram stain of bacteria under oil immersion

bacteria growth in the tea tree oil trials. At first glance, I noticed that the bacteria were white and semi-transparent. It looked almost cloudy or foggy. Looking under a microscope I was able to tell that the bacteria were stained purple therefore were gram positive. The bacteria were shaped in chains of circles, telling me that the bacteria were cocci and the margin was entire. Overall, the biggest observation I had was how quickly and vastly the bacteria grew.

Surface Area of Growth

Morphology Form

Morphology Elevation

Morphology Margin

Gram +/-

Trial 1 22.85 cm² Circular Raised Entire PositiveTrial 2 35.55 cm² Circular Raised Entire PositiveTrial 3 21.3 cm² Circular Raised Entire PositiveTrial 4 26.2 cm² Circular Raised Entire PositiveAverage 26.5 cm² Circular Raised Entire Positive

Data Tables

Antibiotics:

Tea tree oil:

Surface Area of Growth

Morphology Form

Morphology Elevation

Morphology Margin

Gram +/-

Trial 1 0 cm² Circular Raised Entire PositiveTrial 2 0 cm² Circular Raised Entire PositiveTrial 3 0 cm² Circular Raised Entire PositiveTrial 4 0 cm² Circular Raised Entire PositiveAverage 0 cm² Circular Raised Entire Positive

Control:

Surface Area of Growth

Morphology Form

Morphology Elevation

Morphology Margin

Gram +/-

Trial 1 35.0 cm² Circular Raised Entire PositiveTrial 2 35.8 cm² Circular Raised Entire PositiveTrial 3 24.5 cm² Circular Raised Entire PositiveTrial 4 23.1 cm² Circular Raised Entire PositiveAverage 29.6 cm² Circular Raised Entire Positive

Conclusion

This experiment disproved my hypothesis. The tree tea oil didn’t allow for any growth of staphylococcus, while the antibiotics only killed off a very small percentage of bacterial growth.

Sources of Error

Like most inquiries, there were many errors that occurred in the process of my experiment. The first error that occurred was with my agar plates. Because agar was vital for this experiment, I had to focus on fixing that error first. When I tried to make the agar the first time, I didn’t transfer it to the fridge carefully enough, and it spilt all over the tray, making it un-useable. When I tried again to make agar plates, I was very careful not to spill any, and the next day, it seemed like it had set properly so I was able to swab. After my swab had been incubated overnight, I realized that the agar that I had used to make the plates was bad. It hadn’t actually set properly and so I had to remake the agar plates again. This error set my inquiry back quite a few days because I also had to re-swab all the bacteria and get more of my variables (antibiotics, tea tree oil). Another error that occurred, was out of my control, but still affected the inquiry. My original question had to do with expired antibiotics vs non-expired, but I quickly realized that it wasn’t as easy to get expired amoxicillin as I’d hoped. This error forced me to re-think my inquiry and come up with alternate questions and solutions. Once I had resolved these errors and continued on with my inquiry, more errors occurred. While I was swabbing for the last time, I didn’t properly use the Q-tips, and ended up using the same side of one twice. Luckily I hadn’t done a full swab before realizing so I was able to swab over it properly. This could have really set me back had I continued swabbing wrongly because I would have had to remake the agar and swabs again. After having swabbed and incubated my agar plates, the control came out with almost a foggy layer covering the bacteria. As it turned out, this was because the distilled water that I had used for the agar plates, wasn’t completely distilled. Luckily this didn’t have a huge effect on my data because all the bacteria that grew was the same, so I was able to swab and gram stain from the other agar plates. After having gram stained the bacteria, I examined it under a microscope that hadn’t been used properly by the last person, so I originally thought that I hadn’t grown the right type of bacteria. Had I not tried another microscope this incorrect data would have greatly affected my data and results. My final source of error, was due to time and resources. I was only able to do 4 trials of each variable, which gave me enough data, but if I were to do this again, I would want to do many more trials to collect more concrete data.

Discussion questions

The results of my inquiry impacted what I know about bacteria by giving me a new understanding on how quickly bacteria can grow. The results also showed me that all bacteria are completely different, and things that we thought would kill off bacteria might not have. Bacteria definitely

cannot be generalized based on the name or morphology. Every single type of bacteria is different in some way. I learned more about how bacteria reacts differently depending on the environment and variables. I didn’t originally realize how such seemingly similar variables, could have such a high impact on the difference in growth of bacteria. Every little change within the different variables and trials, greatly affects the data that we find.The research that I was able to do, was very minimal compared to what can be done. I think that one of the best ways to take this idea farther would be to further study and analyze the variables. If we were to look deeper into tea tree oil, we could research what parts of it exactly are able to prevent the growth of staphylococcus. Is staph the only bacteria that tea tree oil would affect? In my experiment, I found that tea tree oil didn’t allow for any growth of staph, but to take it farther, I think it would be beneficial to see the effects of this oil, after there has already been bacteria growth. For example, taking the control and then applying tea tree oil. The same thing could be done with the amoxicillin. It would be beneficial to research what elements of it actually fight staph, and then to take that data and compare it to other antibiotics to see if there is a more effective one to use. Just like with the tree tea oil, the research could also be taken farther by applying the antibiotic after there has already been bacterial growth.I think that the data I collected could have a positive affect on society by demonstrating that in some cases, such as with staphylococcus, it can be more beneficial to use natural oils and medications and opposed to antibiotics. My experimental data proved tea tree oil to be much more affective against staph than amoxicillin. In our society, I believe that this information can be used to improve the care of certain cases dealing with staph, and this data could be used to further research other natural medications and how they can improve our health.