Fight BAC!

	Project title: Fight BAC!
Difficulty level:	Middle school
Main link:	None

Hypothesis:

Based on knowledge aquired from a Microbiologist, i hypothosize that various types of bacteria will be found on different regions of teh human skin -namely, the palm of the hand, the skin behind the ear, the skin between the toes, and teh axilla- all of which most bacteria will be resistant.

Materials:

20mL of Buffered Saline Solution
33 cotton tipped Applicators
33 Petri Dishes
490mL of Tryptic soy Agar
82 disposable Inoculating loops
21 Microscopic slides
100mL of Disinfectant
Incubator
Microscope
21 Penicillin discs
Grease pencil
Hot plate
Staining rack
Forceps
Gloves
50mL of Crystal violet
50mL of Iodine
100mL of Alcohol
50mL of Safranin
Vitek card reader
6 Vitek cards (gram + bacterium)
1 vitek card (gram – bacterium)
7 test tubes
Hot water bath
lab coat
ruler
Protective eyewear (goggles)
Draining rack
21 Mueller Hinton Agar plates
Biohazardous waste container
Digital Camera

The Procedure:

SAFTEY NOTES:

1) Ensure that a lab coat, safety goggles, and latex or vinyl gloves are worn throughout the experiment.
2) Ensure that all materials used during the experiment are sterile.
3) All pipets, disposable inoculating loops, cotton-tipped applicators, and any other materials that come in contact with bacteria, must be placed into a disinfectant container before they are disposed of.
4) Ensure that hands are kept away from the face while handling bacteria.
5) Ensure that work areas are disinfected with rubbing alcohol at the beginning and end of each lab period.
6) Ensure that the Universal Precautions are followed.
7) Ensure that hair is kept well away from working areas.

PART A: CULTURING AND IDENTIFYING SKIN BACTERIA

a) Preparing Agar Plates

1. Loosen the cap on a bottle of Tryptic Soy Agar.
2. Prepare a hot water bath with a temperature of approximately 95ºC.
3. Place the bottle of agar into the hot water bath until the agar liquefies.
4. Lift one side of a Petri dish lid about 1 inch. Pour approximately 15-20mL of the melted agar into the dish.
5. Close the lid of the dish and gently move it around so that the melted agar evenly covers the whole surface.
6. Once the agar has solidified, turn it upside down and leave it in a refrigerator until you are ready to place bacteria inside.
7. Prepare 31 additional plates, using additional bottles of agar when necessary. These dishes will be needed in order to do 3 trials of the experiment.

b) Culturing Bacteria

1. Dip a cotton-tipped applicator into a sterile phosphate buffered saline solution so that the tip is wet.
2. Gently swab the palm of your hand.
3. Next carefully open the lid of a Petri dish. With a twisting motion, gently rub the applicator on the agar plate in one place. Upon doing so, place the used applicator into the biohazardous waste container.
4. With a sterile inoculating loop, make a zigzag pattern on 1/3 of the Petri dish containing the nutrient agar. Begin the zigzag pattern where the above-mentioned applicator made contact with the agar. Refer to figure 1: First Streak. Once this step is complete, discard the used applicator into the biohazardous waste container.
5. With another sterile inoculating loop, dilute out the first zigzag by streaking the bacteria in another zigzag pattern as seen in Figure 1: Second Streak.
6. Upon completing this step, discard the used inoculating loop in the biohazardous waste container.
7. Using a new sterile inoculating loop, dilute out the second zigzag by streaking the bacteria from where it was left off, to another clean area, thereby covering the remaining third of the plate. Refer to Figure 1: Third Streak.

NOTE: AVOID TEARING THE AGAR SURFACE WHILE CONDUCTING THE STREAK PLATE TECHNIQUE. USE THE INOCULATING LOOP ON A SLANT.

8. Cover the Petri dish with its lid and carefully turn it upside down.
9.
10. Using a grease pencil, label the dish with the date, the time, and the location of the body where the bacteria were obtained from.
11. Place the dish in an incubator set at 35ºC for 24 hours.
12. Repeat the previous 8 steps with two other prepared agar plates.
13. Repeat steps 1-10, swabbing the skin between the toes instead the palm of your hand.
14. Repeat steps 1-10, swabbing the skin behind the ear instead of the palm of your hand.
15. Repeat steps 1-10, swabbing the axilla instead of the palm of your hand.

c) Sub-Culturing

1. Take the plates out of the incubator. Examine all 12 Petri dishes that were cultured from the previous part of the experiment, in order to identify similar looking colonies that represent identical types of bacteria. (look at there shape, size, and colour)
2. With an inoculating loop, transfer a small amount of one bacteria type on to another clean agar plate by streaking it in the 3-streak plate technique described earlier.
3. Upon completing this step discard the inoculating loop.
4. Label the dish with the date and time, using a grease pencil.
5. Place the dish in an incubator that is set at 35ºC for 24 hours.
6. Repeat steps 1-6 with 6 other identical looking colonies representing same type of bacteria.

d) Identifying Skin Bacteria: Gram Stain Technique

1. With the aid of a pipet, prepare a good smear by placing one drop of sterile phosphate buffered saline solution on a microscope slide.
2. Examine all 12 petri dishes that were sub-cultured from the previous part of the experiment, in order to obtain a pure culture of the type of bacteria.
3. With a sterile inoculating loop, transfer a small amount of bacteria from a Petri dish sub-cultured earlier. Place the bacteria in the drop of saline and mix it so the suspension on the microscope slide is milky.
4. Place the slide on a hot plate that is set at 80ºC.
5. After 10 minutes, the suspension should be dried, and ready to be gram stained.
6. To stain, pick up the slide off the hot plate using forceps, and lay the slide on a staining rack in a sink.
7. Then, squirt Crystal violet stain on the slide, so that the slide is flooded with purple dye.
8. Leave the dye on for a minute, and then rinse carefully.
9. Repeat steps 7 and 8 one more time using the same slide; however substitute Crystal violet stain, with iodine.
10. Then repeat steps 1 and 8 once again, this time substituting iodine with alcohol, and immediately rinse without the one minute delay.

11. Finally, repeat steps 7 and 8 for the last time using safranin instead of alcohol and rinse after 30 seconds.
12. Once the test has been completed, leave the microscope slide on a slant, so the excess water can drain off.
13. Once the slide is completely dry, observe it under a microscope. The slide will either be purple, indicating a gram positive bacterium, or light pink representing a gram negative bacterium.
14. Discard all materials that came in contact with bacteria.
15. Repeat steps 1-14, with other commonly found colonies that were on the precultured Petri dishes.

e) Identifying Skin Bacteria: Vitek test

1. Take 5 mL of sterile phosphate buffered saline solution and place it in a test tube.
2. Then with an inoculating loop, transfer a small amount of bacteria from on of the sub-cultures (prepared earlier), and gently mix the solution.
3. After that, introduce the solution to a vitek card through a micro straw, which will suck the solution into the wells located in the card.
4. Place the card in a vitek card reader/machine.
5. Wait for the card reader to identify the type of bacteria. This may take 15 minutes to a few hours, depending on the type of bacteria.
6. Discard the inoculating loop, into the biohazardous waste container.

PART C: SUSCEPTIBILITY TO PENICILLIN

1. First, take a sterile cotton tip applicator and dip it in the saline/bacterial solution that was prepared earlier for vitek test in the test tube.
2. After that has been done, gently squeeze the tip of the applicator against the side of the test tube to drain the excess bacterial solution.
3. Then take the same applicator and apply a streak technique in a new Mueller Hinton agar plate, which has been labeled, dated and timed.
4. To streak the plate, hold the inoculating loop on a slant and streak on the plate in every direction. Refer to figure 3 to apply the streak.
5. Then, using a pair of forceps, place a penicillin disc in the center of a Mueller Hinton agar plate.
6. Replace the lid, and leave the plate at 37°C in an incubator.
7. After 24 hours, take the plate out of the incubator, and observe thoroughly.
8. You will notice a clear zone (a circle) around the disc. The bigger the zone, the more antibiotic is effective to the bacteria, and the smaller the zone the less effective the bacteria is. If you notice there is no clear zone, it means the bacteria are completely resistant to the penicillin.
9. Using a ruler, measure the approximate diameter of the zone of inhibition.
10. After that has been done, the diameter will show you which bacteria is the most susceptible to penicillin and which is the least susceptible.
11. Record results.
12. To finish the experiment, dispose everything used into a disinfectant container.

Results:

By conducting this experiment, I determined that numerous types of bacteria can be commonly found on different parts of the skin, such as, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus anthracis etc. Many bacteria were hard to find because they are very rare. Different people carry different bacteria on their skin, but most of them are the same. I also found out that most of the skin bacteria that I cultured were resistant to penicillin, except for Streptococci pyogenes. I also noticed that most of the skin bacterium are getting resistant to penicillin every. Over the years, Staphylococcus aureus is modifying itself against antibiotics and is, according to microbiologists, getting resistant to almost all antibiotics. Consequently, research is needed in order to develop new antibiotics which many bacteria will be susceptible to. Without new antibiotics, many diseases can be caused to people who carry those types of bacteria on their skin, especially hospitalized elders.