BIO 105 Science of Biology w/Lab

Experiment 1Antibiotic Sensitivity Materials Candle

Matches

Yeast Packet

7 mL Sterile Saline

1 Tsp. Sugar

Nutrient Agar

*Distilled Water

*Bleach

*Microwave or Boiling Water Bath

*Warm Water

*Isopropyl Alcohol

*Permanent Marker

Experiment Inventory

Labware Parafilm®

(4) 9 cm Petri Dish

*3 Ampicillin Disks (Marked AM10; disk potency

= 10 ug)

250 mL Beaker

Forceps

Ruler

3 Standing Test Tubes

1 Pair of Disposable Gloves

Reusable Metal Inoculation Loop

*3 Kanamycin Disks (Marked K30; disk potency

= 30 ug)

Mueller-Hinton Agar

Test Tube Rack

6 Sterile Cotton Swabs

*3 Tetracycline Disks (Marked Te30; disk potency

= 30 ug)

2 Sterile Transfer Pipettes

*Hot Pad

*Measuring Spoons

*Scissors

*Lab Notebook (optional)

Note: You must provide the materials listed in *red.

*Antibiotic disks and Mueller-Hinton agar should be stored at 40 – 45 ºF until use. Allow to reach room temperature

before use.

EXPERIMENT 1: ANTIBIOTIC SENSITIVITY The Kirby-Bauer disk diffusion test is a method for determining the resistance of pathogens to various antibiotics. The test is a multistep process that begins with culturing pathogens of interest, such as bacteria, and then creating a suspension in saline. The suspension is then plated onto Mueller-Hinton agar followed by the addition of antibiotic disks (filter paper impregnated with determined amounts of antimicrobics). Antibiotic resistance is then observed based on the inhibition of growth in zones around the disks. This cleared area is known as “zones of inhibition” and is measured in millimeters across the diameter of the zone, including the disk. Larger zones of inhibition indicate that the pathogen of interest has a low resistance (high sensitivity) to the antibiotic of interest. In contrast, smaller zones of inhibition are indicative of high antibiotic resistance (low sensitivity). The results are used to determine the appropriate antibiotic or method of treatment of the infection. In this experiment, you will perform a disk diffusion test on various pathogens to determine resistance to three antibiotics.

Staphylococcus is a genus of bacteria commonly found on the skin of humans. In healthy humans, the presence of some species on the skin such as Staphylococcus epidermidis is normal and harmless; but Staphylococcus epidermis and other species such as Staphylococcus aureus can be harmful or evenly deadly when they cause infection. Affected individuals, individuals with a deep wound, or individuals who ingest food contaminated with Staphylococcus are susceptible to a staphylococcal infection. Since Staphylococcus is a bacteria, an infection can often be treated with antibiotics, but many species of Staphylococcus have shown an increase in antibiotic resistance. In this experiment, you will sample microbes from your skin to test for the antibiotic resistance of a bacterial lawn. Your skin may or may not contain Staphylococcus; any results indicating antibiotic resistance from your skin sample should not be cause for alarm, as these may be from other unrelated species.

PROCEDURE

Note: If you are keeping a lab notebook, record the date, time, and experiment title on a fresh page before you begin.

Prepare Agar Plates 1. Loosen or remove the cap on the nutrient agar bottle.

2. Place the bottle in a microwave. You will need to remove the bottle from the microwave and swirl the contents every 10 seconds to evenly distribute the heat. If you do not have a microwave, place the bottle in a heat-safe bowl, create a water bath by pouring boiling water into the bowl (around the bottle), and heat until the entire bottle of agar is liquefied.

Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. Once boiling has stopped, use a hot pad to protect your hands and remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!

3. With a hot pad protecting your hands, remove the agar bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!

4. Gently swirl the bottle a final time to mix the solution.

5. Pour the agar solution into the bottom half of one 9 cm Petri dish. USE THE AGAR SPARINGLY. YOU ONLY NEED ENOUGH TO COAT THE BOTTOM OF THE PETRI DISH.

Note: Approximately 5 – 10 mL should be enough agar to cover the entire bottom of the dish. If not, continue to add agar in approximately 1 mL increments until the bottom of the petri dish is completely covered. The agar should be only 1 or 2 millimeters deep. If too much agar is poured, there will be no space under the cover for microbial growth.

6. Place the lid onto the dish slightly off-center to create a small space to allow condensation to evaporate and allow the agar to solidify undisturbed. This should take approximately 30 – 60 minutes. If you will not be using the dish immediately, you can store it upside down in the refrigerator after it has fully gelled. Remove the dish from the refrigerator, and allow it to sit at room temperature for at least 1 hour prior to use.

Prepare the Yeast Sample 1. Measure 230 mL of warm water into a 250 mL beaker.

2. Add 1 tsp. of sugar and the yeast packet to the beaker. Gently stir the solution to dissolve the ingredients. The solution should begin to foam/froth after a few minutes.

Obtain Samples and Inoculate Nutrient Agar 1. If the nutrient agar has been refrigerated, allow it to sit at room temperature before obtaining samples.

2. Use a permanent marker on the bottom of the nutrient agar plate to section it into three equally sized areas. Label one section “Skin,” the second “Local,” and the third “Yeast.”

3. Use a sterile cotton swab to obtain a microbial sample from your skin such as your forearm. To do this, moisten the tip of the swab with a small amount of distilled water.

4. Firmly rub the damp cotton swab on your skin multiple times in a back-and-forth motion, swirling the swab as you rub.

5. Remove the lid from the nutrient agar plate, but hold it closely over the top of the plate to use as a shield to prevent airborne contamination of the plate.

6. Carefully streak the skin-sample cotton swab onto the gelled nutrient agar in the section labeled “Skin.” To do this, start at edge of the plate and move the swab along the surface of the agar in a zig-zag motion until you reach the narrow end of the “Skin” section. Ensure that you do not press too hard when streaking the swab on the agar to prevent cutting into the agar surface.

7. Place the lid onto the agar plate, and set it aside. Dispose of the skin cotton swab in a trash receptacle.

8. Use a new sterile cotton swab to obtain a sample from your local environment such as the surface of a doorknob, the handle of a phone or a countertop. To do this, pour a small amount of distilled water over the swab without touching the lip of the distilled water container to the swab.

9. Firmly rub the damp cotton swab on the surface of the local environment in a back-and-forth motion multiple times, swirling the swab as you rub.

10. Repeat Steps 5 - 7 for the sample from your local environment using the section of the nutrient agar plate labeled “Local.”

11. Use a disposable transfer pipette to add 8 – 10 drops of the yeast solution to the palm of your nondominant, gloved hand. Make a fist with your yeast-covered hand to spread the yeast over the surface of the palm your hand.

12. Using a sterile swab and your dominant hand, firmly rub the cotton swab on your yeast covered hand in a back and forth motion multiple times, swirling the swab as you rub.

13. Hold the yeast swab with your index finger and thumb. Be careful not to let it come into contact with any surface. Use the free fingers of your dominant hand to remove the glove from your non-dominant hand. Do this by inserting your free fingers under the lip of the glove and pulling up and away from your body to remove the glove.

14. Using the free fingers of your dominant hand, put a new glove on your non-dominant hand. Do this by using your free fingers to guide a new glove over your non-dominant hand.

15. Repeat Steps 5 - 7 for the yeast sample using the section of the nutrient agar plate labeled “Yeast.”

16. Seal the closed nutrient agar plate with Parafilm®, let it incubate in a warm location (not to exceed 37.7. °C or 100 °F), and observe the growth. You should see colonies form in two to five days, depending on the temperature at which the plates are incubated (the warmer the incubation temperature, the quicker the colonies will form).

17. In preparation for inoculating the Mueller-Hinton agar plates, make sure to allow the agar plates and antibiotic disks to come to room temperature if they have been stored in a refrigerator or freezer. The suspension to inoculate the plates must be used within 15 minutes of preparation. Please plan accordingly for the next steps of the procedure.

Prepare Mueller-Hinton Agar Plates 1. Loosen or remove the cap on the Mueller-Hinton agar bottle.

2. Place the bottle in a microwave. You will need to remove the bottle from the microwave and swirl the contents every 10 seconds to evenly distribute the heat. If you do not have a microwave, place the bottle in a heat-safe bowl, create a water bath by pouring boiling water into the bowl (around the bottle), and heat until the entire bottle of agar is liquefied.

Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. Once boiling has stopped, use a hot pad to protect your hands and remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!

3. With a hot pad protecting your hands, remove the agar bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!

4. Gently swirl the bottle a final time to mix the solution.

5. Pour the agar solution into the bottom half of three 9 cm Petri dishes. USE THE AGAR SPARINGLY. YOU ONLY NEED ENOUGH TO COAT THE BOTTOM OF THE PETRI DISH.

Note: Approximately 5 – 10 mL should be enough agar to cover the entire bottom of the dish. If not, continue to add agar in approximately 1 mL increments until the bottom of the petri dish is completely covered. The agar should be only 1 or 2 millimeters deep. If too much agar is poured, there will be no space under the cover for microbial growth.

6. Place the lid onto the dish slightly off-center to create a small space to allow condensation to evaporate and allow the agar to solidify undisturbed. This should take approximately 30 – 60 minutes. If you will not be using the dish immediately, you can store it upside down in the refrigerator after it has fully gelled. Remove the dish from the refrigerator, and allow it to sit at room temperature for at least 1 hour prior to use.

Prepare Samples in Suspension 1. Use a permanent marker to label three standing test tubes “Skin,” “Local,” and “Yeast.” Leave the caps of the test tubes on

at all times unless transferring samples into them.

2. Use a transfer pipette to measure and pour 2 mL of sterile saline into each standing test tube.

3. Remove the Parafilm® from the nutrient agar plate.

4. Sterilize your inoculation loop with your candle.

a. Pour 70% isopropyl alcohol into a 100 mL beaker to a depth of 2 – 4 cm. Place the cap back on the bottle, and position it far out of the way.

b. Light your candle, and set it aside. Be very cautious that your candle is safely positioned away from the beaker of isopropyl alcohol.

c. Dip the inoculation loop into the isopropyl alcohol for 10 seconds. *Once you’ve put the inoculation loop in the alcohol, keep it angled down so that no alcohol drips back onto your hand*.

d. Without touching the inoculation loop to anything, carefully pass the end of the inoculation loop through the flame several times.

e. Extinguish the flame when complete.

5. Transfer an isolated colony of the “Skin” sample growth from the nutrient agar plate using the sterile inoculating loop.

6. Remove the cap of the standing test tube labeled “Skin” and dip the inoculated loop into the sterile saline. To ensure as much of the sample as possible is transferred, swirl the loop, and tap it against the inside of the tube before removing it. Replace the cap immediately.

7. Repeat Steps 2 - 6, for the “Local” sample and the “Yeast” sample. Make sure to resterilize the inoculating loop for each sample.

8. Use the suspensions within 15 minutes of preparation.

9. Once you have created the suspensions, pour approximately 5 mL of the bleach onto the surface of the nutrient agar plate, allowing it to cover the entire surface. Let it incubate for 20 minutes, and then pour the bleach down the sink with running water.

10. Seal the nutrient agar plate with Parafilm® and dispose of it in the trash.

Inoculate Mueller-Hinton (MH) Agar Plates 1. Use a permanent marker to label the bottom of one MH plate “Skin,” one “Local,” and the third “Yeast.”

2. Remove the cap of the “Skin” standing test tube.

3. Dip a sterile cotton swab into the “Skin” sample suspension, and press the swab firmly against the inside of the tube before removing it to remove any excess suspension.

4. Remove the lid of the MH plate labeled “Skin.”

5. Inoculate the MH plate by starting at one end of the plate and running the swab in a close zig-zag motion across the entire plate.

6. Rotate the plate about 90 degrees, and repeat Step 40. Repeat this step one more time, for a total of three inoculations.

7. Run the swab around the outer edge of the of the agar.

8. Close the lid and set the plate aside. Dispose of the swab in a trash receptacle.

9. Repeat Steps 2 - 8 for the “Local” suspension and the “Yeast” suspension and their corresponding MH plates, using a new swab for each sample.

Place the Antibiotic Disks onto Samples 1. The antibiotic disks are marked as follows:

Ampicillin = AM10

Kanamycin = K30

Tetracycline = Te30

2. Sterilize the forceps with your candle.

a. Pour 70% isopropyl alcohol into a 100 mL beaker to a depth of 2 – 4 cm. Place the cap back on the bottle, and position it far out of the way.

b. Light your candle, and set it aside. Be very cautious that your candle is safely positioned away from the beaker of isopropyl alcohol.

c. Dip the forceps into the isopropyl alcohol for 10 seconds. ONCE YOU’VE PUT THE FORCEPS IN THE ALCOHOL, KEEP THEM ANGLED DOWN SO THAT NO ALCOHOL DRIPS BACK ONTO YOUR HAND.

d. Without touching the forceps to anything, carefully pass the end of the forceps through the flame several times.

e. Extinguish the flame when complete.

3. Remove the lid of the “Skin” MH plate.

4. Use the forceps to place an AM10, K30, and Te30 disk on the “Skin” plate evenly spaced out in a triangle configuration, resterilizing the forceps between each disk. Use the forceps to press the disks into the agar, Disks not firmly pressed into the agar may fall onto the lid when stored upside-down. Do not press too hard, as this may damage the agar.

5. Replace the lid of the “Skin” MH plate.

6. Repeat Steps 2 - 5 for the “Local” and “Yeast” suspensions and MH plates.

7. Incubate the plates upside-down.

8. Seal the closed MH agar plates with Parafilm®, and let them incubate in a warm location (not to exceed 37.7 °C or 100 °F). You should see growth form in two to five days, depending on the temperature at which the plates are incubated (the warmer the incubation temperature, the quicker the colonies will form).

Measuring Zones of Inhibition 1. After incubation, measure the zones of inhibition to the nearest millimeter.

2. To do this, place the plate labeled “Skin” on a dark surface, and view the back of the plate. Use a ruler to measure the zone around the AM10 disk from end to end. Include the disk in the measurement. If the zone cannot be measured end-to-end due to overlap with other zones or interference with the edge of the plate, find a place on the zone where a measurement from the center of the disk to the edge of the zone (the radius) can be obtained. Then, multiply that measurement by two.

3. If there is growth all the way to the disk (no zone of inhibition), then record that measurement as 0 mm.

4. Record the zone of inhibition for the “Skin” AM10 disk in Table 3.

5. Repeat Steps 1 - 4 with the K30 and Te30 disks on the “Skin” plate.

6. Repeat Steps 1 - 4 for the “Local” plate and record measurements in Table 4.

7. Repeat Steps 1 - 4 for the “Yeast” plate and record measurements in Table 5. If keeping a lab notebook, print out the tables and tape them into your lab notebook or re-create them by hand.

8. Pour approximately 5 mL of the bleach onto each surface of the three agar plates, allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour the bleach down the sink with running water.

9. Seal the Petri dishes with Parafilm®, and dispose of them in the trash.

Data Sheet Experiment 1 Data Sheet Table 1: Zone of Inhibition for Skin Sample

Antibiotic Disk Diameter of Zone (mm)

AM10

K30

Te30

Table 2: Zone of Inhibition for Local Sample

Antibiotic Disk Diameter of Zone (mm)

AM10

K30

Te30

Table 3: Zone of Inhibition for Yeast Sample

Antibiotic Disk Diameter of Zone (mm)

AM10

K30

Te30

Table 4: Zone of Inhibition Interpretative Standards

Antibiotic Sensitive (mm) Moderately Resistant (mm) Resistant (mm)

Ampicillin (AM10) Zone of Inhibition

Diameter

Enterobacteriaceae species: ≥ 17

Enterococcus: ≥ 17 Staphylococcus: n/a

Enterobacteriaceae species: 14 – 16

Enterococcus species: n/a Staphylococcus species: n/a

Enterobacteriaceae species: ≤ 13 Enterococcus species: ≥ 16

Staphylococcus species: n/a

Kanamycin (K30) Zone of Inhibition

Diameter

Enterobacteriaceae species: ≥ 18

Staphylococcus species: ≥ 18

Enterobacteriaceae species: 14 – 17

Staphylococcus species: 14 – 17

Enterobacteriaceae species: ≤ 13 Staphylococcus species: ≤ 13

Tetracycline (Te30) Zone of Inhibition

Diameter

Enterococcus species: ≥ 19 Staphylococcus species: ≥

19

Enterococcus species: 15 – 18 Staphylococcus species: 15 –

18

Enterobacteriaceae species: ≤ 14 Staphylococcus species: ≤ 14