Experiment 2: Selection and Differentiation of Body Inhabiting, Gram-Positive Bacteria

Mannitol Salt Agar (MSA) is both selective and differential for gram-positive bacteria, containing a high (7.5%) salt (sodium chloride) concentration, which makes it selective, and mannitol as the carbohydrate source for fermentation, which makes it differential. Most bacteria cannot grow in the high salt environment; however, Staphylococcus species have adapted to high salt environments, such as human skin. The differentiation agent mannitol can be effectively fermented by Staphylococcus aureus, but not by other Staphylococcus species. MSA also includes the pH indicator dye phenol red, which is red in basic conditions and yellow in acidic conditions. Fermentation of mannitol generates an organic acid which lowers the pH of the agar and changes the dye from red to yellow.

Procedure

1. Turn four petri plates over, draw a line down the center of each plate, and label the bottom of two plates as “MSA” and the bottom of two plates as “Nutrient”. Return the lids to the plates and set them aside.
2. Loosen or remove the cap on the MSA agar bottle. Place the bottle in the microwave (if you do not have a microwave, place the bottle in a heat-safe cup and pour boiling water into the bowl around the bottle).
3. Heat the bottle in 10 second increments until the agar is liquefied. You may need to remove the bottle from the microwave and swirl it every 10 seconds to evenly distribute the heat.
   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. After boiling has stopped, use a hot pad to remove the bottle from the microwave. Remember, the bottle will be HOT!
4. Gently swirl the bottle to ensure that the contents are fully liquefied.
5. Slowly pour the liquefied MSA agar into the bottom half of the two MSA plates so that it covers the entire bottom of the dish. Return the lids to the petri dishes and set aside.
6. Repeat Steps 2 - 5 for the nutrient agar. Be sure to loosen or remove the cap on the bottle to allow for heat expansion.
7. Pour the agar into the bottom of the two nutrient petri plates. It is important that the entire bottom is coated and that the agar is given time to spread out over the plate.
8. Place the lids onto the plates and allow the agar to gel undisturbed.
   Note: If you will not be using the dishes immediately, store them upside down in the refrigerator after they have fully gelled. Remove from the refrigerator and allow them to sit at room temperature for at least one hour prior to use.
9. Use a sterile cotton swab to gently rub a portion of your skin (such as your arm, cheek, etc.).
10. Lightly rub the exposed swab over half the surface of one MSA plate and half of the surface of one nutrient plate.
11. Use a permanent marker to label the respective half of each dish as “Skin”.
12. Use a new sterile cotton swab to gently swab the inside of your nose.
13. Lightly rub the exposed swab over the other half of the same MSA plate and half of the same nutrient plate.
14. Use a permanent marker to label the respective half of each dish as “Nose”.
15. Use a new sterile cotton swab to gently swab a non-porous surface (such as a countertop).
16. Lightly rub the exposed swab over half of the second MSA plate and half of the second nutrient plate.
17. Use a permanent marker to label the respective half of each dish with surface you swabbed.
18. Lightly rub a new sterile cotton swab over the remaining halves of the second MSA plate and second nutrient plate. Do not touch the cotton swab to anything!!
19. Use a permanent marker to label this half of each dish as “Control”.
20. Seal the plates with Parafilm™ and incubate them at room temperature (up to 37.7 °C or 100°F) for 1 - 2 days, or until colonies appear.
21. Examine the plates for amount of growth and colony/agar color. Record your results in Table 4.
22. When you are finished with the experiment, flood the plates with a 10% bleach solution, incubate for 20 minutes, then pour the bleach down the sink with running water.
23. Seal the plates with Parafilm™ and dispose of them in the trash.