Experiment 1: Importance of Hand Hygiene

Microorganisms are tiny, typically only microns in length--much too small to be seen without a microscope. Our bodies harbor bacteria that do not normally cause any disease (that is, they are not pathogenic), but they can cause disease if transmitted to a person with an impaired immune system. Since they are so small, it is difficult to know whether we have bacteria on our hands that we could easily transfer to another person by direct contact (e.g., by shaking their hand) or indirectly (e.g., by touching a counter top which someone else then touches). In this experiment, you will assess how easy it is to transfer a microorganism with your hands, and how to prevent this transmission by hand washing. Baking yeast (Saccharomyces cerevisiae) is an unicellular microorganism that does not cause disease but is an effective model for how microorganisms can be easily transferred.

Notes About Working With Agar Plates

• Prepared agar dishes should be stored upside-down in the refrigerator until used. This will prevent condensation from disrupting the growing surface.
• Before inoculating, let agar plate come to room temperature (about one hour).
• After inoculating, replace the cover on the dish, seal with Parafilm™, and store upside-down in a warm location (not to exceed 37.7 °C or 100 °F).
• You should see growth within a few days. The plate will start to smell once microorganisms are growing.
• Before disposing plates, kill the microorganisms by pouring bleach solution onto the agar surfaces and let sit for 20 minutes.

Procedure:

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 beforehandling. Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. After boiling has stopped, use a hot pad protecting your hands and remove the bottle from the microwave. Use caution when removing the bottle from the microwave as it will be HOT!



Figure 4: Image of yeast colonies growing on culture media. Colonies should be small, round, and white to slightly yellow in color. Fuzzy appearing growth indicates mold contamination and should not be counted.



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 four petri dishes.
   Note: Approximately 5 mL should be enough agar to cover the entire bottom of the dish. If not, continue to add agar in approximately1 mL increments until the bottom of the petri dish is completely covered.
6. Place the lids onto the dishes and allow the agar to solidify undisturbed. This should take approximately 30 - 60 minutes. If you will not be using the dishes immediately, you can store them upside down in the refrigerator after they have fully gelled. Remove them from the refrigerator and allow them to sit at room temperature for at least one hour prior to use.
7. Use a permanent marker to label the bottom of the plates as #1, #2, #3, and #4.

Prepare the Yeast Solution:

8. Measure 230 mL of warm water into a 250 mL beaker.
9. 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.

Collecting Hand Bacteria:

10. Put a disposable glove on your non-dominant hand (i.e., your left hand if you are right handed; your right hand if you are left handed).
11. Use a disposable transfer pipette to add 8 - 10 drops of deionized water to the palm of your gloved hand. Rub your hands together to spread the water over the surface of your hand. Remove and dispose of the glove.
12. Wipe the surface of your dominant hand with the water on it with a clean cotton swab then gently rub the cotton swab on the surface of nutrient agar plate #1. Put the lid on the plate and discard the cotton swab.

Aseptic Hand Washing:

13. Hold your hands under warm water.
14. Use soap and rub your hands together to form a visible lather. Continue rubbing for at least 20 seconds (use your timer for this).
15. Rinse the soap off your hands, and dry them with clean paper towels.
16. Repeat Steps 10 - 11
17. Wipe the surface of your hand that had the water on it with a clean cotton swab then gently rub the cotton swab on the surface of nutrient agar plate #2. Put the lid on the plate and discard the cotton swab.
18. Repeat Steps 13 - 15.
19. Put a disposable glove on your non-dominant hand.
20. Use a disposable transfer pipette to add 8 - 10 drops of the yeast solution to the palm of your nondominant hand. Rub your hands together to spread the yeast over the surface of your hand. Remove and dispose of the glove.
21. Wipe the surface of your hand with the yeast on it with a clean cotton swab then gently rub the cotton ball on the surface of nutrient agar plate #3. Put the lid on the plate and discard the cotton swab.
22. Repeat Steps 13 - 15.
23. Wipe the surface of your dominant hand with a clean cotton swab then gently rub the cotton swab on the surface of nutrient agar plate #4. Put the lid on the plate and discard the cotton swab.
24. Seal your plates with Parafilm™, let them incubate in a warm location (not to exceed 37.7. °C or 100 °F) and observe the growth. You should see colonies form in 2-5 days, depending on the temperature at which the plates are incubated (the warmer the incubation temperature, the quicker the colonies will form).
25. Count the number of colonies that grew and record the growth results in Table 1. Record growth as: (0) = no growth; (+) = little growth (less than or equal to 5 colonies); (++) = moderate growth (6-20 colonies); (+++) = heavy growth (20 - 50 colonies); (++++) = confluent growth (no individual colonies, rather they grow as a “lawn” and are uncountable).
26. Pour approximately 5 mL of the 10% bleach solution onto each surface of the remaining 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.
27. Seal the petri dishes with Parafilm™ and dispose of them in the trash.