Ubiquity of Microorganisms Lab Activity
Introduction
Microorganisms are organisms that are too small to be seen without the aid of magnification. Viruses and prions
are included even though they aren’t necessarily alive. We will also include helminths (worms) in our studies
even though the adults are not microscopic. This is because the infective stages of parasitic helminths are
microscopic.
The phrase, “Ubiquity of Microorganisms”, refers to the concept that microorganisms are everywhere. This is
not entirely true, but microorganisms (especially prokaryotes) occupy an amazing diversity of niches. For
example, some Archaea live in hot springs where temperatures can exceed boiling. Other microbes can live in
solid rock. Some bacteria have even been revived after being suspended in amber (fossilized tree sap) for tens of
millions of years!
In our everyday common experiences, microbes are virtually ubiquitous. They are in the air we breathe, the
foods we eat, and the water we drink. Microorganisms contaminate the surfaces of objects that we use (like this
sheet of paper) as well as the skin of our fingers. In practical terms, the only places where microorganisms are
not found are places where we intentionally exclude them. For example, microorganisms are usually not found
in our blood or cerebrospinal fluid. We invest a lot of energy into immune defenses that keep these fluids sterile
(free of microbial contamination) and we get very sick when our immune defenses fail. In silicon wafer
manufacturing plants, millions of dollars are spent on “clean rooms” designed to eliminate contamination.
In microbiology labs, we expend lots of time and energy sterilizing growth media so that we only grow the
microbes that we want to grow. A growth medium is a laboratory preparation that contains nutrients that are
appropriate for growing microorganisms. The mixture of nutrients in a particular growth medium determines
the range of microbes that can grow. Growth media may be liquid—these are called broths. Adding a gelatinous
substance called agar to liquid medium results in a solid medium.
Day 1 Activities
In this lab, we will investigate the abundance and diversity of microorganisms in our local environments. We will also
learn basic lab protocols including the use of solid media, labeling lab cultures, and procedures for disinfecting lab
benches and for washing our hands.
In this lab module, as well as most other modules, you can work in teams of two students.
Activity 1: What’s in the air we breathe?
Materials needed for each team:
 One TSA plate
 Grease pencil or permanent ink marker
Procedure
1. Label the NAYE plate with your names, the date, the type of medium (TSA in this case), the incubation temperature
(30°C in this case), and the inoculum (“Air” in this case). The plate should be labeled around the perimeter of the agarside (Figure 1).
a. We label the agar side for two reasons. First, the microbes will grow on the agar surface (not on the plastic
Petri lid). So by labeling the agar-side, we will always know the identity of the plate, even if the lid and the agar-
side become separated. Second, we incubate agar plates agar-side up. If condensation (water accumulation)
occurs during incubation, it will accumulate on the lid, not on the agar surface.
b. We use TSA for this activity because it allows the growth of a large diversity of microorganisms.
TSA stands for Tryptic Soy Agar. TSA contains a variety of nutrients that are especially good for growing
fastidious organisms (organisms that live in the human body and have finicky nutrient requirements).
c. Most common microorganisms grow optimally at warm temperatures. We generally grow microbes that exist
outside our bodies at 30°C. What incubation temperature should we use for organisms that grow inside our
bodies?
d. The inoculum is the source of microorganisms that we add to the growth medium.
2. Open the plate and place it, agar exposed to the air, on your lab benchtop. Leave the agar exposed to the air for 15
minutes.
3. After 15 minutes, close the plate.
Activity 2. What gets on your arms when you put your arms on the benchtop?
Materials needed for each team:
• Two TSA plates
• Grease pencil or permanent ink marker
• One test tube containing two sterile cotton swabs in saline solution
• Table disinfectant
Procedure:
1. Label the two plates as described in Activity 1. The inocula are “Benchtop Before” for plate 1 and “Benchtop After” for
plate 2. Both will be incubated at 30°C.
2. Swab the benchtop with one of the cotton swabs. Twirl the tip of the swab as you go over the entire benchtop.
3. Transfer cells to the “Benchtop Before” plate by gently swabbing the agar surface. (Tip: hold the swab so that you are
using the side of the cotton (not the very tip).
4. Close the “Benchtop Before” plate.
5. Spray table disinfectant on your benchtop. Use paper towels to spread the disinfectant all over the benchtop. You are
trying to wet down the bench, not dry it off. Allow the liquid to air-dry.
6. Inoculate the “Benchtop After” plate by repeating steps 2 through 4.
Activity 3. What’s on your hands?
Materials needed for each team:
• Two TSA plates
• Grease pencil or permanent ink marker
Procedure:
1. Label the two plates as described in Activity 1. The inocula are “Hands Before” for plate 1 and “Hands After” for plate
both will be incubated at 30°C.
2. One person in your team will volunteer to be the guinea pig. She/he will inoculate the “Hands Before” plate by
rubbing the middle three fingers from one hand on the agar surface.
3. The guinea pig will then wash hands using the same procedure as demonstrated by the instructor.
4. The guinea pig will then inoculate the “Hands After” plate in the same manner as in step 2 (using the same three
fingers for the same amount of time).
Activity 4. When you kiss your significant other, what exactly are you giving him/her?
Materials needed for each team:
• One TSA plate
• Grease pencil or permanent ink marker
Procedure:
1. Label the plate as described in Activity 1. The inoculum is “Lips”. The plate will be incubated at 30°C.
2. One person in your team will volunteer to be the kisser. She/he will inoculate the plate by giving it a passionate,
parted-lipped kiss.
Activity 5. Listerine kills germs on contact, or does it?
Materials needed for each team:
• One TSA plate
• Grease pencil or permanent ink marker (located in your lab drawer)
Procedure:
1. Label the plate as described in Activity 1. The inoculum is “Tongue”. The plate
will be incubated at 37°C.
2. In addition to the label, draw a line down the middle of the back of the plate. Label one side “Before” and the other
side “After”.
3. One person in your team will volunteer to be the “tongue”. She/he will lick the “Before” side of the agar (being careful
not to cross over the dividing line).
4. The “tongue” will then use Listerine mouthwash for 30 seconds.
5. The “tongue” will then lick the “After” side of the plate (again, being careful not to cross the dividing line).
Stack your 30°C plates from Activities 1-4 (you should have six NAYE plates) and tape them together. Place them on the
tray labeled 30°C.
Place your one TSA plate (from Activity 5) on the tray labeled 37°C.
Day 2 Activities
Today, you will make observations of the agar plates that you inoculated on Day 1. Each bump that has formed on the
agar surface is called a colony (Figure 2). Fungal colonies tend to be furry; bacterial colonies tend to have rather
smoothly-textured surfaces (there are many exceptions to these tendencies). Each bacterial colony is made of tens to
hundreds of millions of cells—all descendents of one cell that was placed there during inoculation on Day 1. The millions
of cells in a colony are all genetically-identical to one another having arisen through the process of bacterial
reproduction called binary fission (essentially one cell splitting into two identical daughter cells).
Observation 1. Abundance and Diversity of Microbes
1. Estimate the number of colonies on each plate. Your estimation needs only to be very rough (e.g., “lots of colonies” or
“ a gabillion colonies”). Fill in the table below.
2. Estimate the number of different kinds of colonies (the diversity). See Figure 2.
a. Two colonies that look different from one another are probably different species (although we can’t be sure
without performing further identification tests).
b. Characteristics that are important indicators of differences are:
i. Colony color
ii. Colony translucency vs opacity
iii. Colony texture (rough vs smooth)
iv. Margin (outer edge of the colony): entire (round), lobed, toothed, irregular.
v. Colony size
vi. Elevation (how tall off the agar the colony rises)
Questions
1. What is benefit of solid agar medium?
2. What is a colony?
3. How do you tell the difference between a bacterial colony and a fungal colony?
4. Why do bacterial colonies reach a certain size and then stop growing?
5. How do you reduce airborne contamination?
6. Does table disinfection work? What is the purpose of allowing the table disinfectant to air dry?
7. Does hand washing work?
8. For many people, the “hands after” plate has more colonies than the “hands before” plate. Why does this happen?
What happens to the diversity of colonies on these plates?
9. Does antiseptic mouthwash work?
10. Was there a difference in colony abundance and/or diversity between the “lips” plate and the “tongue before”
plate? What factors cause these differences?