Lab Module 1: Ubiquity of Microorganisms

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1 Lab Module 1: Ubiquity of Microorganisms INTRODUCTION Microorganisms are organisms that are too small to be seen without magnification. Viruses and prions are included in this group even though they aren t alive. We will also include helminths (worms) in our studies even though the adults are not microscopic; however, their infective stages 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 the boiling point of water. Other microbes can live inside of 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, cerebrospinal fluid, muscle, bone, or nervous tissues. We invest a lot of energy into immune defenses that keep these fluids and tissues 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 a liquid medium results in a solid medium. DAY ONE ACTIVITIES (Performed by pairs of students) In today s 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, procedures for disinfecting lab benches, and procedures for washing our hands. In this lab module, as well as most other modules, you will work in teams of two students. Activity 1: What s in the air we breathe? One Nutrient Agar with Yeast Extract (NAYE) plate, found on the cart/table in the front of the lab. 1. Label the NAYE plate with your names, the date, the type of medium (NAYE in this case), the incubation temperature (30C in this case), and the inoculum ( Air in this case). The plate should be labeled around the perimeter of the agar-side (See Figure 1).

2 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 the plates agar-side up. If condensation (water accumulation) occurs during incubation, it will accumulate on the lid, not on the agar surface. Figure 1. A Properly Labeled Petri Plate. b. We use NAYE for this activity because it has a broad mixture of nutrients that allows the growth of a large diversity of microorganisms. c. Most common microorganisms grow optimally at warm temperatures. We generally grow microbes that exist outside our bodies at 30C. What incubation temperature should we use for organisms that grow inside our bodies? d. The inoculum is the source of the microorganisms (where they came from) that we add to the growth medium, OR the scientific name of the organism we are working with, if it is known. 2. Open the plate and place it, agar exposed to the air, on top of your lab bench. 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 bench? Two NAYE plates. Test tube rack. One test tube containing two sterile cotton swabs in saline solution. Table disinfectant (located on your bench).

3 1. Label the two plates as described in Activity 1. The inocula are Bench Before for plate 1 and Bench After for plate 2. Both will be incubated at 30C. 2. Swab the top of the bench with one of the cotton swabs. Twirl the tip of the swab as you go over the entire surface of the bench. 3. Transfer cells to the Bench 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 Bench Before plate. 5. Spray table disinfectant on the top of your bench. Use paper towels to spread the disinfectant all over the top of the bench. You are trying to wet down the bench, not dry it off. Allow the liquid to air-dry. 6. Inoculate the Bench After plate by repeating steps 2 through 4. Activity 3. What s on your hands? Two NAYE plates. 1. Label the two plates as described in Activity 1. The inocula are Hands Before for plate 1 and Hands After for plate 2. Both will be incubated at 30C. 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 their 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? One NAYE plate. 1. Label the plate as described in Activity 1. The inoculum is Lips. The plate will be incubated at 30C. 2. One person in your team will volunteer to be the kisser. She/he will inoculate the plate by giving it a closed-lipped kiss (no tongue).

4 Activity 5. Listerine kills germs on contact, or does it? One TSA plate. 1. Label the plate as described in Activity 1. The inoculum is Tongue. The plate will be incubated at 37C. a. 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). 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). Activity 6. Are there microorganisms in soil? One NAYE plate. One test tube containing a sterile cotton swab in saline solution. 1. Label the plate as described in Activity 1. The inoculum is soil. 2. Use the cotton swab to transfer a small amount of soil into the test tube. 3. Mix the soil suspension by gently swirling the saline in the tube. 4. Gently swab the cotton swab across the agar surface. Tip: Hold the swab so that you are using the side of the swab (not the very tip). 6. Incubate the plate at 30C. Stack your 30C plates from Activities 1-4 and Activity 6 (you should have seven NAYE plates) and tape them together. Place them on the tray labeled 30C, with the agar sides up. Place your single TSA plate from Activity 5 on the tray labeled 37C, with the agar side up. DAY TWO 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 (see Figure 2). Fungal colonies tend to be furry; bacterial colonies tend to have rather smoothly-textured surfaces (there are many exceptions to these tendencies).

5 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 geneticallyidentical 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. Colony Characteristics (10-15 minutes): When you look at your agar plates, you ll see that there are many different looking colonies. Differences in colony appearance often indicate different bacterial species. Developing the ability to distinguish among different colony types (which takes careful observation) will be very useful as you proceed through the term (especially when you do the Unknowns Project). 1. Select any two colonies from any two of your agar plates. Describe the following characteristics for each colony: a. Colony color: white or off-white, or pale yellow on an uncolored agar (like NAYE and TSA), is considered unpigmented. Any bright or noticeable coloration is considered pigmented. b. Colony translucency (see-through) vs. opacity (not see-through). c. Colony texture (rough vs. smooth). d. Margin (outer edge of the colony): entire (round), lobed, toothed, irregular. e. Colony size (measured in mm). f. Elevation (how tall off the agar the colony rises). g. Any other characteristics. Observation 2. Abundance and Diversity of Microbes: 1. Estimate the number (abundance) of colonies on each plate. Your estimation only needs to be very rough (e.g., a few colonies or lots of colonies or tons of colonies ). Fill in Table 1 below. 2. Estimate the number of different kinds of colonies (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 (as described in Observation 1 above) are: i. Colony color, translucency vs. opacity, texture, margin, size, and elevation. Table 1. Abundance and Diversity of Bacterial Colonies. Abundance of Colonies Airborne Bench Before Bench After Hands Before Hands After Lips Tongue Before Tongue After Diversity of Colonies

6 Colony Type A: 2 mm, entire margin, smooth texture, opaque yellow color. Colony Type B: 3-5 mm, irregular margin, margin is translucent and of lower elevation than the colony center 5 mm Figure 2. Examples of Bacterial Colonies. QUESTIONS: 1. What is the benefit of a solid agar medium compared to a broth 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 of your sterile media? 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? 11. How did the colonies from the soil compare with those from the hands (before washing) plate? Was there greater abundance of colonies from the soil? Was the diversity different between the soil and the hands?