S E C T I O N Bacterial Cultivation III Part A: Aseptic Technique Exercise 16: Bacteria and Fungi in the Laboratory Environment Part B: Culture of Bacteria Exercise 17: Preparation and Inoculation of Growth Media Exercise 18: Culture Characterization of Bacteria 109
ale87445.un16.qxd 1/30/03 10:05 AM Page 110 E X E R C I S E 16 Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique Background Bacteria and Fungi in the Laboratory Environment Bacteria and fungi occur widely in the natural environment in association with air, water, soil, plants, and animals. These microorganisms find their way into our homes, offices, and buildings in a variety of ways: (1) through open doors and windows; (2) on the bot- toms of shoes; (3) on the surfaces of plants, pets, and food; and (4) on the surfaces of our hands and clothes. Bacteria and fungi also find their way into our laboratory environment, where they can be found in the air and on countertops (figure 16.1). We must be aware of these microorganisms in the laboratory environment when working with laboratory cultures. To demonstrate their presence, you will use two types of media to culture bacteria and fungi from the laboratory environment: nutrient agar and Sabouraud Bacteria Fungi (a) Bacteria and fungi from laboratory air. (b) Fungi from laboratory air. (c) Bacteria and fungi from laboratory countertop. (d) Fungi from laboratory countertop. Figure 16.1 Bacteria and fungi from the laboratory environment. 110
Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique EXERCISE 16 111 Table 16.1 Components of Nutrient Agar and Sabouraud Dextrose Agar Nutrient agar* (bacteria) Sabouraud dextrose agar (fungi) Peptone 5 g Peptone 10 g Beef extract 3 g Dextrose 40 g Agar 15 g Agar 15 g Distilled water 1,000 ml Distilled water 1,000 ml Final ph= 6.8 Final ph= 5.6 Source: The Difco Manual. Eleventh Edition. Difco Laboratories. *Nutrient broth has the same formula, but does not contain agar. Inhibiting organism (chemical producer) Inhibited organism Zone of inhibition Figure 16.2 Evidence of inhibition of one microbe by another. The inhibiting organism is producing a chemical that is active against the other organism. dextrose agar (table 16.1). Nutrient agar contains organic compounds, which support the growth of a wide variety of bacteria, and agar as a solidifying agent. The final ph of the medium is 6.8. Sabouraud dextrose agar also contains organic compounds and agar, but the high dextrose content (4%) and low ph (5.6) favor the growth of fungi over bacteria. The medium can be made even more selective for fungi through the addition of an antibiotic, such as chloramphenicol. Together, these two media will demonstrate the number and variety of bacteria and fungi in our laboratory environment. Excluding Environmental Contaminants from Laboratory Cultures Once your examination of culture media has revealed the existence of bacteria and fungi in our laboratory environment, you will be asked to consider how this relates to working with pure cultures in the laboratory. For example, can these environmental bacteria and fungi contaminate our laboratory cultures? Can their entry be prevented by using certain techniques designed to exclude them? If such techniques exist, what are they? Searching for Examples of Antibiosis The primary focus of this exercise is to demonstrate bacteria and fungi in the laboratory environment and to consider techniques to exclude them from cultures. A secondary focus is to find an example of antibiosis on the media you inoculate with samples from the laboratory environment. What is antibiosis? When environmental microorganisms grow in close proximity to one another, as occurs naturally in soil or unnaturally in a culture medium, one microbe may produce a chemical substance that inhibits the growth of another microbe nearby. This phenomenon is called antibiosis. Antibiosis is identified in culture media by a zone of inhibition around the chemical-producing organism (figure 16.2). Examples of antibiosis in culture media are not common, since the odds are low that you will inoculate in close proximity an organism that produces a chemical substance inhibitory to another. You and other laboratory students may collectively find only one example on all your plates, but finding that one example is the objective. When you see this example, you will understand what Alexander Fleming saw in 1928 when he examined a plate in his laboratory. He found that the growth of Staphylococcus aureus was inhibited by a mold. The mold was identified as Penicillium, and the chemical substance it produced was later isolated and named penicillin. Penicillin proved to be effective in treating infections caused by Staphylococcus aureus in the human body. It became the first antibiotic, an antimicrobial chemical agent of microbial origin put into the human body to treat disease. Since the introduction of penicillin, many other antibiotics of microbial origin have been discovered. Streptomyces and Bacillus, Examples of Antibiotic-Producers Streptomyces is a genus of bacteria that is common in soil. These bacteria are Gram-positive and produce rods in branching filaments similar to those of fungi, but
112 SECTION III Bacterial Cultivation Streptomyces colonies Figure 16.3 Swab results from the laboratory floor. A number of white, powdery colonies of Streptomyces are present. the filaments have a much smaller diameter than those of fungi. Streptomyces produces a small, white, powdery colony on culture media (figure 16.3) and gives off an earthy, soil-like odor. Bacillus is a genus of bacteria also common in soil. This organism is a Gram-positive, endospore-forming rod. Bacillus generally produces a large, flat colony that is typically white or cream-colored. Streptomyces and Bacillus are both sources of useful antibiotics. Species of Streptomyces are the source of more than half of all antibiotics effective against bacteria, including streptomycin, tetracycline, and chloramphenicol. They are also the source of the polyenes, such as amphotericin B and nystatin, effective against fungi. Species of Bacillus are the source of antibiotics such as bacitracin, effective against Gram-positive bacteria, and polymyxin B, effective against Gram-negative bacteria. A third focus of this exercise is to find one or both of these common soil bacteria in the laboratory environment. One of these bacteria may provide the example of antibiosis in your culture media. Materials Media 4 nutrient agar plates 4 Sabouraud dextrose agar plates 3 nutrient broth (or water) tubes, sterile Stains Gram stain Crystal violet Gram s iodine Ethanol (95%) Safranin Equipment Dissecting microscope Incubator (set at 35 C) Light microscope Miscellaneous supplies Bibulous paper Bunsen burner and striker Clothespin Cotton-tipped swabs, sterile (3) Disposable gloves (optional) Glass slides Immersion oil Inoculating needle Lens paper Staining tray Wash bottle with tap water Wax pencil Procedure First Session: Inoculation of Nutrient Agar and Sabouraud Dextrose Agar Plates 1. Remove the lids from a nutrient agar plate and a Sabouraud dextrose agar plate. Leave these two plates open to the laboratory air for 30 60 minutes.
Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique EXERCISE 16 113 Sample surface Swab Line 1 (start) Line 2 (end) Line 2 (start) Line 1 (end) (a) A moistened swab is first rubbed back and forth across the sample surface to pick up microorganisms. (b) Microorganisms are then transferred to an agar plate by rubbing the swab back and forth along lines 1 and 2. Figure 16.4 Swab inoculation of an agar plate. 2. Dip the cotton-tipped end of a sterile swab into a tube of sterile water or nutrient broth. Blot the excess liquid against the test tube wall. Use the wetted end to rub back and forth across the countertop of your work area; then inoculate a nutrient agar plate (figure 16.4). Repeat this process to inoculate a Sabouraud dextrose agar plate. 3. Wet a second sterile, cotton-tipped swab with sterile water or broth, and use it to rub back and forth across the floor below your work area. Inoculate a second nutrient agar plate as before. Repeat to inoculate a second Sabouraud dextrose agar plate. 4. Wet a third sterile, cotton-tipped swab with sterile water or broth, and use it to rub back and forth across the skin on the inside of your left hand. Inoculate a third nutrient agar plate as before, and repeat to inoculate a third Sabouraud dextrose agar plate. 5. Label the plates, and incubate the air, countertop, and floor plates at room temperature (22 C). Place the skin plates in an incubator set at 35 C. Incubate all plates at least 3 4 days before examining. Second Session: Examination of Nutrient Agar and Sabouraud Dextrose Agar Plates Number and Variety of Bacteria and Fungi in the Laboratory Environment 1. After incubation, examine all plates for growth. Sketch a typical nutrient agar plate and Sabouraud dextrose agar plate in the laboratory report. 2. Record the number and variety of bacteria and fungi on your plates in the table of your laboratory report. Note: When determining number, count only bacterial colonies on nutrient agar plates and fungal colonies on Sabouraud dextrose agar plates. Bacterial colonies are smooth and round, while fungal colonies are large and cottony in appearance (see figure 16.1a). Note: When determining variety, look for bacterial and fungal colonies that are different in appearance. Colonies that look different (based on size, shape, margin, texture, elevation, pigmentation, etc.) represent different types. The use of a dissecting microscope may help you count and differentiate colonies.
114 SECTION III Bacterial Cultivation Examples of Antibiosis on Plates 1. After completing the drawings and table and considering the implications of these results, go back through your plates searching for examples of antibiosis. Refer to figure 16.2 to determine if a zone of inhibition is present on any of your plates. If you find none, examine the plates of other students. Generally, at least one example can be found. 2. In the laboratory report, draw the example of antibiosis you see. If time permits, Gram-stain the two organisms in order to get an idea of what antibiotic may be involved. Presence of Streptomyces and Bacillus 1. Examine your plates for signs of Streptomyces and Bacillus. Their colony characteristics were described previously in the Background section of this exercise. 2. If you find suspect colonies, do a Gram stain to verify your identification. Also, do a spore stain on the suspect Bacillus colony if it turns out to be a Gram-positive rod. If you have isolated species of one or both of these bacteria, you have isolated important antibiotic-producers. Did one of these bacteria provide your example of antibiosis?
LABORATORY REPORT NAME LAB SECTION DATE E X E R C I S E 16 Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique 1. a. Draw a typical nutrient agar plate and Sabouraud dextrose agar plate inoculated with a laboratory sample. Nutrient agar plate (bacteria) Sample Total bacterial colonies Total colony types Sabouraud dextrose agar plate (fungi) Sample Total fungal colonies Total colony types b. Record your results for all plates in the following table. Laboratory Nutrient agar (bacteria) Sabouraud dextrose agar (fungi) sample Total colonies Colony types Total colonies Colony types Air Countertop Floor Skin Which sample had the highest number of bacteria? Why? c. Based on your results, does the laboratory environment contain a large number and variety of bacteria and fungi? 115
116 SECTION III Bacterial Cultivation d. Describe several techniques you might use to keep these environmental bacteria and fungi from contaminating your laboratory cultures. 2. a. Did you see any zones of inhibition on your plates? (yes or no) Any zones on other students plates? (yes or no) b. If yes, draw a representative result indicating a zone of inhibition. Draw only the region of the interacting organisms. Label the chemical-producing organism, the zone of inhibition, and the inhibited organism. c. Explain how your result is similar to that observed by Alexander Fleming in 1928. d. Why was Fleming s observation historically important? e. Gram-stain results: Antibiotic-producer in the drawing in (b): Gram reaction Cell shape Bacteria or fungi? Inhibited organism in the drawing in (b): Gram reaction Cell shape Bacteria or fungi? Do these results give you any clues as to what antibiotic is being produced? If so, describe the possibilities.
Bacteria and Fungi in the Laboratory Environment: The Necessity of Aseptic Technique EXERCISE 16 117 3. a. Did you or another student have an isolate from the countertop or floor with the following characteristics: Small, white, powdery colony? (yes or no) Gram-positive rods in branching filaments? (yes or no) If you answered yes on both lines, you may have isolated Streptomyces, a common bacterium in soil. Can you explain how this organism gets into the lab? What is the medical significance of this organism? b. Did you or another student have an isolate from the countertop or floor with the following characteristics: Large, flat colony, white or cream-colored? (yes or no) Gram-positive endospore-forming rod? (yes or no) If you answered yes on both lines, you may have isolated Bacillus, a common bacterium in soil. Can you explain how this organism gets into the lab? What is the medical significance of this organism? 4. Answer the following questions based on these photographs: (2) (1) a. Bacteria or fungi? b. Type of medium? c. Bacteria from air or skin? (1) How do you know? (2) How do you know?
118 SECTION III Bacterial Cultivation Gram-negative rod Fungi (1) (1) Gram-positive, endospore-forming rod Gram-positive rods in branching filaments d. Name area of no growth (1) e. Name area of no growth (1) Given these results, what group of antibiotics may Given these results, what group of antibiotics may be indicated? be indicated? f. These two photographs indicate what type of bacterium? Where does this organism occur naturally? What is the medical significance of this organism?