Week 6-7 TC Unknown Microbiology Lab Manual PDF

Table of Contents EXERCISE 14: SELECTIVE AND DIFFERENTIAL MEDIA........................................................................................... 2 OBJECTIVES.....................................................................................................................................

Table of Contents EXERCISE 14: SELECTIVE AND DIFFERENTIAL MEDIA........................................................................................... 2 OBJECTIVES................................................................................................................................................................... 2 INTRODUCTION.............................................................................................................................................................. 2 MATERIALS.................................................................................................................................................................... 3 PROCEDURE.................................................................................................................................................................. 3 REVIEW QUESTIONS........................................................................................................................................................ 4 FOR FURTHER THOUGHT.................................................................................................................................................. 4 EXERCISE 25: DETERMINATION OF BACTERIAL PROPERTIES-EXOENZYMES AND ENDOENZYMES.......................... 5 OBJECTIVES................................................................................................................................................................... 5 INTRODUCTION.............................................................................................................................................................. 5 PART 1 - LIPID HYDROLYSIS............................................................................................................................... 5 MATERIALS.................................................................................................................................................................... 6 PROCEDURE.................................................................................................................................................................. 6 RESULTS........................................................................................................................................................................ 6 PART 2 - STARCH HYDROLYSIS........................................................................................................................... 7 MATERIALS.................................................................................................................................................................... 7 PROCEDURE.................................................................................................................................................................. 7 RESULTS........................................................................................................................................................................ 7 PART 3 - DNASE ACTIVITY................................................................................................................................. 8 MATERIALS.................................................................................................................................................................... 8 RESULTS........................................................................................................................................................................ 9 PART 4 – COAGULASE ACTIVITY......................................................................................................................... 9 MATERIALS.................................................................................................................................................................... 9 PROCEDURE.................................................................................................................................................................. 9 RESULTS...................................................................................................................................................................... 10 PART 5 – CATALASE ACTIVITY.......................................................................................................................... 10 MATERIALS.................................................................................................................................................................. 10 PROCEDURE................................................................................................................................................................ 10 RESULTS...................................................................................................................................................................... 11 PART 6 – OXIDASE ACTIVITY............................................................................................................................ 11 MATERIALS.................................................................................................................................................................. 11 PROCEDURE................................................................................................................................................................ 11 RESULTS...................................................................................................................................................................... 12 PART 7 - NITRATE REDUCTION......................................................................................................................... 12 MATERIALS.................................................................................................................................................................. 13 PROCEDURE................................................................................................................................................................ 13 RESULTS...................................................................................................................................................................... 14 REVIEW QUESTIONS...................................................................................................................................................... 14 FOR FURTHER THOUGHT................................................................................................................................................ 15 1 Laboratory Safety Procedures (Week 6-7) Exercise 14: Selective and Differential Media Objectives Upon completion of this laboratory exercise you will be able to: 1. Distinguish between selective media and differential media. 2. Define the term: hemolysis. 3. Explain the difference between alpha, beta, and gamma hemolysis. 4. Perform the procedures to observe the growth patterns of eight cultures on selective and differential media. 5. Distinguish organisms based on their appearance and ability to grow on various media. Introduction Selective and differential media are commonly used in microbiology to isolate and distinguish microorganisms. A selective medium contains chemicals to encourage the growth of desired organisms, while inhibiting the growth of others. A differential medium allows all organisms to grow but distinguishes these organisms based on how they metabolize a particular component in the medium. Colonies of a particular organism will display a distinctive appearance related to their particular properties. In this laboratory session, you will use the following selective and differential media to isolate or distinguish microorganisms: 1. Blood Agar is a differential medium that distinguishes organisms based on their ability to lyse, or destroy, red blood cells. This property is known as hemolysis. Some organisms break down red blood cells completely, while others only partially degrade red blood cells or cannot break down red blood cells at all. There are three types of hemolysis: a) alpha ( ), or partial hemolysis. Red blood cells are partially degraded. Colonies or the areas surrounding the colonies appear a green-gray color. b) beta (β), or complete hemolysis. Red blood cells are completely lysed. A clear zone is seen around the colonies. c) gamma (), or no hemolysis. Colonies are-their characteristic color. 2. Phenylethanol Agar (PEA) is a selective medium that encourages the growth of Gram- positive organisms. Growth of Gram-negative organisms is inhibited because the ethanol in this medium destroys the lipoprotein in the Gram-negative cell wall. 3. MacConkey Agar has properties of both selective and differential media. The medium contains crystal violet, which inhibits the growth of Gram-positive organisms, so the medium will only support the growth of Gram-negative bacteria. This medium also contains lactose, bile salts, and a pH indicator called neutral red. These components 2  differentiate organisms based on their ability to ferment lactose. Acid is produced when lactose is fermented, which causes the colonies to turn a pink-red color. 4. Eosin-methylene blue (EMB) is another medium with properties of both selective and differential media. In this medium, the dyes eosin and methylene blue inhibit the growth of Gram-positive organisms and support the growth of Gram negatives. This medium also contains lactose, so organisms can be distinguished based on their ability to ferment lactose. lf lactose is fermented, acid is produced, precipitating the dyes in the medium. The lactose-fermenting colonies turn a green-black color, with a metallic sheen or brightness. 5. Mannitol salt agar (MSA) is both a selective and differential medium. The medium contains 7.5% NaCl which only supports the growth of staphylococci. Other organisms are unable to grow in this medium. MSA also contains a pH indicator, phenol red, and mannitol. Mannitol can be metabolized to generate energy through fermentation. Acid is produced as a result of mannitol fermentation, causing the phenol red in the medium to turn yellow. Materials cultures of Bacillus subtilis, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa a mixed culture of Staphylococcus epidermidis, Escherichia coli, and Proteus vulgaris 2 blood agar plates 2 phenylethanol agar plates 2 MacConkey agar plates 2 Eosin methylene blue plates 2 Mannitol salt agar plates Procedure 1. Each group of 4 will be assigned one culture, and all groups will work with the mixed culture. 2. Label the bottom of each plate with the name of the medium, organism, date, and your initials. 3. For each type of medium, streak one plate with your culture, and the other with the mixed culture. 4. Incubate plates at 37°C for 24-48 hours. 5. After incubation, examine the plates for growth, and then record the color and appearance of any growth you observe on the plates. 3  Name of Bacterium Blood PEA MacConkey EMB MSA Review Questions 1. If you wanted to isolate all the bacteria found in a soil sample, would you use a selective or differential medium? Explain your answer. 2. What property is used to distinguish microorganisms on blood agar? 3. Name one medium that is used to isolate Gram-negative bacteria. 4. What are the two components of EMB that allow it to be a selective medium? 5. What would happen if the crystal violet was removed from MacConkey agar? 6. Both E. coli and P. vulgaris grow on MacConkey agar. Describe how you could distinguish these 2 bacteria from each other. 7. Explain why mannitol salt agar has properties of both selective and differential media. 8. If you wanted to isolate a Gram-positive, beta-hemolytic bacterium, name the media you would use to do so. 9. Stretpococcus. pyogenes, the cause of strep throat, is beta-hemolytic. Describe what it would look like on blood agar. 10. Name the component of PEA that makes it a selective medium and explain how this component inhibits the growth of certain bacteria. For Further Thought 1. How would you develop a new type of medium, one that is different from the media used in this laboratory exercise? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 2. Could computers be programmed to carry out the laboratory procedures in this exercise? Explain your answer. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 4 Exercise 25: Determination of Bacterial Properties- Exoenzymes and Endoenzymes Objectives Upon completion of this laboratory exercise, you will be able to: 1. Distinguish between exoenzymes and endoenzymes. 2. Name the end products of each biochemical reaction described in this exercise. 3. Name the indicator for each reaction, and describe the appearance of a positive and a negative test result. 4. State a purpose for glucose in cellular metabolism. 5. Perform accurately and record the results of the various tests for exoenzymes and endoenzymes. Introduction The cytoplasm of cells contains many enzymes that carry out specific biochemical reactions. Since these enzymes function within the inner boundaries of cells, they are known as endoenzymes (endo = within). Certain endoenzymes, such as those involved in glycolysis and Krebs cycle, are used to identify organisms. Many microbes also possess enzymes that catalyze biochemical reactions outside of the cells. These are called exoenzymes (exo = outside). Exoenzymes catabolize, or break down, large molecules into smaller molecules that can permeate a cell's membrane and cell wall. Once inside a cell, these molecules can be used to synthesize cellular components, such as molecules of the cell membrane and cell wall, or take part in energy-producing biochemical pathways, such as the Krebs cycle. Lipase and amylase are examples of exoenzymes. The presence of these enzymes can be used to identify certain bacteria. Part 1 - Lipid Hydrolysis Lipids, also known as triglycerides or fats, are hydrolyzed by the exoenzyme lipase. Lipase breaks down lipids to form fatty acids and glycerol, which can then enter the pathways of cellular respiration. Energy, in the form of ATP molecules, is produced for various cellular activities. To test for lipase activity, spirit blue agar is used. This medium contains the lipid vegetable oil. A lipase-producing microorganism secretes lipase to degrade the vegetable oil. As a result, a clear area surrounds the growth on the plate. Some bacteria secrete lipase that breaks down the lipids on the skin's surface. These microbes use the fatty acids and glycerol generated as energy sources for continued growth. Clostridium perfringens, the pathogen responsible for gas gangrene, produces a type of lipase that damages the cell membranes of human cells. This leads to destruction and death of the infected tissue. 5 Materials Cultures of Bacillus subtilis, Enterobacter aerogenes, Escherichia coli, Proteus vulgaris and Psuedomomas aeruginosa 1 spirit blue agar plate Procedure 1. Obtain the culture you have been assigned to work with, and label a spirit blue agar plate with the name of your culture. 2. Inoculate the plate with one line of culture down the center of the plate as shown below. 3. Incubate the plate at 37°C for 24-48 hours. 4. After incubation, examine the appearance of the plate. Based on your observations, record the results. If the microorganism produces lipase, it will degrade the vegetable oil in the medium and a clear zone will appear around the growth on the plate. If the microorganism does not contain lipase, the plate will remain blue and no clear zone will appear around the growth on the plate. 5. Observe the tubes containing other organisms studied by other students, and record the results in the table. Results Complete the table below. Describe the appearance of the plate by writing its color. In the column labeled "interpretation" write a brief explanation for the observed appearance. Organism Appearance Interpretation 6 Part 2 - Starch Hydrolysis Starch is a high molecular weight carbohydrate that is acted on by the exoenzyme amylase. Through a series of enzymatic reactions, amylase breaks down starch into shorter polysaccharide chains known as dextrins, and then into the disaccharide maltose. This disaccharide is broken down to yield the monosaccharide glucose. Glucose can enter the cell to be the starting molecule of glycolysis, which will generate energy in the form of ATP. To test for amylase activity, starch agar is used. Once growth appears on the starch agar plate, iodine is added to the plate. Iodine reacts with starch to produce a characteristic blue-black color. If the microorganism produces amylase, however, the amylase diffuses into the medium and degrades the starch so that no blue-black color results. Instead, a clear zone is seen around the growth on the plate. Materials cultures of Bacillus subtilis, Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa 1 starch agar plate Gram's iodine Procedure 1. Obtain the culture you have been assigned to work with. and label a starch agar plate with the name of your culture. 2. Inoculate the plate with one line of culture down the center of the plate. 3. Incubate the plate at 37°C for 24-48 hours. 4. After incubation. add Gram's iodine to the plate. Add enough iodine to cover the entire agar surface. 5. Examine the appearance of the plate. Based on your observations. record the results. If the microorganism produces amylase, it will degrade the starch in the medium and a clear zone will appear around the growth on the plate. If the microorganism does not produce amylase, the entire agar surface will appear blue-black due to the reaction between iodine and starch. 6. Observe the plates containing other organisms studied by other students, and record the results in the table. Results Complete the table below. Describe the appearance of the plate by writing its color. In the column labeled "interpretation" write a brief explanation for the observed appearance. 7  Organism Appearance Interpretation Part 3 - DNase Activity DNase catalyzes the hydrolysis of DNA into deoxyribose, phosphate (P04), and nitrogen- containing bases. Many pathogenic microbes produce this enzyme because it breaks down the host's DNA and then these cells die. To test for DNase activity, DNase test agar, which contains DNA, is used. After an organism has grown on DNase test agar, hydrochloric acid (HCl) is added to the plate. HCl reacts with DNA to produce a cloudy appearance on the agar surface. If DNase is produced by the organism, however, the DNA in the medium will be degraded and a clear zone will appear next to the growth on the plate. DNase activity is used to distinguish two organisms, Staphylococcus aureus and Staphylococcus epidermidis, commonly found on the skin. Both of these organisms can cause skin infections. Materials 1. Obtain the culture you have been assigned to work with, and label a DNase test agar plate with the name of your culture. 2. Inoculate the plate with one line of culture down the center of the plate. 3. Incubate the plate at 37°C for 24-4.8 hours. 4. After incubation, add HCI** to the plate. Add enough HCI to cover the entire agar surface. Once you have added the HCl, do not move the plate. 5. Examine the appearance of the plate. Based on your observations, record the results. If the microorganism produces DNase, it will degrade the DNA in the medium. A clear zone will surround the growth on the plate. If there is no DNase activity, the. agar surface will have a cloudy appearance due to the reaction between DNA and HCI. 6. Observe the plates containing other organisms studied by other students, and record the results in the table. ****Your lab may use DNAse test agar with Methyl Green added instead. In that case, you do not need to add HCl, but instead observe clearing in the green color as a positive result. 8 Results Complete the table below. Describe whether the plate has a clear area around the growth or a cloudy appearance. Jn the column labeled "interpretation" write a brief explanation for the observed appearance. Organism Appearance Interpretation Part 4 – Coagulase Activity Coagulase is an enzyme that causes fibrin, a protein found in blood plasma, to clot. A clot of fibrin forms around the bacterium, which then protects the bacterium from the host's immune system. For this reason, microbes that produce coagulase are more pathogenic than other microbes. Coagulase activity is determined using tubes of rabbit plasma. Coagulase-producing microbes form a visible clot of fibrin in the plasma. Materials Cultures of Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis 1 tube of citrated rabbit plasma Procedure 1. Obtain the culture you have been assigned to work with, and label a tube of rabbit plasma with the name of your culture. 2. Inoculate the tube of rabbit plasma with 0.3 ml of your culture. 3. Incubate the tube at 37°C for 1-3 hours. 4. Examine the appearance of the tube each hour during the laboratory session and record your results. A clot will form if the microbe produces coagulase. No clot will form if the microbe does not produce coagulase. 5. Observe the tubes containing other organisms studied by other students, and record the results in the table. 9 Results Complete the table below. State whether or not a clot formed in the tube during the incubation period. In the column labeled "interpretation” write a brief explanation for the observed appearance Organism Appearance Interpretation Part 5 – Catalase Activity Some bacterial cells produce hydrogen peroxide (H₂O₂) during aerobic respiration. H₂O₂ destroys cellular components rapidly if it is not broken down into nontoxic compounds. The enzyme catalase degrades hydrogen peroxide into nontoxic water and oxygen. To test for catalase activity, cultures are exposed to H₂O₂. The appearance of O₂ bubbles on the surface of the medium indicates that the organism produces catalase. Materials cultures of Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis 1 nutrient agar slant hydrogen peroxide (H₂O₂) in a dropper bottle Procedure 1. Obtain the culture you have been assigned to work with, and label a nutrient agar slant with the name of your culture. 2. Inoculate the nutrient agar slant with your culture. 3. Incubate the tubes at 37 C for 24-48 hours. 4. Add 5-10 drops of H₂O₂ to the growth on the slant. 5. Immediately examine the appearance of the tube. Based on your observations, record the results. If the microorganism produces catalase, you will observe a bubbling on the surface of the agar slant. 6. Observe the slants containing other organisms studied by other students, and record the results in the table. 10 Results Complete the table below. Indicate whether or not you observed bubbling on the agar slant. In the column labeled "interpretation" write a brief explanation for the observed appearance. Organism Appearance Interpretation Part 6 – Oxidase Activity Cytochrome c oxidase plays a vital role in aerobic respiration. It is involved in the transport of electrons from cytochrome c to oxygen, which, as you recall, is the final electron acceptor for aerobic respiration. To test for oxidase activity, the reagent tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) is added to bacterial cultures on solid medium. An organism that produces oxidase causes this pink reagent to be oxidized and turn black. Materials cultures of Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa 1 tryptic soy agar plate oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride) Procedure 1. Obtain the culture you have been assigned to work with, and label a tryptic soy agar plate with the name of your culture. 2. Inoculate the tryptic soy agar plate with your culture. 3. Incubate the plate at 37°C for 24-48 hours. 4. Add the oxidase reagent to the growth on the plate. 5. Examine the appearance of the plate. Based on your observations, record the results. If the microorganism produces oxidase, the reagent and the colonies on the plate will turn from pink, to red, to maroon, and finally to black. You will not observe a color change if the organism does not produce oxidase. 11  6. Observe the plates containing other organisms studied by other students, and record the results in the table. Results Complete the table below. Describe the appearance of the plate by writing its color. In the column labeled "interpretation" write a brief explanation for the observed appearance. Organism Appearance Interpretation Part 7 - Nitrate Reduction Some organisms use an inorganic molecule other than oxygen, such as nitrate (NO₂), as the final electron acceptor in respiration. When an organism uses NO₃ as a final electron acceptor, it is reduced to nitrite (NO₂). The enzyme nitritase (also called nitrate reductase) catalyzes this reaction. Some organisms can further reduce NO₂ to nitrogen gas (N₂). This reaction is known as denitrification and is catalyzed by the enzyme nitritase (also called nitrite reductase). To test for nitrate reduction, nitrate broth is used. There are three possible results to this test (see Figure 25.7): (1) the organism produces nitratase only; (2) the organism produces both nitratase and nitritase; and (3) the organism produces neither nitratase nor nitritase. To perform this test, the reagents sulfanilic acid (Nitrate A)-and alpha-naphthylamine (Nitrate B) are added to cultures inoculated in nitrate broth. If a red color develops in the tube of nitrate broth, the enzyme nitratase has reduced NO₃ to NO₂ If there is no red color, the organism either produces both enzymes (nitratase and nitritase) or no enzymes. To distinguish between these two, possible results, zinc dust is added to the tube. Zinc is a strong reducing agent. If a red color appears after the addition of zinc dust, then zinc reduced NO₃ to NO₂ and no enzymes are produced by the organism. If no red color develops after adding zinc dust, then N₂ is present in the tube and the organism produces both enzymes. (Note: Since N₂ is already reduced, the zinc dust cannot reduce it any further.) 12 Materials cultures of Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa 1 tube of nitrate broth Nitrate A (sulfanilic acid) Nitrate B (a-naphthylamine) Zinc dust Procedure 1. Obtain the culture you have been assigned to work with, and label a tube of nitrate broth with the name of your culture. 2. Inoculate the tube of nitrate broth with your culture. 3. Incubate the tubes at 37°C for 24-48 hours. 4. Add 5 drops of Nitrate A and 5 drops of Nitrate B. If a red color develops, skip to step #6. If no red color develops, proceed with step #5. 5. Add zinc dust to the test tube. 6. Examine the appearance of the tube. Based on your observations, record the results. 7. Observe the tubes containing other organisms studied by other students, and record the results in the table. 13 Results Organism Appearance Interpretation Review Questions 1. Name the medium used to test for lipid hydrolysis. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ a. Describe the appearance of a lipase-producing organism on this medium. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 2. What is the product of starch hydrolysis? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ a. Why is iodine used to test for starch hydrolysis? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 3. Why is HCI used to test for DNase activity? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ a. Explain how DNase-producing bacteria damage host cells. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 4. Name the medium used to test for coagulase activity. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ a. Describe the appearance of a coagulase-positive organism in this medium'. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 14  5. Why does hydrogen peroxide bubble when it is added to cuts on the skin? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ a. Name the enzyme that breaks down hydrogen peroxide into nontoxic products. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ b. Can an anaerobe produce catalase? Yes or No. Explain your answer. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 6. What is the function of the enzyme cytochrome c oxidase? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 7. Explain how to test whether a particular microbe has cytochrome c oxidase. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 8. What type of energy producing pathway is detected using nitrate broth? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ For Further Thought 1. Microbes produce a wide variety of enzymes that allow them to successfully break down many different compounds to yield energy. Do you think it is possible for the energy produced by microbes to be harvested so it can be used as an alternative energy source for humans? Provide evidence for your answer. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 2. Clostridium perfringens, the causative agent of gas gangrene, produces several enzymes that increase its virulence. Name and describe the function of at least three of these enzymes. In addition, explain how these enzymes affect the virulence of this bacterium. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 15