Microbio - Lab Exam 2 (17-23) PDF

Chapter 17 1. Purpose of red phenol broth as a differential medium: - To differentiate different gram negative bacteria by determining their ability to ferment different carbohydrate sugars. The tube with glucose also has a durham tube which shows if gas...

Chapter 17 1. Purpose of red phenol broth as a differential medium: - To differentiate different gram negative bacteria by determining their ability to ferment different carbohydrate sugars. The tube with glucose also has a durham tube which shows if gas is produced during fermentation. 2. Fermentation: if bacteria can ferment carbs they produce a weak acid causing the pH level to drop and the color to change from red to yellow. This means it is positive for fermentation. Deamination: Bacteria that are negative for fermentation can use this method instead. They break down the peptones in the broth into amino acids then remove the amino group. They will be broken down into ammonia and excreted as waste. The broth will turn pink as the ammonia accumulates. This means it is negative for fermentation Cellular respiration: Bacteria can utilize carbs but uses cellular respiration instead of fermentation. End products are neutral and will not produce a color change. Neutral broth means the results are negative. 3. Recall the substrates and pH indicator of phenol red broth, identifying the role of each in differentiation. - The substrates are the lactose, sucrose, glucose, and mannitol broths while the pH indicator is the phenol red. The broths differentiate by showing which carbohydrate sugar is broken down for which bacteria. The phenol changes to yellow or bright pink when a positive result has occurred while red means the result is negative. 4. What is the purpose of the durham tube? - To indicate if the inoculum produces gas during fermentation. 5. Identify the detectable products and interpret the results of the phenol red broth test, differentiating organisms on their ability to utilize fermentation with or without gas production, cellular respiration or deamination. Don’t think this is the type up kind of answer. 6. Identify mistakes that might lead to a false positive. - Do not incubate for over 24 hrs at 37 degrees C the bacteria will use up all available sugar and start breaking down peptones. This will produce a false positive as the broth would have been yellow before reversion took place. Following reversion, the broth would be pink from the accumulation of ammonia. Chapter 18 1. Identify the purpose of nitrate broth and recognize it as a differential medium. - Nitrate broth is used to differentiate bacteria based on their ability to reduce nitrate and/or nitrite during anaerobic respiration. It’s purpose is to identify bacteria that can use nitrate as the final electron acceptor. This helps differentiate bacteria from the Enterobacteriaceae family. 2. Contrast aerobic and anaerobic cellular respiration via nitrate reduction in terms of final electron acceptor and enzyme complex. - In aerobic respiration, oxygen is the final acceptor with water and oxygen being reduced. In anaerobic respiration via nitrate reduction the final acceptor is nitrate and nitrate/nitrite are reduced. 3. Recall the substrate in nitrate broth and its role. - The substrate used in nitrate broth is potassium nitrate. This acts as the nitrogen source that bacteria can reduce to nitrite allowing for detection of nitrate reduction. 4. Identify the reagents added to the nitrate reduction test, their role, and when to apply to each. - Reagent A ( sulfanilic acid) tests for the presence of nitrite - Reagent B (alpha-naphthylamine) tests for the presence of nitrite - Reagent C (powdered zinc) is only added if the results after adding reagent a&b is inconclusive, a chemical catalyst that reduces nitrate to nitrite - Add A&B to incubated broth and look for pink or red precipitate to form. If this happens, the test is finished and the bacteria has utilized anaerobic respiration and reduced the nitrate. It is positive for anaerobic respiration and partial nitrate reduction. - If no reaction after 30 seconds, then add reagent C and watch for a gradual production of pink to red precipitate. This indicates a positive result for the presence of nitrite but means it was chemically induced. The bacteria is negative for anaerobic respiration and nitrate reduction. 5. Identify the enzyme complex detected by the nitrate test. - It detects nitrate reductase which is responsible for reducing nitrate to nitrite. 6. Interpret the results of the nitrate reduction test and relate the results to the organism’s ability to utilize anaerobic cellular respiration via partial or complete nitrate reduction (denitrification). -The organism cannot utilize nitrate as a final electron acceptor and the nitrate supplied by the broth remains unchanged. -The organism can utilize nitrate as the final acceptor, reducing it to nitrite (partial nitrate reduction). -The organism can utilize nitrate as a final electron acceptor but has rapidly reduced it to nitrite and then further reduced it to ammonia or nitrogen gas (complete nitrate reduction or denitrification). Chapter 19 1. Identify the purpose of motility medium and recognize it as a differential medium. - A motility medium is a differential media that allows us to distinguish bacteria based on their ability to engage in cellular respiration (either aerobic or anaerobic) and if they are motile 2. Interpret the results of motility medium, differentiating motility and respiration results. - Look at the picture on page 196 in the lab book 3.Identify the redox indicator in motility medium, contrasting the reduced and oxidized forms of the indicator. - Redox indicator is tetrazolium salt (TTC) - Oxidized form – TTC is colorless - Reduced form – when TTC is reduced by actively metabolizing bacteria, it forms formazan which is red or pink in color Chapter 21 1.Identify the purpose of mannitol salt agar and recognize it as a selective and differential medium. - MSA is a selective and differential medium that is used to isolate and differentiate the staphylococci 2.Recall the selective ingredient in mannitol salt agar, identifying its role. - The selective ingredient is 7.5% sodium chloride (NaCl) which allows the growth of (selects for) the staphylococci which are salt-tolerant, and inhibits the growth of (selects against) other gram-positive bacteria that are salt-intolerant 3.Recall the substrate, products produced, and pH indicator in mannitol salt agar, identifying the role of each in differentiation.  - Substrate: mannitol - Sugar alcohol used as the primary carbohydrate source is the medium. Bacteria that can ferment mannitol will do so, producing acid as a byproduct - Products produced: acid - When mannitol is fermented, acid is produced as a metabolic byproduct. This acid lowers the pH of the medium - pH indicator: phenol red - Red (neutral pH): when there is no acid production (no fermentation of mannitol), the phenol red remains at a neutral pH - Yellow (acidic pH): when mannitol is fermented, acid is produced, and the pH of the medium drops 4.Interpret the selective results of mannitol salt agar, differentiating staphylococci from other Gram-positive catalase-positive cocci. - Organisms that grow poorly or not at all on this media are not staphylococci and the test is complete at this point as the results for the differential portion of this media cannot be interpreted if the bacterium didn’t grow on the media 5.Interpret the differential results of mannitol salt agar, differentiating pathogenic and non-pathogenic staphylococci. - Pathogenic staphylococci grow on the media and ferment mannitol, producing acid(s), which are detected by the pH indicator phenol red - Nonpathogenic staphylococci also grow on the media, but cannot ferment mannitol, resulting in no color change or result in the pH indicator turning pink due to ammonia production from peptone break-down Chapter 22 1.Identify the purpose of the coagulase test and recognize it as a differential test for the staphylococci. - The coagulase test is a differential test used to separate the gram-positive staphylococci into those that produce the enzyme coagulase (coagulase positive) and those that do not (coagulase negative) - A fast, reliable test for separating Staphylococcus aureus, which is coagulase positive, from other pathogenic staphylococci 2.Recall the substrate, enzyme detected, and product produced in the coagulase test. - Substrate: citrated rabbit plasma  - Enzyme detected: coagulase - Product produced: fibrin clot - When coagulase is present, it catalyzes the conversion of fibrinogen (in plasma) to fibrin, forming a gel-like clot 3.Identify the reagent used for the coagulase test. - Citrated rabbit plasma 4.Interpret the results of the coagulase test, identifying pathogenic staphylococci such as Staphylococcus Aureus. - Coagulase positive: has a clot formation and is Staphylococcus aureus (pathogenic) - Coagulase negative: has no clot formation and is Staphylococcus epidermidis (nonpathogenic) Chapter 23 1.Identify the purpose of blood agar and recognize it as a differential and enriched medium. - The hemolysis test is a differential test that allows the microbiologist to determine if bacteria can produce the toxin hemolysin and if so, what type of hemolysin - Useful for differentiating bacteria that are gram-positive cocci 2.Relate virulence to hemolysin production. - Virulence refers to how harmful or capable a microorganism is in causing disease. One way bacteria can become more virulent is through the production of hemolysins - Hemolysin production makes bacteria stronger by breaking down RBCs to access nutrients and cause more damage to the host. This increases their virulence or disease-causing ability 3.Contrast negative hemolysis (gamma), incomplete hemolysis (alpha), and complete hemolysis (beta). - Beta: complete destruction of the red blood cells in the agar, or complete hemolysis - Alpha: incomplete destruction of the red blood cells, or partial or incomplete hemolysis and is the result of partial breakdown of hemoglobin in the red blood cells  - Gamma: if no change occurs in the appearance of the blood agar, the organism is said to be nonhemolytic 4.Interpret the results of blood agar, differentiating organisms based on their ability to produce hemolysin. - Look on pages 228-229 in lab book to see results of blood agar and what they look like