Microbio Lab Exam 2 (Labs 24-29) PDF

Chapter 24 1.Identify the purpose of Lancefield grouping and hemolysis testing as it relates to distinguishing streptococci. - Lancefield grouping differentiates the streptococci and enterococci into Groups A through T based on the specific carbohydrate composition of antigens on their...

Chapter 24 1.Identify the purpose of Lancefield grouping and hemolysis testing as it relates to distinguishing streptococci. - Lancefield grouping differentiates the streptococci and enterococci into Groups A through T based on the specific carbohydrate composition of antigens on their cell wall surface - Group A: Streptococcus pyogenes - Group B: Streptococcus agalactiae - Group D: various streptococci and enterococci 2.Contrast bacitracin sensitivity testing with optochin sensitivity testing, interpreting the results of each,and distinguishing between the streptococcal groups these tests help to differentiate. - Bacitracin sensitivity test: differentiates Group A Streptococcus (GAS) (sensitive) from Group B Streptococcus (GBS) (resistant) - Grow bacteria on blood agar; place a bacitracin disc on the agar; check for a clear zone around the disc where bacteria can’t grow (zone of inhibition) - Positive test: a zone of inhibition measuring 10 mm surrounding the bacitracin disc is interpreted as sensitive - Negative test: no zone of inhibition; bacteria is resistant - Optochin sensitivity test: distinguishes Streptococcus pneumoniae (sensitive) from other alpha-hemolytic streptococci, like Streptococcus viridans (resistant) - Grow bacteria on blood agar; place optochin disc on the agar; look for a zone of inhibition around the disc - Positive test: zone of inhibition (Streptococcus pneumoniae) – sensitive - Negative test: no zone of inhibition (Streptococcus viridans) – resistant 3.Identify the purpose of bile esculin agar and recognize it as a selective and differential medium. - A selective and differential medium that can separate Group D streptococci and enterococci from other streptococci 4.Recall the color indicator, selective ingredient, and differential ingredient in bile esculin agar, identifying the role of each. - Selective ingredient: bile – a substance normally found in the digestive tract (and normal habitat of Group D bacteria) that selects against most gram-positive organisms but selects for the Gram-positive enteric streptococci and enterococci - Differential ingredient: esculin – a sugar compound that only certain Group D streptococci can chemically alter  - Color indicator: when the product esculin is released, it will bind to ferric citrate (color indicator), present in BEA to produce a dark brown-black iron precipitate 5.Identify the detectable product and interpret the selective and differential results of bile esculin agar,differentiating Group D streptococci from other streptococci. - Detectable product: esculetin - Selective results: bile salts in the agar make it selective; bile inhibits most non-group D streptococci, allowing Group D streptococci (like Enterococcus) to grow - Differential results: based on the ability to break down esculin; Group D streptococci can hydrolyze esculin into esculetin, producing a dark color - Positive result: black/dark brown color in the agar - Indicates the organism can grow in the presence of bile and hydrolyze esculin - Suggests Group D streptococci - Negative result: no color change - Indicates the organism cannot hydrolyze esculin, suggesting non-Group D streptococci 6.Identify the purpose of 6.5% salt broth, recalling the selective ingredient. - A selective medium due to high concentration of sodium chloride (NaCl) which select against the non-enterococci such as Streptococcus bovis but select for enterococci such as Enterococcus faecalis - Selective ingredient: 6.5% sodium chloride (NaCl) 7.Interpret the selective results of 6.5% salt broth, differentiating Group D enterococci from non-enterococci. - Positive result: turbid or sedimentary growth in the broth - Enterococci such as Enterococcus facecalis - Negative result: a broth that remains clear, with no growth Chapter 26 1.Identify the purpose of MacConkey agar and recognize it as a selective and differential medium. - A selective and differential medium that is used to select for and differentiate members of the family Enterobacteriaceae 2.Recall the selective ingredient in MacConkey agar, identifying its role.  - Bile salts: normally found in the intestines (the habitat of enteric bacteria) that inhibit the growth of any non-enteric bacteria - Crystal violet: inhibitory to gram-positive bacteria that are enteric 3.Recall the substrate, products produced, and pH indicator in MacConkey agar, identifying the role of each in differentiation. - Substrate: lactose – helps differentiate bacteria based on their ability to ferment lactose - Products produced: acid from lactose fermentation – acid production lowers the pH, causing color changes in the pH indicator - pH indicator: neutral red – indicates whether the bacteria fermented lactose - Pink/red colonies: acidic pH (E. coli) - Colorless colonies: neutral pH (Salmonella) 4.Interpret the selective results of MacConkey agar, differentiating bacteria in family Enterobacteriaceae From non-enteric bacteria. - MacConkey agar’s selective properties allow Gram-negative bacteria (including Enterobacteriaceae) to grow while inhibiting gram-positive bacteria. This makes it useful for isolating and identifying enteric bacteria in mixed samples 5.Interpret the differential results of MacConkey agar distinguishing between coliforms and non-coliforms. - Coliforms (lactose fermenters) produce pink/red colonies due to acid production - Non-coliforms (non-lactose fermenters) produce colorless colonies because they don’t ferment lactose 6.Identify mistakes that might lead to a false test result for MacConkey agar. - Incubating MacConkey agar past 24 hours - Incorrect incubation temperature - Contamination Chapter 27 1.Identify the purpose of triple sugar iron agar and recognize it as a differential medium. - A differential medium that distinguishes the members of family Enterobacteriaceae by differences in their ability to ferment glucose, lactose, and sucrose, produce gas while fermenting one or more of these sugars, and produce hydrogen sulfide (H2S) 2.Recall the substrates and indicators in triple sugar iron agar and the role of each. - Substrates: - Lactose: used by bacteria that can ferment lactose to produce acid - Sucrose: another sugar that can be fermented by some bacteria, producing acid and helping to distinguish them based on sugar fermentation - Glucose: simple sugar that can also be fermented by bacteria. However, it is present in a lower concentration that lactose and sucrose in TSIA - Peptones (proteins): serve as the source of amino acids. If a bacterium cannot ferment any of the sugars, it will use these proteins for growth, which can lead to alkaline (basic) conditions and no color change or a red color in the slant - Thiosulfate: helps identify bacteria that can produce hydrogen sulfide (black color in the medium) - Indicators: - Phenol red: a pH indicator that is yellow bellow pH 6.8 and pink at pH 8.2 and higher - Ferrous ammonium sulfate: a color indicator which turns black in the presence of hydrogen sulfide (H2S) 3.Relate relative concentration of each sugar to metabolic preference, identifying which sugar will be utilized first. - Glucose is used first due to its lower concentration and easier metabolism - Once glucose is consumed, bacteria that can ferment lactose or sucrose will use these sugars, but at a slower rate due to their higher concentration 4.Interpret the carbohydrate results of triple sugar iron agar and relate the results to the organism's ability to ferment each carbohydrate with or without gas production. - A/A (yellow slant, yellow butt): glucose fermentation, lactose and/or sucrose fermentation - K/A (red slant, yellow butt): glucose fermentation only (exhausted supply in slant after 12 hours, bacterium switched to peptone catabolization) - K/K or K/NC (red slant, red butt or no change): no fermentation, peptones catabolized and organism is not in family enterobacteriaceae; no change in the butt indicates organism cannot grow anaerobically - +G (cracks, bubbles, or lifting of agar): gas produced during fermentation of carbohydrates - +H2S (black precipitate in butt): sulfur reduction (always assume butt is yellow beneath precipitate) 5.Interpret the thiosulfate reduction results of triple sugar iron agar. - Black precipitate indicates the production of hydrogen sulfide from the reduction of thiosulfate, signaling the presence of bacteria that can produce this compound - The bacteria have reduced thiosulfate to hydrogen sulfide 6.Identify mistakes that might lead to a false test result for triple sugar iron agar. - A false negative for fermentation of a carbohydrate may be observed if the test is read later than 24 hours Chapter 28 1.Identify the purpose of urea broth and recognize it as a differential medium. - A differential medium that distinguishes Proteus species from other lactose nonfermenters in Family Enterobacteriaceae on their ability to express (synthesize) the enzyme urease and hydrolyze urea, using it as their sole source of nitrogen, producing carbon dioxide and ammonia 2.Recall the substrate, products produced, and pH indicator in urea broth, identifying the role of each indifferentiation. - Substrate: urea – tested for breakdown by bacteria. If a bacterium produces urease, it will break down the urea into ammonia - Products produced: ammonia – increases the pH of the medium, making it more alkaline (basic) - pH indicator: phenol red – if ammonia is produced, the medium turns pink which is a positive result for urease activity 3.Identify the enzyme detected by the urea hydrolysis test. - Urease 4.Interpret the results of the urease test and relate the results to the organism’s ability to produce urease, contrasting rapid and slow production. - Positive result: bright pink – rapid production - Negative result: the tube will remain the same color as an uninoculated tube (control) or turn slightly yellow – slow production 5.Identify mistakes that might lead to a false test result for the urease test.  - Incorrect incubation time, contamination, inoculating too much bacterial culture, etc. Chapter 29 1.Identify the purpose of the IMViC series of tests as they relate to distinguishing among members of family Enterobacteriaceae. - The tests in this panel can be used to differentiate many bacteria, but they are typically run together as a series to distinguish between members of Family Enterobacteriaceae, especially when identifying the coliform members of this family (Escherichia, Citrobacter, and the Enterobacter-Klebsiella group) 2.Identify the purpose of the indole test and the differential medium used for this test, recalling the substrate, detectable product, and reagent added. - Purpose: Differentiates bacteria on their ability to synthesize the enzyme tryptophanase - Differential medium used: tryptone broth - Substrate: tryptophan (amino acid) - Detectable product: indole - Reagent added: Kovac’s reagent – allows for the indirect detection of tryptophanase as it reacts with the end product indole, producing a red ring at the surface of the broth 3.Identify the purpose of the methyl red test and the differential medium used for this test, recalling the substrate, detectable product, and reagent added. - Purpose: detects bacteria that utilize the mixed acid fermentation pathway by their production of large amounts of a mixture of stable acidic end products (acetic, lactic, and succinic acids) - Differential medium: MR-VP broth - Substrate: glucose - Detectable product: acid (like lactic acid, acetic acid, formic acid, etc) which lowers the pH - Reagent added: methyl red, turning red if the pH is acidic (positive result) 4.Identify the purpose of the Voges-Proskauer test and the differential medium used for this test, recalling the substrate, detectable product, and reagent added. - Purpose: Identifies bacteria that utilize the 2,3 butanediol fermentation pathway by detection of acetoin, an intermediate in the pathway - Differential medium: MR-VP broth - Substrate: glucose  - Detectable product: acetoin - Reagent added: VP reagent A (alpha-naphthol) and VP reagent B (potassium hydroxide) 5.Identify the purpose of the citrate hydrolysis test and the selective and differential medium used for this test, recalling the substrate, pH indicator, and products produced. - Purpose: detects bacteria that can synthesize the enzyme citrase, allowing them to hydrolyze citrate a sa source of carbon and energy when no other nutrients are supplied - Selective and differential medium used: simmon’s citrate agar - Selective: it selects for bacteria that can use citrate as their sole carbon source - Differential: it differentiates bacteria based on their ability to utilize citrate - Substrate: citrate - pH indicator: bromthymol blue - Products produced: alkaline products (like ammonia), which raise the pH of the medium 6.Identify the enzymes detected by the indole and citrate hydrolysis tests. - Indole test: tryptophanase - Citrate hydrolysis test: citrate permease 7.Interpret the results of the indole test, methyl red test, Voges-Proskauer test, and citrate hydrolysis test. - Indole test: - Positive result: pink-ish color - Negative result: cloudy, yellow color - Control media: clear, slightly yellow - Methyl red test: - Positive result: pink/red color - Negative result: appears the same color as uninoculated (control) media - Voges-Proskauer test: - Positive result: pink color on top and middle, yellow-ish color on bottom - Negative result: a ring of pink color on the very top of the media; color is mainly yellow - Citrate test: - Positive result: a color change to blue must occur in at least half of the slant - Negative result: appears the same color as control media (dark green color)  - look on pages 290-292 in lab book 8.Identify mistakes that might lead to a false test result for the methyl red test. - Incubating for under 48 hours at 37 degrees Celsius - Needs to be incubated for a minimum of 48 hours at 37 degrees Celsius