Biochemical Tests for Microbiology

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macdegnan
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Biochemical Tests for Microbiology
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2014-12-02 17:48:49
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Biochemical tests involved in Microbiology
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  1. The Western Blot test is used to identify what?
    The Western Blot test is used to identify antibodies in a patients serum
  2. Describe the Western Blotting process.
    • 1.Proteins from a known bacterium or virus are separated by an electric current in electrophoresis.
    • 2. The proteins are then transferred to a filter by blotting.
    • 3. Patients serum is then washed over the filter. If the patient has antibodies to one of the proteins in the filter the antibodies and protein combine. Anti-human serum linked to an enzyme is then washed over the filter
    • 4. This will be made visible as a colored band on the filter after addition of the enzymes substrate.
  3. Name two diseases that may be diagnosed by Western blotting
    • HIV
    • Lyme Disease
  4. What is a selective medium?
    • A selective medium is one that selects a particular group of bacteria.
    • Example: gram negative
  5. What is a differential medium?
    • A differential medium is one that further tells you something about members within a particular group. 
    • Example: MAC agar selects for Gram negative bacteria. Now within its group some utilize lactose and some cannot.
  6. The MacConkey agar medium is selective, differential, or both?
    Both selective and differential
  7. The MAC agar medium is commonly used to select  _____ and differentiate ____.
    The MAC agar medium is commonly used to select  Gram + and differentiate lactose fermenters.
  8. The key ingredients for MAC agar differentiation are ___ and a pH indicator, ____ ___.
    The key ingredients for MAC agar differentiation are lactose and a pH indicator, neutral red.
  9. How does a MAC agar inhibit growth of bacteria?
    There are ingredients (crystal violet and bile salt) in the medium that prevent Gram positive bacteria from growing but these ingredients do not discourage the growth of Gram negative bacteria. 
  10. Lactose fermenters are ___ colored and non-lactose fermenters are ____ colored on a MAC agar medium.
    Lactose fermenters are pink colored and non-lactose fermenters are white/colorless colored on a MAC agar medium.
  11. Are the following organisms lactose fermenters or non-lactose fermenters?

    Salmonella typhimurium, Shigella dysenteriae, Proteus vulgaris, Alcaligenes faecalis
    non-lactose fermenters
  12. Are the following organisms lactose fermenters or non-lactose fermenters?

    Escherichia coli
    , Enterobacter aerogenes, Klebsiella pneumoniae
    lactose fermenting organisms
  13. How do lactose fermenters retain a purplish-pink color?
    The drop in the pH from the catabolic activity of breaking down lactose causes ion flow from the MAC agar medium into the bacterial colonies turning the colonies a purplish-pink.
  14. What happens when carbohydrates such as lactose are broken down?
    acids result causing the pH to drop from uninoculated pH 7.0.
  15. Triple sugar iron (TSI) is designed to test what?
    The triple sugar- iron agar test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish them from other gram negative intestinal bacilli.
  16. TSI agar contains which three fermentative sugars?
    • glucose
    • sucrose
    • lactose
  17. TSI slants contain __% glucose, __% sucrose, and __% lactose.
    0.1% glucose, 1.0% sucrose, and 1.0% lactose
  18. When is the TSI slant inoculated?
    It is inoculated when you "stab the butt" then inoculating up the slant.
  19. Why is it that we do not "stab the butt" all the way to the bottom in a TSI slant?
    We do not stab all the way down because we gather data from this area that deals with anaerobic processes, so stabbing to the bottom could lead to oxygen contamination
  20. The blackening of the slant and the breaks in the agar indicates
    Hydrogen sulfide and carbon dioxide respectively. H2S is colorless but it turns black when it reacts with iron
  21. If the entire butt is black you won't see the carbohydrate utilization result. What is the butt alkaline or acidic?
    acidic
  22. If a bacteria produces H2S then the iron in the TSI medium will react with it to create
    ferrous sulfide (FeS)
  23. Result for TSI (slant/butt): K/A
    • Red/Yellow 
    • Glucose fermentation only, peptone catabolized.
  24. Result for TSI (slant/butt): A/A
    • Yellow/Yellow 
    • Glucose and lactose and/or sucrose fermentation
  25. Phenol red turns ___ color at low (acidic) pH, ___ color at pH 7.0, and ___ color at high (alkaline) pH
    Phenol red turns yellow color at low (acidic) pH, orange color at pH 7.0, and red color at high (alkaline) pH
  26. Result for TSI (slant/butt): K/K
    • Red/Red
    • No fermentation, Peptone catabolized.
    • In other words the bacteria doesn't use any carbohydrates, instead it went straight to protein
  27. Result for TSI (slant/butt): A/NC
    Red/No Change

    • Utilizes glucose + lactose and/or sucrose
    • AEROBIC
  28. Result for TSI (slant/butt): K/NC
    Yellow/ No Change

    • protein only
    • AEROBE
  29. Some non-enteric bacteria are unable to metabolize glucose, lactose or sucrose. Instead they utilize peptones which causes the release of ammonia resulting in a pH indicator phenol red
  30. What enzyme catalyzes the reaction in a  urease test?
    Urease
  31. The pH indicator in urease test is
    phenol red
  32. When urease is produced by the organism, _______ is released and the pH of the urea slant becomes________
    When urease is produced by the organism, ammonia is released and the pH of the urea slant becomes alkaline
  33. Urease acts on urea to produce ammonia and drive the pH up or down?
    drive the pH up
  34. True or False: The formation of pink colour is the negative result of urease test
    FALSE: The formation of pink colour is the positive result of urease test
  35. Lysine Iron Agar (LIA) tests for what?
    Lysine iron agar (LIA) slants test organisms for the ability to deaminate lysine or decarboxylate lysine.
  36. What is the AEROBIC process which occurs on the slant of the media?
    Lysine Deamination
  37. What is the ANAEROBIC process which occurs in the butt of the media?
    Lysine Decarboxylation
  38. What does the dark red color on the slant of the media indicate?
    Lysine Deamination (AEROBIC)
  39. What does the purple color on the slant of the media indicate?
    • Lysine Decarboxylation (ANAEROBIC)
    • It produces an amine end-product which reacts with the pH indicator Bromcresol Purple and the media remains purple.
  40. What does a yellow color at the butt of the tube indicate?
    A negative result for Lysine DecarboxylationThe yellow indicates an acidic reaction
  41. What is the pH indicator for Lysine Decarboxylation (ANAEROBIC)?
    Bromcresol Purple
  42. In a LIA test a purple slant means the pH is __ and a yellow slant means the pH is ____
    • purple= alkaline
    • yellow= acidic
  43. Another name for the Voges-Proskauer Test is
    MRVP
  44. What is the objective of a Voges-Proskauer Test?
    To differentiate among the enteric organisms such as Escherichia coli, Enterobacter aerogenes andKlebsiella pneumoniae.
  45. What does the MR and the VP stand for in a MRVP test?
    • MR=mixed acid fermentation 
    • VP= butylene glycol fermentation
  46. Why does it take 3-5 days for a MRVP test to show results?
    It takes 3-5 days for a MRVP test to show results because the acid pathways are very long
  47. The reagent use in Voges-Proskauer test is
    Barrits reagent
  48. What colour is a positive result for VP test?
    red
  49. The VP test determines the ability of the organisms to produce neutral end products _________ after glucose metabolism
    acetyl methyl carbinol
  50. True or False: Barrits reagent must be added to the test culture before incubation
    FALSE
  51. Citrate allows you to determine if your bacterium utilizes ___ as a sole source of ____.
    Citrate allows you to determine if your bacterium utilizes citrate as a sole source of carbon
  52. The pH indicator in a citrate test ____
    bromothymol blue 

    the medium is green at neutral pH and blue under alkaline pH
  53. Citrate utilization requires what two enzymes?
    citrate permease  and citrase
  54. Give the function of citrate permease and citrase
    citrate permease brings citrate into the cell through it selectively permeable cell membrane while citrase catabolizes citrate.
  55. If the bacterium lacks citrate permease and citrase, then the medium usually remains this color
    green
  56. If the citrate is positive it will be this color
    blue
  57. If the citrate test is negative it will be this color
    green
  58. If the bacteria is positive for a mixed acid fermentative pathway it will be this color
    red
  59. In a MIO medium test ornithine is demonstrated by first, utilization of glucose. This will turn the medium what color?
    yellow because of the acidic pH
  60. Why do we give MIO glucose when testing Ornithine decarboxylase?
    because the enzyme decarboxylase of ornithine only works at a low pH
  61. What is the alkaline by-product after the enzyme is decarboxylase of ornithine is demonstrated?
    putresine
  62. The presence of putresine changes the pH of the medium to more acidic or alkaline? Resulting in this color
    alkaline resulting in a purple to bluish-grey color
  63. A negative result of MIO would be this color
    yellow
  64. The pH indicator in an Motility Indole Ornithine test is
    bromcresol purple
  65. How do you test for indole in a MIO medium test?
    By adding Kovac's reagent to your tube. A crimson ring will form if indole is present.
  66. A negative indole result would be this color and a positive indole result would be this color
    a negative indole result is yellow and a positive indole result is red
  67. Name the three carbohydrates in TSI medium and give their proper concentrations
    • 0.1% glucose 
    • 1.0% sucrose
    • 1.0% lactose
  68. What does it mean in terms of carbohydrate utilization when a bacterial species gives a K/A reaction + H2S in a TSI tube?
    Alkaline top and acidic bottom K/A and a black precipitate. The only carbohydrate utilized was glucose and the catabolism of the protein peptone.
  69. If a bacterium utilizes citrate, the bacterium contains citrase. What is the name of the other enzyme it must possess in order to utilize citrate?
    citrate permease
  70. Name one product of citrate utilization
    CO2- sole source of carbon
  71. What does it mean when a bacterium presents a pink colony on a MAC plate?
    It means the bacteria is a lactose fermenter. The colony turns pink due to the decrease in pH. The pH drop creates an ion flow of selective agents from the medium to flow to the bacteria colonies.
  72. If a MIO tube is yellow 24 hours after inoculation and then turns to purple 24 hours later, what does that mean in terms of ornithine utilization?
    Ornithine decarboxylase positive. We detected the enzyme that takes away the carboxyl group leaving the NH4 on. 
  73. How can you tell a virus has either lysine decarboxylase or deaminase in a LIA test?
    If the pH goes up (turning the slant purple) then we are able to interpret that the carboxyl group was taken off the lysine cell leaving NHturned on. Decarboxylase positive; Deaminase negative.

    If the pH goes down (leaving the slant yellow) then we are able to interpret that the ammonia group was taken off leaving the carboxyl group turned on. Decarboxylase negative; Deaminase positive
  74. If hydrogen sulfide gase (H2S) is colorless, how do you know your bacterial species produces H2S? (be specific, i.e., provide the chemistry)
    Iron will react with hydrogen sulfide to produce the black precipitate, Ferrous sulfide a.k.a Iron III Sulfide. 

    Fe+H2S= FeS
  75. If you inoculated LIA with an enteric bacterium and the slant was purple in color and the butt was yellow, what does this mean in terms of lysine utilization?
    • Butt (anarobic) Lysine decarboxylase negative
    • Slant (aerobic) Lysine deaminase negative
  76. What is the formula for converting tryptophan to indole?
    tryptophan → (triptophanase) → indole + pyruvate + CO
  77. What is the name of the reagent that you use to detect indole in a MIO tube?
    Kovac's reagent
  78. How old should your bacterial cells be when you preform a catalase and oxidase test?
    18 - 24 hours "fresh cells"
  79. Why do you need 40% KOH in the VP test?
    In order to determine the presence of acetone we must use 40% KOH to lyse open the cell. Acetone (the intermediate compound) is on the cytoplasm (where metabolic reactions occur, right?). Once you lyse open the cell add alpha-napthol. If VP turns red your cell follows a butylene glycol fermentation pathway.
  80. What protein is in the TSI medium?
    peptone
  81. What is the enzyme catalase?
    • Catalase is an enzyme that takes toxic peroxides and catalyzes them
    • H2O2→(catalase)→ H2O + O2
  82. How will you know if the bacterium has the enzyme catalase?
    You will see bubbles.
  83. Give the reason behind why you should not perform a catalase test on a blood agar plate
    There is catalase in blood so you will get a false positive.
  84. What is it you are trying to determine when performing an oxidase test?
    If the bacterium has the enzyme cytochrome c oxidase. Blue and black means your bacteria has this enzyme. No color means your enzyme does not.
  85. Why is it important to use a wooden stick instead of the nichrome wire loop when performing an oxidase test?
    Because you run the risk of getting a false negative in your results. The wire is made of nichrome and the enzyme contains cytochrome.
  86. For the following bacteria determine whether they are oxidase positive/negative and catalase positive/negative.

    E. coli, Enterococcus faecalis, S. aureus, Pseudomonas aeruginosa
    Escherichia coli and Staphylococcus aureus are the two bacteria that are catalase positive while Pseudomonas aeruginosa is the only bacteria oxidase positive.
  87. Name the selective agent and the differential agent in a MSA medium
    • selective agent is salt, 7.5% NaCl, and the 
    • differential agent is mannitol
  88. The medium Mannitol Salt Agar (MSA) is selective for ____ and differential for ____.
    The Mannitol Salt Agar is selective for Gram positive bacteria and differential for mannitol fermenters and non-mannitol fermenters. 
  89. What is the pH indicator in a Mannitol Salt Agar (MSA)?
    phenol red. The plate will turn yellow if the bacterium is a mannitol fermenter. If the bacterium is a non-mannitol fermenter the plate will be red.
  90. Give one example of a mannitol fermenting bacteria and one example of a non-mannitol fermenting bacteria?
    Staphylococcus aureus is a mannitol fermenter. This strain is harmful because it can cause toxic shock syndrome

    Staphylococcus epidermitis is an example of a non-mannitol fermenter. This strain is benign.
  91. What color is the MSA medium if it is Staphylococcus aureus and what color will it be if it is Staphylococcus epidermutis?
    S. aureus will be yellow

    S. epidermitus will be pink
  92. What is the cellular morphology and the cellular arrangement of Staphylococcus aureus?
    The cellular morphology is cocci and the cellular arrangement is in clusters.
  93. Why are S. aureus and S. epidermitus incubated at 37 degrees C? And what happened to the rabbits plasma when it solidified?
    Because 37 degrees C is the average temperature in the human body. The rabbit plasma contained a coagulase- positive organism.
  94. There are two types of coagulase produced by most strains of S. aureus, bound coagulase also called clumping factor and free coagulase. Explain bound coagulase:
    Bound coagulase or clumping coagulase is attached to the bacterial cell wall and can enzymatically convert fibrinogen in plasma to insoluble fibrin and cause the bacterial cells to clump.

    fibrinogen → coagulase → fibrin
  95. What is a BAP?
    a BAP (Blood Agar Plate) is enriched with blood.
  96. How is a Blood Agar Plate (BAP) selective and differential?
    Selective for bacteria that love blood and differential for degree of hemolysis,
  97. What are hemolysins?
    Hemolysins are the proteins that cause hemolysis.
  98. What is the main function for extracellular enzymes?
    Extracellular enzymes break large molecules into smaller molecules so they can pass through the semipermeable cell membrane.
  99. State the function of the following extracellular enzymes; proteus, lipase, and amylase.
    Proteus breaks down protein. Use a casein plate, a milk protein, if there is a clearing then the bacteria has this enzyme. 

    Lipase breaks down fats and lipids. Use tributyrin and look for clearing

    Amylase breaks down starch. Use a starch plate, add iodine around border of the colony. Will turn blue.
  100. Why do we add iodine to a starch plate to see if it contains the amylase enzyme?
    We add iodine because iodine + starch react to create a blue color.
  101. If the bacterium is amylase positive what color will it be?
    Brown because the iodine is all thats left. Iodine reacts with starch to produce a blue color. If there is no more starch on the plate then the iodine will have nothing to react with. This is all because amylase breaks down starch.
  102. In the Wright's Stain neutrophils phagocytized the S. epidermitus but not Streptococcus pneumoniae. How come?
    S. pneumoniae has a capsule which disguised it from the bodies neutrophils.
  103. How is an API strip test selective?
    It only selects Gram negative bacteria.
  104. Give an example of a beta complete hemolysis strain of Streptococcus ssp.
    Group A i.e., Streptococcus pyogenes 

    *flesh eating bacteria which can cause toxic shock- like syndrome such as Scarlett fever
  105. A CAMP test is used to determine what infection?
    Strept B but can help you determine if you have a Group A.
  106. Give an example of a bacterium that can cause toxic shock syndrome and an example of a bacterium that can cause toxic shock-like syndrome.
    toxic shock syndrom- Staphylococcus aureus

    toxic shock-like syndrom- Streptococcus pyogenes 
  107. Name the four Gram negative bacteria that are lactose fermenters in a MAC agar
    • Escherichia coli
    • Klebsiella pneumoniae
    • Citrobacter freundii
    • Serratia marcescens
  108. Name the four Gram negative bacteria that are non-lactose fermenters in a MAC agar
    • Shigella flexneri
    • Salmonella typhimurium
    • Proteus vulgaris
    • Enterobacter aerogenes
  109. Name four bacteria who have a A/A Triple Sugar Iron test result with gas: A/A + gas
    • Klebsiella pneumoniae 
    • Serratia marcescens 
    • Escherichia coli
    • Enterobacter aerogens
  110. Name four bacteria who have a A/A Triple Sugar Iron test result with gas: A/A + H2S + gas
    • Citrobacter freundii
    • Proteus vulgaris
  111. In a Lysine Iron Agar (LIA) name two gram negative bacteria that are lysine deaminase negative (purple) and lysine decarboxylase negative(yellow) [K/A]
    Citrobacter freundii and Shigella flexneri
  112. In a Lysine Iron Agar (LIA) name two gram negative bacteria that are lysine deaminase positive (red) and lysine decarboxylase negative (yellow): [A/A]
    Proteus vulgaris and Salmonella typhimurium 
  113. In a Lysine Iron Agar (LIA) name two gram negative bacteria that are lysine deaminase negative (purple) and lysine decarboxylase positive (purple) [K/K]
    • S.E.E.K
    • Serratia marcescens 
    • Enterobacter aerogenes
    • Escherichia coli
    • Klebsiella pneumoniae 
  114. Name the four gram negative bacteria we worked with that are citrate positive
    • Enterobacter pneumoniae
    • Salmonella typhimurium
    • Citrobacter freundii
    • Serratia marcescens 
  115. Name the four gram negative bacteria we worked with that are citrate negative
    • Shigella
    • E. coli
    • Klebsiella
    • Proteus
  116. Is a blood agar plate (BAP) selective or differential?
    Differential
  117. A blood agar plate allows distinction among bacteria based on their ability to lyse red blood cells and hemoglobin. What happens in Beta hemolysis and how can you tell this is happening?
    Beta hemolysis is the complete hemolysis of red blood cells and hemoglobin. The results show complete clearing of the blood around the colony.
  118. A blood agar plate allows distinction among bacteria based on their ability to lyse red blood cells and hemoglobin. What happens in Alpha hemolysis and how can you tell this is happening?
    In alpha hemolysis there is partial hemolysis of the red blood cells and hemoglobin. This results in a greenish grey discoloration of the blood around the colonies.
  119. Is a Mannitol Salt Agar (MSA) selective or differential?
    both
  120. What is the pH indicator in a MSA plate?
    phenol red
  121. What is the selective agent in a MSA (mannitol salt agar)?
    7.5 % NaCl
  122. Organisms that are able to ferment the mannitol in a MSA plate will produce acid fermentation products which lower the pH, causing the phenol red indicator to turn this color
    yellow
  123. Does Pseudomonas aeruginosa possess the enzymes lipase, amylase and protease?
    Pseudomonas aeruginosa has the ability to break down lipids/fats and proteins.
  124. Does Escherichia coli  possess the enzymes lipase, amylase and protease?
    Escherichia coli has the ability to break down proteins.
  125. Does Bacillus cereus possess the enzymes lipase, amylase and protease?
    Bacillus cereus has the ability to break down starch and proteins.

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