Microbiology Lab

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Microbiology Lab
2012-12-16 09:43:11
Micro Lab Study Notes

Lab Study Notes
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  1. Aseptic Technique
    • Any procedure that is performed under a sterile condition
    • Inc. Disinfecting Lab Bench and Microscope Before and After
  2. Media/Medium
    • Food, Nutrients or Home in which Bacteria grow in
    • Agar plate (Solid Media)
    • Luria Broth or LB (Liquid Media)
  3. Describe cell wall of a gram + bacteria
    Thick peptidoglycan cell walls
  4. Describe the cell wall of a gram - bacteria
    • Thin peptidoglycan cell wall with a L
    • LPS layer
  5. Describe E. Coli
    Gram negative pink rod bacilli
  6. Describe Staphylococcus epidermidis
    Gram + purple round cocci
  7. Describe Bacillus Sphaericus
    Gram + purple rod bacilli
  8. Describe gram variable
    : staining irregularly or inconsistently by Gram's stain
  9. Purpose of gram staining
    • Divides bacterial cells into two major groups, gram+ and gram-
    • Essential toll for classification and differentiatinon of microorganisms
  10. Primary stain in gram staining is
    Crystal Violet, Stains all cells purple
  11. Mordant
    Grams IODINE, increases the cells affinity to a stain 
  12. Decolorizing agent in gram staining
    95% Ethyl Alcohol, Its actions are determined by the thickness of the Peptidoglycan layer in the bacterial cell wall
  13. Counter stain in Gram Staining
    Safranin, Final reagent. Stains Pink/Red cells that have been previousl decolorized gram negative
  14. Most critical phase of gram staining
    Delorization step
  15. Affects of over decolorization
    will result in loss of primary stain causing gram + organism to appear gram -.
  16. Affects of under decolorization
    Will not decolorize gram - organisms from primary stain causing them to appear gram +
  17. Purpose of Subculturing
    The transfer of microorganisms from one media type to another by asptic technique
  18. Purpose of Fourway Streak Technique of mixed culture
    Isolation of pure or discrete colonies from a mixed culture
  19. First step in focusing a microscope
    Always start focusing from low power objective, first set your coarse adjustment followed by fine adjustment.
  20. When should you never use youre coarse adjustment
    When focusing on medium to high power objective lens
  21. What is the most widely used microscope and how does it work
    • Compound microscope
    • Works by letting light pass through an object and then through two or more lenses can magnify upto 2000x
  22. Describe a  fail agar plate microbial test
    After 24hrs of incubation there is growth of 11 or more microbial colonies on the agar plate
  23. Microorganism are transferred from one medium to another by?
    Is used routinely in preparing and maintaining stock cultures.
    Is used in microbial test procedure
    • Subculturing
  24. Purpose of Subculturing
    • Microorganism are transferred from one medium to another
    • Is used routinely in preparing and maintaining stock cultures.
    • Used in microbial test procedure
  25. Why flame the inoculating instrument prior to and after each inoculation?
    Heat sterilization prevents exposure/contamination of one bacteria from one inoculation to the next
  26. Pure culture are suitable for the study of their
    • Cultural
    • Morphological Properties
    • Biochemical Properties
  27. What technique can be used to produce discrete colonies
    Fourway streak plate technique
  28. Describe Streak Plate
    (Rapid qualitative isolation method) Dilution technique that involves spreading a loopful of culture over the surface of an agar palte is called
  29. Instrument used to Heat Fix
    Bunson Burner
  30. How are microorganisms transferred from one media to another
    By Subculturing 
  31. How are stock cultures prepared and maintained 
    By Subculturing
  32. How are bacteria isolated into pure and discrete colonies from a mixed culture 
    B y Fourway Streak Technique
  33. The final reagent in gram staining 
  34. How is exposure contamination of one bacteria from one inoculation to the next inoculation prevented
    By Flaming the inoculating instrument (Heat Sterilization) prior to and after each inoculation 
  35. When focusing medium to high power objective lens what should be avoided
    course adjustment focusing 
  36. The actions of what component are by the thickness of the Peptidoglycan layer in the bacterial cell wall 
    95% Ethyl Alcohol 
  37. What affect will result in loss of primary stains causing gram + organism to appear gram -
    Over decolorization 
  38. What classification of bacteria have thick peptidoglycan cell walls 
    Gram + Bacteria 
  39. What component increases the cells affinity to a stain
  40. Inconsistent or Irregular staining by Grams stain is known as 
    Gram variable
  41. Thin peptidoglycan cell wall with a LPS layer are known as 
    Gram - Bacteria 
  42. Describe Bacillus
     Gram positive rod shaped bacteria
  43. Describe Bacillus Sphaericus
    Gram + purple rod bacilli
  44. What affect will cause gram - bacteria to appear gram + in gram staining
    Under decolorization of primary stain crystall violet
  45. Transfer of a liquid media to a solid media or solid media to a liquid media can be done by 
  46. What microscope works by letting light pass through an object and then through two or more lenses `
    Compound microscope
  47. E. Coli
    Gram negative pink bacilli
  48. Bacillus
    Any rod shape bacteria
  49. Cocci
    Spherical or ovoid bacterium (Round)
  50. Staphylococcus Epidermis 
    Gram + Purple Cocci
  51. Bacillus sphaericus
    Gram + Purple Bacilli
  52. Purpose of Sugar Fermentation Laboratory 
    To determine if a bacterium can ferment a particular carbohydrate and determine the end products of that fermentation. 
  53. Define Fermentation
    The conversion of carbohydrates into alcohols or acids under energy production in a cell under anaerobic conditions.
  54. Phenol red fermentation medium contains
    Peptone, phenol red (a pH indicator) and the carbohydrate to be tested.
  55. Phenol red is yellow at what pH level
    pH level less than 6.8
  56. Phenol red is red in what Ph level
    pH level greater than 7.4
  57. If a bacterium ferment a sugar it breaks it down into?
  58. Phenol red tubes contain an inverted small tube inside named
    Durham tube
  59. If a bacterium produces a gas during fermentation what happens to the gas
    It is trapped in the Durham tube will float and a bubble will be vissible
  60. Sometimes an orange color will develop in the the tube this represent a pH level
    greater than 6.8 and should not be considered positive.
  61. describe characteristic of oxidative in a Phenol red fermentation medium
    Tubes initially produce acid then turn red or pink as nitrogenous products are produced.
  62. What is the color in a negative phenol red tube (negative for fermentation)
    Red and Orange
  63. What is the color in a positive phenol red tube (Positive for acid)
  64. What happens to the Phenol red tube when its positive for gas formation
    bubbles are trapped in the durham tube causing it to float
  65. Purpose of Catalase Test
    Prove that staph has catalase
  66. MacConkey agar is a
    Culture medium designed to grom gram negative bacteria and stain them for lactose fermentation
  67. MacConkey agar contains
    • Bile salts
    • Crystal Violet Dye 
    • Neutral red dye 
    • Lactose 
    • Peptone
  68. Purpose of bile salts in MacConkey agar
    Inhibit most Gram positive bacteria, except Enterococcus and some species of Staphylococccus
  69. Purpose of Crystal violet dye in MacConkey agar
    Inhibits certain Gram-positive bacteria
  70. Purpose of Neutral red dye in MacConkey agar
    Stains microbes fermenting lactose
  71. Neutral red dye in MacConkey agar serves as a
    Visual pH indicator
  72. Visual pH indicator in ManConkey agar distinguishes those
    Gram negative bacterium that can ferment the sugar lactose (Lac+) from those that cannot (Lac-)
  73. In MacConkey agar when Lac+ bacteria utilize the lactose available in the medium what happens in the agar
    Lowers pH level below 6.8 and results in the appearance of red/pink colonies
  74. In MacConkey agar when Lac- bacteria cannot utilize the lactose available in the agar it will use peptone instead what will happen in the agar
    This forms amonia, which raises the pH of the agae, and leads to formation of white/colorless colonies.
  75. In MacKonkey agar gram negative bateria that ferment lactose grow in 
    Pink colonies
  76. In MacKonkey agar gram negative bateria that do not ferment lactose grow in colorless colonies
  77. If MacConkey agar is innoculated and results in no growth after 24hr incubation what does it indicate 
    It was a gram + bacteria. MacConkey agar is selective medium for gram negative bacteria
  78. Mannitol salt agar (MSA) contains a
    high concentration (7.5-10%) of salt (NaCl) sodium chloride
  79. What make Mannitol salt agar a selective medium for Staphylococci and Micrococcaceae
    Its high level of sodium chloride. Since this level of NaCl is inhibitory to most other bacteria. Most bacteria cannot grow in extremely salty enviroment
  80. What make mannitol salt agar a differential medium
    Its mannitol and the indicator phenol red
  81. In mannitol salt agar what happens to species that can ferment the mannitol
    They will produce yellow colonies with yellow zones
  82. In mannitol salt agar what happens to species that cannot ferment the mannitol
    They will produce small pink or red colonies with no color change to medium
  83. Mannitol salt agar contains
    pH sensative dye and a sugar mannitol alcohol
  84. What causes the pH sensative dye in Mannitol salt agar to turn bright yellow (previously Red)
    When bacteria that can ferment eat mannitol they creat acidic waste product when they break it down. This acidic waste products triggers the pH sensative dye to change colors
  85. What happens to the mannitol agar plate when bacteria are not mannital fermenters
    The medium remains pink
  86. What type of agar is used to determine the ability of bacterial colonies to induce hemolysis
    Blood agar plate
  87. Alpha hemolysis
    Incomplete hemolysis. This is sometimes called green hemolysis because of the color change in the agar
  88. Beta hemolysis
    Complete hemolysis
  89. Gama hemolysis
    does not induce hemolysis. no hemolysis
  90. Bubbling upon the addition of hydrogen peroxide in indicative of the presence of 
    catalase for the organism
  91. Caltalase enzyme is responsible for protecting the bacterium from ?
    H2O2 accumulation, which can occur during aerobic metabolism
  92. Catalase breakdown H2O2 into
     water and O2.
  93. Positive bubbling on a test organism after adding H2O2 indicates 
    presence of catalase and is considered a Positive test 
  94. Are starch plate selective media and why
    No because its an all purpose media that will grow anything
  95. The breadown of starch in a starch plate indicates
    Lets us know that the organism has the ability to produce amylase, the enzyme that breaksdown amylose, the main chain of starch.
  96. After inoculation and incubation of a starch plate what is the next step
    The plate is flooded with grams iodine, a reactive ingredient with starch.
  97. In a starch plate grams iodine stains any starch molecule as 
    blue/black color
  98. In starch plate any area or clearings near the culture that are not dyed by iodine indicates 
    That the organisms can produce amylase and breakdown starch. Since stach is broken down there is no starch left on the plate in those clearing showing no blue or black color.
  99. In a starch plate a bacteria is positive for starch hydrolysis if we see a 
    clearing in the area 
  100. The eosin methlyene blue (EMB) plate is a selective media for 
    Gram negative bacteria
  101. EMB plate contains what 2 dyes 
    Eosin and methylene blue ina ratio if 6:1.
  102. In EMB plate what is the purpose of eosin and methylene blue dye
    They inhibit the growth of gram positive as well as differentiates bacteria that can ferment lactose
  103. EMB plate as the ability to distinguish microbes that are 
    Pathogenic in a very short amount of time 
  104. What happens 
  105. In EMB plate lactose positive bacteria will be able to produce acid and 
    lower the pH of the plate allowing for the dye to be absorbed by colonies leaving a purplish color
  106. In EMB plate lactose negative bacteria will not be able to produce acid hence not allowing the dyes to
    be absorbed by the colonies leaving the colorless
  107. The metachromatic properties of the dyes in the EMB plate will cause E. Coli to have a distinct
    Metallic green color
  108. Describe Bacillus Subtilis
    Gram + Purple Bacillus