Microbiology Lab

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Al810024
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250626
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Microbiology Lab
Updated:
2013-12-04 03:35:00
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Microbiology
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Practical
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  1. Sterilization
    • Heat 172C 2 hours 
    • - all of the water will evaporate if boiled 
    • - so dry heat is best
  2. Autoclave
    121C,15 minutes, 15 PSI
  3. Why do we need a media?
    A nutrient food to help it reproduce and multiply
  4. What does sterilization mean?
    Process of getting rid of all forms of life and viruses
  5. At what temperature will nutrients not survive?
    121 C
  6. Ultraviolet (UV)
    -Cabinets are where you place the dishes, so best place to put UV light to decontaminate 

    -It cannot penetrate through the container, so UV is not strong
  7. Enthylene Oxide (gas)
    • -Used in industry or in hospital 
    • -It penetrates through materials
  8. Pasteurization
    It doesn't kill everything, it is partial sterilization. 

    -Vegetative cell (spores)
  9. Vegetative Cells
    • -If the environment is not good it will stop producing but it will survive but eventually die
    • - They sporulate, hibernate
    • - slows down metabolism and use remaining energy to create a shield, barrier
  10. Classic
    • 63 C
    • 20-30 min
  11. Flash
    • 72 C
    • 1-2 min
  12. Media (Physical Properties)
    • Liquid 
    • Solid 
    • Semi-solid
  13. Media (Solid)
    • 1.5% Agar 
    • ~add to 100 ml liquid
  14. Semi-solid agar (Media)
    • 0.4-0.8% Agar 
    • to dissolve agar, it has to be at 100 C
    • at 42-45 C it will start solidifying and dissolve
  15. Agar
    • Plate 
    • deep 
    • slant
  16. Deep
    Anaerobic/aerobic organism
  17. Slant
    Used as storage
  18. Complex medium
    Give all nutrient, amino acid and sugar
  19. Defined synthetic medium
    A medium in which specific nutritional requirements or chemical compositions needed for microbial growth
  20. Complete (rich) medium
    Gives everything, it has all the nutrients
  21. Selective medium
    ass something or take away that some organism will grow or don't
  22. Differential medium
    -a medium which is used to differentiate different types of microorganisms based on their different colours or colony shapes.

    -Differential media or indicator media distinguish one microorganism type from another growing on the same media
  23. Phase Contrast
    • Used for wet mount- not for stained prep
    • Great for spores/capsules 

    Phase contrast microscopy allows the viewing of unstained specimens
  24. Light refraction
    Refractive index 1.2 

    Use Immersion oil on the slide it will
  25. Immersion Oil
    The function of immersion oil is to direct the light from the microscope directly to the slide and stop it from refracting.
  26. Gram Positive
    Thick peptidoglycan layer
  27. Gram Negative
    Thin peptidoglycan layer
  28. What is the purpose of gram staining?
    • Staining gives you visualization of the cell
    • Direct staining and Negative staining
  29. Direct staining
    staining of the organism and cells
  30. Negative staining
    Staining the background
  31. Simple staining
    -stain on all the cells 

    -Simple stains highlight an entire microorganism so that cellular shapes and basic structures are visible. Simple stains commonly used include methylene blue, carbolfuchsin, crystal violet, and safranin.
  32. Chromophore (+)
    • stain that are color 
    • positively charge will only stain
  33. Differential Stain
    It allows you to differentiate the different groups or individual around it
  34. Spore staining (Specific)
    Spore staining is used to identify bacteria that have endospores, or hard outer shells that do not absorb dye. Spores are heated to break the shells, then malachite green dye is applied to dye the spores.
  35. Primary stain
    A primary stain is a technique in which certain types of dyes are used to stain tissue and bacteria so that they become easily visible under the microscope.

    It stains the outer membrane

    crystal violet
  36. Decolorizing agent
    • removes excess primary dye and/or removes the primary dye and outer
    • membrane if it is unable to attach to the cell (is negative)
  37. Counter stain
    attaches to the inner membrane in the event that the primary stain doesn't (is negative)

    safranin
  38. Mordant (means biting, bite)
    It keeps the color of the cell more permanent

    gram iodine
  39. what are the steps of gram staining?
    • 1. smear
    • 2. air dry
    • 3. heat fix
    • 4. crystal violet (primary stain) 60 sec
    • 5. water rinse 
    • 6. gram iodine (mordant)
    • 7. water rinse
    • 8. decolorizer (80% isopropyl alcohol + acetone)
    • 9. water rinse 
    • 10. safranin (counter stain)
    • 11. water rinse 1 min 
    • 12. blot dry
  40. Acid-fast stain
    The acid-fast stain is a differential stain used to identify cells capable of retaining a primary stain when treated with acid alcohol.
  41. What does acid fast positive contain?
    Mycolic acid is a waxy substance which does not allow the cells to be stained by simple stains, but when stained by carbolfuchsin can retain this stain even acid alcohol decolorizer is used.
  42. what are the three methods for acid fast staining?
    • 1.Ziehl-Neelsen (ZN)
    • 2.Kinyoun (K)
  43. Ziehl-Neelsen (ZN)
    which uses heat to drive the carbolfuchsin in the cells
  44. Kinyoun (K)
    which uses a more concentrated, more lipid soluble form of carbolfuchsin.
  45. There are three components in the acid-fast procedure. They are:
    • 1. Carbolfuchsin 
    • 2. Acid alcohol 
    • 3. Methylene blue
  46. Carbolfuchsin
    A primary stain that is a phenolic compound that is lipid soluble.

    Stains cells reddish purple.
  47. Acid-alcohol
    A decolorizer that decolorizes non acid-fast cells.
  48. Methylene Blue
    A secondary stain that stains non acid-fast cells blue
  49. PROCEDURE (Kinyoun Procedure)
    1. Prepare a smear, air dry and heat fix.

    2. Flood slide for 5-10 minutes with carbolfuchsin prepared with Tergitol No. 7 (heat is not required)

    3. Decolorize with acid-alcohol (30 sec) and rinse with tap water. Repeat this step until no more color runs off slide.

    4. Counterstain with alkaline methylene blue for 2 min. Rinse with water and blot dry.

    5. Examine by brightfield microscopy using the 100X oil-immersion objective. Acid-fast organisms stain red; the background and other organisms (non-acid fast organisms) stain blue.
  50. what is the purpose of Endospore staining?
    A differential stain used to detect the presence and location of bacterial endospores.

    Endospore-forming cells initiate differentiation into endospores in response to low nutrient conditions.An endospore is a dormant form of the bacterium that allows it to withstand harsh environmental conditions, even for hundreds or thousands of years.
  51. Malachite green
    is used to stain the spore because it has a low affinity for cellular material. Therefore,vegetative cells and endospore mother cells can be decolorized with water and counter stained with saffranin.
  52. What is the purpose of the Starch Hydrolysis?
    The purpose is to see if the microbe can use starch, a complex carbohydrate made from glucose, as a source of carbon and energy for growth.
  53. What is the enzyme for starch?
    Amylase
  54. What is the medium used in starch hydrolysis?
    Starch Agar
  55. What is the reagent for starch hydrolysis?
    Iodine reagent 

    iodine reagent is added to detect the presence of starch. Iodine reagent complexes with starch to form a blue color in the culture medium.
  56. What was the result for starch hydrolysis?
    • Bacillus subtilis (clear)
    • Escherichia coli (Not clear) 

    If the area is clear starch is hydrolyzed
  57. what is the purpose of casein hydrolysis test?
    Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. Casein is a large protein that is responsible for the white color of milk.

    It is a source of carbon and energy and degrades into amino acids
  58. What is the medium for Casein hydrolysis?
    Milk a1% skim milk
  59. what is the enzyme for casein hydrolysis?
    Casinase
  60. what is the substrate for Casein hydrolysis?
    Casein and protein
  61. What is the reagent for Casein hydrolysis?
    15% TCA Trichloroacetic acid 

    it reacts with starch and make it precipitate and react with protein and peptide to give white color precipitate
  62. what is the results for casein hydrolysis test?
    • Bacillus subtilis: clear
    • Escherichia coli (not clear)

    • if presence 
    • protein such as casein will be opaque
  63. What is the purpose of Gelatin hydrolysis test?
    The purpose is to see if the microbe can use the protein gelatin as a source of carbon and energy for growth
  64. what is the enzyme for gelatin hydrolysis test?
    Gelatinase
  65. What is the medium for Gelatin Hydrolysis test?
    1.5% Gelatin
  66. What is the substance for Gelatin hydrolysis test?
    Gelatin and protein
  67. what is the product of Gelatin Hydrolysis test?
    Amino acid
  68. what is the reagent for Gelatin hydrolysis?
    15% TCA 

    sit for 15 minutes
  69. What is the results for Gelatin Hydrolysis?
    • Bacillus subtilis clear
    • Escherichia coli not clear
  70. what is the purpose of lipid hydrolysis?
    Lipase allows the organisms that produce it to break down lipids into smaller fragments. Triglycerides are composed of glycerol and three fatty acids.
  71. What is the enzyme for lipid hydrolysis?
    Lipases
  72. What is the medium for Lipid hydrolysis?
    pH indicator SPIRIT BLUE agar plate
  73. How is the result for Lipid Hydrolysis?
    • Escherichia coli clear (light blue)no lipid hydrolyzed 
    • Bacillus subtillis Dark blue (hydrolyzed)
  74. What is the purpose of Hydrogen sulfide production?
    This test determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism.
  75. What is the medium for H2S?
    SIM (Sulfide indole motility)
  76. What is the enzyme for H2S?
    Cysteine Desulfurase
  77. What is the substrate for H2S?
    Cysteine
  78. What is the purpose of Catalase test?
    The purpose is to see if the microbe has catalase, a protective enzyme capable of destroying the dangerous chemical hydrogen peroxide
  79. what is the substrate for catalase test?
    H2O2
  80. What is the reagent for Catalase test?
    3% H2O2
  81. what is the product for Catalase test?
    Water and Oxygen
  82. what is the results for catalase test?
    • Staphylococcus epidermidis (oxygen bubble)
    • Enterocuccus faecalis (neg)

    No bubble there is no catalse it means no oxygen making water
  83. what is the purpose of Oxidase test?

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