MICROBIOLOGY-1.txt

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lalalesley
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MICROBIOLOGY-1.txt
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MICROBIOLOGY LAB PRACTICAL ONE SCHRIEBER
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LAB GUIDE FOR EXAM
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  1. Aseptic Technique: Work Area Disinfection
    Work area is treated with a disinfectant that will kill any microorganisms that may be present, but not endospores
  2. Aseptic Technique: Loops and Needles
    A loop or needle is sterilized by inserting it into a Bunsen burner flame until it is red hot, ensuring that all contaminating organisms will be incinerated. Allow the loop/needle to cool completely before picking up any inoculum, this will ensure that all viable cells are transferred.
  3. Aseptic Technique: Culture Tube Flaming and Inoculation
    The cap is removed, and the mouth of the tube is passed through the flame. If the tube is a broth tube, the loop is inserted into the tube and twisted several times to ensure the organisms on the loop are deliverd to the liquid. If the tube is an agar slant, the surface of the slant is inoculated by drawing the loop up the surface of the slant from the bottom of the slant to the top. For stab cultures, a needle is inserted into the agar medium. After the culture is inoculated the mouth if the tube is reflamed, and the tube is recapped.
  4. Aseptic Technique: Final Flame of the Looop or Needle
    After completing the innoculation the needle/loop is flamed in the bunsen burner, to destroy any organsims. Iteams should be returned to their appropriate place, never laid on the desk.
  5. Aseptic Technique: Petri Plate Inocultaions
    Loops are used to inocutlate or streak petri plates. The plate cover is raised andheld diagonally over the plate to protect the surface from any contaminationin the air. The loop containing the inoculom is then streaked gently over the surface of the agar
  6. EX. 6 Bacterial Ubiquity
    Basic Morphology and Arrangements of Bacterial Cells
  7. EX. 6 Bacterial Ubiquity
    Pouring Agar Plates
  8. EX. 6 Bacterial Ubiquity
    Physical Properties of Agar
    Liquid(broth), Semi-solid(slant), Solid (plate)
  9. EX. 6 Bacterial Ubiquity
    Method of Labeling Plates
    My Initials, Table Number, Instructors Name, Name of Organism, Media
  10. EX. 6 Bacterial Ubiquity
    Incubation of Plates
    48 Hours of Incubation at 37 degrees Celsius
  11. EX. 6 Bacterial Ubiquity
    Bacterial and Mold Growth Characteristics on Agar
    0= no growth, + is 1 to 10 colonies, ++ is 11 to 50 colonies, ++++ is over 100 colonies. Bacteria growth appears as small dots, each dot representing a colony. If the dot posses any fur, or fuzziness, it is mold.
  12. EX. 6 Bacterial Ubiquity
    Environmental and Body Sampling Techniques
    With a swab, moisten it in distilled waterand rub the swab over a part of your body. To expose the agar plate, couch into it, or rub the swab over the entire surface of the plate.
  13. EX. 8 Aseptic Technique
    Transfer of Organisms From Broth to Another Broth
    • REMOVING ORGANISMS
    • 1.) Inoculating loop is heated untill red hot.
    • 2.) Organisms in broth culture are dispersed by shaking tube.
    • 3.) tube enclosure is removed and mouth of tube is flamed.
    • 4.) A loopful of organisms is removed from tube.
    • 5.) Loop is removed from culture and tube mouth is flamed
    • 6.) Tube is enclosure is returned to tube.
    • INOCULATING NUTRIENT BROTH
    • 7.) Cap is removed from steril broth tube mouth is flamed.
    • 8.) Unheated loop is inserted into tube of steril broth.
    • 9.) Loop is removed from broth and tube mouth is flamed
    • 10.) Tube enclosure is returned to tube.
    • 11.) Loop is flamed and retuurned to receptical.
  14. EX. 8 Aseptic Technique
    Transferring of Organisms From Slant to Another Slant
    • 1.) Inoculating loop is heated untill it is red hot.
    • 2.) Cap is removed from slant culture and tube mouth is heated
    • 3.) Organism is picked up from slant with inoculatin loop.
    • 4.) Mouth of tube is flamed. Inoculating loop is not flamed.
    • 5.) Slant culture is recapped and returned to test-tube rack.
    • 6.) Tube of steril agar slant is uncapped and mouth is flamed
    • 7,) Slant surface is streaked with unflamed loop in serpentine manner
    • 8.) tube mouth is flamed, recapped and incubated.
    • 9.) Loop is flamed red hot and replaced in reciptical
  15. EX. 8 Aseptic Technique
    Transferring Organisms From Petri Plate to Slant
    • 1.) Inocluating loop is heated until red hot.
    • 2.) With free hand, raise the lid of the petri plate just enough to access a colony to pick upa loopful of organisms.
    • 3.) After flaming the mouth of a sterili slant, streak its surface, usually in a fishtail method.
    • 4.) Flame the mouth of the tube and recap it.
    • 5.) Flame the inoculating loop and return it to the receptical
  16. EX. 8 Aseptic Technique
    Inoculation Techniques For Agar Slants and Broth
    • 1.) Inoculating loop is heated untill it is red hot.
    • 2.) Cap is removed from slant culture and tube mouth is heated
    • 3.) Organism is picked up from slant with inoculatin loop.
    • 4.) Cap is removed from steril broth tube mouth is flamed.
    • 5.) Unheated loop is inserted into tube of steril broth.
    • 6.) Loop is removed from broth and tube mouth is flamed
    • 7.) Tube enclosure is returned to tube.
    • 8.) Loop is flamed and retuurned to receptical.
  17. EX. 10 Smear Preparation
    Degreasing Slides
    Wash the slide with soap or Bon Ami and hot water. Handle the slide by the edges
  18. EX. 10 Smear Preparation Steps in Smear Prep.
    Aseptic Technique In Removal Of Organism
    • 1.) Shake the culture tube to suspend the organism
    • 2.) Heat the loop
    • 3.) Flame the neck of the tube containing the organism
    • 4.) After allowing the loop to cool for at least 5 secs, remove a loopful of organism.
    • 5.) Flame the mouth if the tube again before replacing the cap
  19. EX. 10 Smear PreparationSteps in Smear Prep.
    From Solid Media
    Corynebacterium diphtheriae
    • 1.) Two loopfuls of water are placed on the target circle of the slide
    • 2.) A very small amount of the organism is dispersed in the water over the entire target circle area using the inoculating loop.
    • 3.) The smear is allowed to air dry at room temperature
    • 4.) Slide is passed throug the flame several times to heat-kill and fix the organisms to slide.
  20. EX. 10 Smear Preparation
    Steps in Smear Prep.
    From Liquid Media
    Escherichia coli
    • 1.) Two loopfuls of liquid containing organisms are placed in the center of the "target circle"
    • 2.) Organisms are over entire area of the target circle
    • 3.) The smear is allowed to dry at room temperature
    • 4.) Slide is passed through flame several times to heat kill and fix organisms to slide.
  21. EX. 11 Simple Staining
    Negatively Charged Bacterial Cells
    The fact that bacteria are negatively charged produces a pronounced attraction between these cationic chromophores and the organism. These dyes are basic dyes
  22. EX. 11 Simple Staining
    Basic Dyes
    The basic dye used in this technique is Methylene blue (methylene+ chloride-) Some other common basic dyes are Basic Fuchsin, Crystal Violet. These dyes posses chromophores, which are color bearing ions that are positively charged
  23. EX. 11 Simple Staining
    Acidic Dyes
    Those dyes that have anionic chromophores are called acidic dyes. Eosin (sodium+ eosinate+) is one. The dye will not stain bacteria because of the electrostatic repelling forces that are involved. Instead it is the background that is dyed rather than the organism
  24. EX. 11 Simple Staining
    Corynebacterium diphtheriae


    • Small, nonmotile, irregularly staining pleomorphic
    • Gram-positive rods with club-shaped swelled ends but no spores; may be straight
    • or slightly curved.
    • Cells tend to lie parallel to one another (palisades) or at acute angles (coryneforms),
    • due to their snapping type of division
    • Vary greatly in dimension, from 0.3 to 1 um in diameter and 1.0 to 8.0 um in length
  25. EX. 11 Simple Staining
    Procedure
    • 1.) A bacterial smear is stained with methylene blue for one minute.
    • 2.) Stain is briefly washed off slide wih water
    • 3.) Water drops are carefully blotted off slide with bibulous paper
  26. EX. 11 Simple Staining
    Escherichia coli
    • average size, is 1.1 to 1.5 um wide by 2.0 to 6.0 um long.
    • Morphology is rod shaped
    • Arrangement is peritrichous
  27. EX. 11 Simple Staining
    Dyes Used
    The dye we use in this exercise is Methylene blue. We could also use Crystal violet or basic fuchsin
  28. EX. 11 Simple Staining
    Simple Staining vs. Differential Staining
    • Simple Stain:
    • A simple stain consists of a solution of a single dye. Some of the most commonly used dyes are methylene blue, basic fuchsin, and
    • crystal violet. Simple stains allow one to distinguish the shape (morphology) of the bacteria. For example, E. coli and Bacillus Subtillus are bacilli or rod-shaped bacteria. Many bacilli occur singularly, but chains may
    • also be observed. Bacilli very greatly in length and diameter. Staphylococcus aureus and Streptococcus pneumoniae are cocci or spherical bacteria. Cocci may occur singularly, in pairs (as in Streptococcus pneumoniae), or in clusters (as in Staphylococcus aureus). R. rubrum is a spirillum or curved bacterium, Spirilla always occur singularly.
    • Differential Stains:
    • Differential stains are more complex than simple ones and use more
    • than one stain to differentiate cellular components. They are used to
    • examine structural differences between bacterial groups or to provide
    • contrast to different structures within the same organism.
  29. EX. 11 Simple Staining
    Palisade Arrangement
    This pertains to a parallel arrangement of rod-shaped cells. This characteristic also called picket fence arrangement is common to many corynebacteria
  30. EX. 11 Simple Staining
    Metachromatic Granules
    These are distinct reddish purple granules within cells that show up when the organisms are stained with methlene blue.They are masses of volutin, a polymetaphosphate
  31. EX. 11 Simple Staining
    Pleomorphism
    This pertains to irregularity of form, that is demonstarting several different shapes.
  32. EX. 12 Negative Staining
    Smear Procedure/ Dye Used
    • Examples of dyes used are nigrosin and india ink. We use nigrosin.
    • 1.) Organisms are dispersed into a small drop of nigrosin. Drop shoud not exceed 1/8 diameter an shoud be near the end of the slide.
    • 2.) Spreader slide is moved toward drop of suspension untill it contacts the drop causing the liquid o spread along its spreading edge,
    • 3.) Once sreader slide contacts the drop on botton slide the suspension will spread out along the spreading edge.
    • 4.) Sprader slide is pushed to the left dragging the suspension over the bottom slide. After the slide has air-dryed, it may be examined under oil immersion
    • SECOUND PROCEDURE USED
    • 1,) A loopful of nigrosin is placed in the center of a clean microscope slide
    • 2.) A steri inoculating wire is used to transfer the organism to the liquid and mix the organisms into the stain
    • 3.) Suspensions of bacteria is spread evenly over an area of one or two centimeters with the straight wire.
    • 4.) Once the preparation has completely air-dryed it can be examined under oil immersion
  33. EX. 12 Negative Staining
    Staphylococcus aureus
    • Morphology: Coccus
    • An average coccus is about 0.5-1.0 micrometer (µm) in diameter
    • Arranged in grape like irregular clusters
  34. EX. 12 Negative Staining
    Bacillus megaterium
    • Morphology: Rod shaped, single
    • Arrangement: Bacillus, one rod
    • Size:An average bacillus is 0.5-1.0 µm wide by 1.0-4.0 µm long.
  35. EX. 12 Negative Staining
    Pros and Cons of Negative Staining
    Negative stains can be useful in studying the morphology of bacterial cells and characterizing some of the external structures, and it is also useful in determing cell dimensions and also in observing spirochaetes. The disadvantage is that little or nothing is learned about the internal structure of the cell
  36. EX. 14 Gram Staining
    Reagent Used
    • Crystal Violet: Stains gram positive bacteria purple
    • Iodine: A mordant that complexes with the Crystal Violet and forms an insoluble complex in gram positive cells.
    • Safranin: Counter stain used to dye gram negative bacteria after slide has been treated with acetone
  37. EX. 14 Gram Staining
    Steps in Procedure
    • 1.) Cover the smear with Crystal Violet and let stand for 20 secs- 1 minute
    • 2.) Briefly wash off the stain using distilled water, drain off excess water
    • 3.) Cover the smear with Gram's Iodine solution and let stand for1 minute
    • 4.) Wash off the grams iodine hold the slide a a 45-degree angle and allow 95% alcohl to flow down the surface of the slide.
    • 5.) Stop decolorization by washing the slide with a gentle stream of water
    • 6.) Cover the smear with safranin for 1 minute.
    • 7.) Wash gently, blot dry,and air dry
    • 8.) Examine slide under immersion oil
  38. EX. 14 Gram Staining
    Escherichia coli
    • Gram Negative
  39. EX. 14 Gram Staining
    Staphylococcus aureus
    • Gram Positive
  40. EX. 14 Gram Staining
    Mycobacterium smegmatis
    • Gram negative
    • Morphology: Bacillus, rod
    • Arrangement: clusters
    • Size:3.0 to 5.0 µm long
  41. EX. 14 Gram Staining
    Bacillus megaterium
    • Gram Positive
    • Morphology: Bacillus, rod
    • Arrangement:Single
    • Size: An average bacillus is 0.5-1.0 um wide by 1.0-4.0 um long
  42. EX. 14 Gram Staining
    Clinical Relevance
    It is a critcal procedure in identifying an unknown bacteria
  43. EX. 14 Gram Staining
    Diagnostic Utility
    It helps in the identification of bacteria, which makes targeting a pathogen much easier
  44. EX. 15 Spore Staining
    Componants of Endospore
    Endospores are very dehydrated structures that are not actively metabolizing. They are resistant to heat, radiation, acids, chemicals due to a protein coat they have called an exosporium.
  45. EX. 15 Spore Staining
    Procedure Schaeffer- Fulton
    • 1.) Cover smear with a small piece of paper toweling and saturate with Malachite green. Steam over boiled water for 10 minuets. Add more stain if stain boils off
    • 2.) After slide has cooled sufficiently, remove the paper toweling and rinse with water for 30 ec.
    • 3.) Counterstain with Safranin for about 1 minute.
    • 4.) Rinse briefly with water to remove safranin
    • 5.) Blot dry with bibulous paper examine slide under immersion oil
  46. EX. 15 Spore Staining
    Dorner Method
    • 1.)Make a heavy suspension of bacteria by dispersing several loopfuls of bacteria in 5 drops of steril water
    • 2.) Add 5 drops of carbolfuchsin to the bacterial suspension
    • 3.)Heat the carbolfuchsin suspension of bacteria in a beaker of boiling water for 10 minutes
    • 4.) Mix several loopfuls of bacteria in drop of nigrosin on the slide.
    • 5.) Spread the nigrosin bacteria mixture on the slide.
    • 6.) Allow the smear to air dry. Examine under oil immersion
  47. EX. 15 Spore Staining
    Bacillus megaterium
  48. EX. 15 Spore Staining
    Spore Forming Bacteria
    Genera Bacillus and Clostridia
  49. EX. 16 Acid Fast Staining Zeihl-Nielson
    Cell Wall Characteristics
    Bacteria in the genus Mycobacterium and some in the genus Nocardia contain a waxy material n their cell walls called mycolic acid
  50. EX. 16 Acid Fast Staining Zeihl-Nielson
    Reagents Used
    Carbolfuchsin. Counterstain is Methylene blue
  51. EX. 16 Acid Fast Staining Zeihl-Nielson
    Procedure
    • 1.) Cover smear with carbolfuchsin Steam over boiling water for 10 minutes Add additional stain if it boils over
    • 2.) After slide has cooled, decolorize wiyth acid-alcohol for 15-20 seconds
    • 3.) Stop decolorization action of acid-alcohol by briefly rinsing with water
    • 4.) Counterstain with Methylene blue for 1 minute
    • 5.) Rinse breifly with water to remove excess methylene blue
    • 6.) Blot dry with bibulous paper Examine dirctly under oil immersion
  52. EX. 16 Acid Fast Staining. Zeihl-Nielson
    Microbes Screened With This Method
    Mycobacterium tuberculosis and Mycobacterium leprae
  53. EX. 16 Acid Fast Staining/ Zeihl-Nielson
    Staphylococcus aureus, Mycobacterium smegmatis
    • Staph is blue because it is negative
    • Myco is pink/purple because it is positive
    • Mycobacterium smegmatis is a rod/ bacillus shape
    • Staphylococcus aureus is coccus
  54. EX. 9 Pure Culture
    Quadrant Streaking
  55. EX. 9 Pure Culture
    Subculturing Techniques
    Quadrant Streaking,
  56. EX. 9 Pure Culture
    Eschericha coli
  57. EX. 9 Pure Culture
    Staphylococcus aureus
  58. EX. 9 Pure Culture
    Serratia marcescens
  59. EX 17 Motility
    Staphylococcus aureus
    • NON MOTILE
  60. EX 17 Motility
    Proteus mirabilis
    Motile, posses flagella. Agar is Motility Medium Triphenyl Tetrazolium Chloride, a semi solid .4% agar with indicator dye
  61. EX 17 Motility
    Semi- solid medium- Inoculation and reading result
    Motility Medium Triphenyl Tetrazolium Chloride
  62. EX 17 Motility
    Motile Microbes
    Proteus mirabilis
  63. EX 17 Motility
    Non-Motile Microbes
    Staphylococcus aureus
  64. EX. 19 Anaerobic Culture
    FTM
    Fluid Thioglycollate MediaResazurin reacts with oxygen and turns pink. Boil the media with sodium thioglycallate removes oxygen
  65. EX. 19 Anaerobic Culture
    TGB
  66. EX. 19 Anaerobic Culture
    Use of Mediums
  67. EX. 19 Anaerobic Culture
    Growth Patterns of Aerotolerance
    This group can grow in the presence of oxygen and are not usually harmed by its presence. Their metabolism does not require oxygen
  68. EX. 19 Anaerobic Culture
    Escherichia coli
    Facultative anaerobe
  69. EX. 19 Anaerobic Culture
    Pseudomonas aeruginosa
    Obligate Aerobe
  70. EX. 19 Anaerobic Culture
    Clostridium sporogenes
    Obligate Anaerobe
  71. EX. 20 Enumeration of Bacteria
    Dilutions: Calculations and Dilution Factors
  72. EX. 20 Enumeration of Bacteria
    Plates for Appropriate Counting
  73. EX. 20 Enumeration of Bacteria
    Cell Density of Original Culture- Formula
  74. EX. 20 Enumeration of Bacteria
    CFU
    Meaning and Concept
    Colony Formng Units
  75. EX. 20 Enumeration of Bacteria
    Spec 20
  76. EX. 20 Enumeration of Bacteria
    Estimate Cell Density Using Standard Curve
  77. EX. 20 Enumeration of Bacteria
    Bacteria Used




    Escherichia coli
  78. EX. 29 Osmotic Pressure
    Obligate Halophile
    An organism that requires high amounts of salt concentrations
  79. EX. 29 Osmotic Pressure
    Facultative Halophile
    An organism that does not require high salt environments, but can live in them
  80. EX. 29 Osmotic Pressure
    Osmophile
    Organisms that can grow in high amounts of sugar
  81. EX. 29 Osmotic Pressure
    M.S.A
    Mannitol Salt Agar
  82. EX. 29 Osmotic Pressure
    Microbes For M.S.A
    • Escherichia coli
    • Staphylococcus aureus
    • Halobacterium salinarium
    • Bacillus megaterium
  83. EX. 34 Disinfectants and Antiseptics
    Disinfectant vs. Antiseptic
  84. EX. 34 Disinfectants and Antiseptics
    Bacteriostatic vs. Bactericidal
  85. EX. 34 Disinfectants and Antiseptics
    Zone of Inhibition
  86. EX. 27 Lethal Effects of Temperature
    Tempeature Used
  87. EX. 27 Lethal Effects of Temperature
    Thermal Death Point
  88. EX. 27 Lethal Effects of Temperature
    Thermal Tolerance
  89. EX. 27 Lethal Effects of Temperature
    Bacillus megaterium
  90. EX. 30 UV Experiment
    Lethal UV Range For Bacteria
  91. EX. 30 UV Experiment
    Why Are Lids Removed Before Exposure?
  92. EX. 30 UV Experiment
    Why Are Exposure Times Different Between Two Lab Microbed Used?
  93. EX. 30 UV Experiment
    Bacterial Lawn
  94. EX. 30 UV Experiment
    Mutagen
  95. EX. 30 UV Experiment
    Thymine Dimers
  96. EX. 30 UV Experiment
    Lab Results
  97. EX. 30 UV Experiment
    Organisms Used
  98. EX. 30 UV Experiment
    Agar Used
  99. EX. 33 Antimicrobial Agents-Kirby-Bauer Method
    Bacterial Lawn
  100. EX. 33 Antimicrobial Agents-Kirby-Bauer Method
    Zone of Inhibition and Interpritating Results
  101. EX. 33 Antimicrobial Agents-Kirby-Bauer Method
    MIC
  102. EX. 1. Microscopy: Brightfield
    Focusing Technique
  103. EX. 1. Microscopy: Brightfield
    Calculation of Total Magnification
  104. EX. 1. Microscopy: Brightfield
    Oil Immersion
  105. EX. 1. Microscopy: Brightfield
    Diopter Adjustment
  106. EX. 1. Microscopy: Brightfield
    Interpupillary Distance Adjustment
  107. EX. 2. Microscopy: Darkfield
    Condenser Settings
  108. EX. 2. Microscopy: Darkfield
    Advantages and Disadvantages
  109. EX. 2. Microscopy: Darkfield
    Microbe Diagnosed With This Method
  110. EX. 3. Microscopy: Phase Contrast
    Condenser Settings
  111. EX. 3. Microscopy: Phase Contrast
    Advantages and Disadvantages
  112. EX. 3. Microscopy: Phase Contrast
    Microbes Typically Studied Using This Method
  113. EX. 4. Microscopy: Measurements
    Technique
  114. EX. 4. Microscopy: Measurements
    Calculations
  115. EX. 4. Microscopy: Measurements
    Constants For Oil Immersion

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