Microlab Final

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  1. BSL 1 & 2
    BSL 1 involves no known agent to consistently cause disease in healthy individuals. 

    BSL 2 involves agents associated with human disease, hazard = percutaneous injury, ingestion, mucous membrane exposure
  2. BSL 3 & BSL 5
    BSL 3 involves indigenous or exotic agents with potential for aerosol transmission, disease may have serious or lethal consequences

    BSL 4 involves dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections, or related agents of unknown risk
  3. what type of microscope do we use
    • bright-field microscope
    • produced image made from light transmitted through specimen
  4. Pros and cons to staining
    • contrast btwn specimen and background are improved
    • However, it usually kills cells
  5. condenser lens
    • lens below stage
    • concentrates light on specimen
  6. objective lens
    • the "tits" on the cow, or microscope
    • above stage
    • as light passes through, it is refracted from specimen to provide a real image
  7. refraction
    means "bending" of light as it passes through one medium to another
  8. ocular lens
    lenses you put your eye up to
  9. how to calculate magnification
    mag. of objective lens x mag of ocular lens
  10. limit of resolution
    • also called resolving power
    • the minimum distance between two objects such that the two objects still appear separate and distinct.
    • resolution improves as limit of resolution is made smaller
  11. how to calculate limit of resolution

    • where D is minimum distance 2 points can be resolved
    • λ is wavelength of light used, NAcondenser and NAobjective are the numerical apertures of the condenser lens and objective lens
  12. Numerical aperture
    ability of a lens to capture light from a specimen and make an image
  13. Bright field microscopy
    • Most common type of light microscope, type we use in lab
    • Illuminates field of view evenly
    • Use visible light for observing objects
    • Visible light passes through specimen and through series of lenses to magnify
    • Can magnify up to 1000x, making it relatively easy to observe cell size, shape &  motility
  14. Darkfield microscopy
    • special condenser is used so only light reflected off specimen enters objective
    • Appearance is of brightly lit specimen against dark background, often better resolution than bright-field
    • Cells stand out bright objects against dark background
    • Light is directed toward specimen at an angle
    • makes unstained cells easier to see
  15. Phase-contrast microscopy
    • Markes cells and other dense material appear darker
    • Increases contrast by amplifying differences in refractive index (parts of specimen appear as various levels of darks and lights)
    • Makes unstained cells easier to see
  16. Simple stain
    Basic Stain/Dye
    • Uses only one dye which has a positive charge that's attracted to negatively charged surface of most bacterial cells
    • Thus cell becomes colored
    • Common basic stains:
    • Methylene blue
    • crystal violet
    • Safranin
    • Malachite Green
  17. Acidic Dye
    • Negative stain
    • Negative charge in dye is repelled from negative charge on bacterial surface, so cell remains unstained against colored background

    Uses Eosin, Congo Red, or Nigrosine
  18. Gram stain
    • a differential stain
    • Can use any medium

  19. Acid-fast stain
    Used to detect organisms that don't easily take up stains, such as members of genus Mycobacterium which cause TB and Hansen's disease (leprosy)

    Primary stain is red, counterstain is Methylene blue
  20. Capsule stain
    • Test for bacteria with capsules,Any medium can be used
    • *Recall capsules repel most stains. Therefore, capsule stain takes advantage of staining around the cells
    • An acidic stain (such as Congo red or nigrosin) stains background red or black and a basic stain like Crystal violet that colorizes the cell Violet
    • The capsule remains unstained and appears as a white halo btwn cells & colored background
  21. Endospore Stain
    • Is a differential stain - only a few genera produce spores.
    • Among them are Bacillus & Clostridium

    • Dyes used are Malachite Green & Safrainin
    • Endospores will be green
  22. Flagella Stain
    • Usually, flagella are too thin to be seen w light 
    • This stain uses a substance that allows staining agent to adhere to and coat thin flagella, making them visible using light microscopy
  23. Motility test
    • Can use SIM tube
    • A tetrazolium salt (TTC) may be included in the medium to make interpretation easier
    • TTC is colorless and soluble in oxidized form, when bacteria reduce it, it turns red and insoluble
    • A positive result for motility is indicated when red TTC is found throughout medium
    • Neg shows red only along stab line
  24. Hydrogen Sulfide Production test (sulfur reduction)
    • Tests for sulfur fermenting anaerobes such as Proteus vulgaris
    • Can use SIM tube or KIA tube; as iron salts are in medium

    • Positive results: black area (H2S) around stab and/or black parcipitate
    • Neg results: no black color
  25. How can you test for O2 requirements
    • Testing for aerobes vs anaerobes
    • Use Thioglycolate Tube

    • Aerobes grow only at surface
    • Facultative anaerobes grow throughout tube
    • Anaerobes grow below oxidized layer
  26. How does UV light kill microorganisms
    • Destroys DNA
    • Prolonged exposure can be lethal to cells because when DNA absorbs UV radiation, energy is used to form new covalent bonds btwn adjacent pryimides: C-C, C-T, or T-T
    • Distorts DNA molecule and interferes with DNA replication
  27. disinfectants
    • are antimicrobial chemicals used for disinfecting inanimate objects 
    • toxic to many life forms and therefore are biocides (cida means to kill)
    • Often called germicides
    • Target microorganisms and viruses
  28. Antiseptics
    • antimicrobial chemicals non-toxic enough to use on skin or other body tissue
    • routinely used to decrease bacterial numbers on skin before invasive procedures such as surgery
  29. Antibiotics vs antimicrobials
    • Antibiotics are natural antimicrobial agents produced by microorganisms 
    • Ex: penicillin is produced by Penicillium notatum

    Today, because many agents that are used to treat bacterial infections are synthetic, the term antimicrobial is used to describe all substances used for this purpose
  30. Differential agar?
    • TSA
    • BAP (Blood Agar)
    • EMB (Eosin Methylene blue)
    • MAC
    • Starch Hydrolysis

    Today Everyone Must Be Sweet
  31. Selective agar
    • Only allows growth of gram neg
    • EMB (Eosin Methylene blue)
    • MAC 
    • BEA (Bile Esculin agar)
    • MSA (MSA)

    Everyone Makes MBarf
  32. What is a test used to determine the resistance/susceptibility of an organism to antibiotics?
    What medium is used?
    • Kirby-Bauer
    • Uses Muller-Hilton agar plate
    • To determine resistance/susceptibility, measure the zone of inhibition in mm and then look up on the chart for zone diameter standards. Will tell you whether it's resistant, intermediate, or sensitive by zone diameter
  33. Describe starch hydrolysis test
    • Test for Bacillus   which is starch hydrolysis positive
    • Uses Starch Agar Plate
    • Testing for enzyme call amylase
    • Incubate for 1 day, flood with iodine
    • Positive result shows halo around bacteria
  34. Describe Carbohydrate fermentation test
    • Uses Glucose broth, Lactose Broth, and/or Sucrose Broth with phenol red indicator
    • Looks to see if organism ferments glucose, lactose, or sucrose
    • Have durham tube to collect gas, have phenol red indicator in broth
    • If they do, broth will turn yellow (meaning they produce acid) & may have bubble if gas produced
    • If red & no gas, neg result

    No reagent added
  35. Urease test
    • testing for enzyme urease
    • Pos bacteria: Proteus

    • Uses urea broth, has phenol red in medium
    • Grow for 24 hrs
    • If positive, will change red or pink 
    • If neg, no color change

    No reagant
  36. Catalase test
    • If positive, Makes Bubbles!! :-)  "Cat's make Cami Happy"
    • Tests organisms that produce the enzyme catalase
    • Grow, and then put hydrogen peroxide on it
    • Differentiates btwn Strepto. can also id Mycobacterium
  37. Oxidase test
    • Id's presence of oxidase, uses square test slide
    • If positive, will turn blue/purple, must read results immediately as false pos can take place after 30 sec
    • Pos bacteria: Neisseria, Pseudomonas, Enterobacteria

    *Remember x in Oxidase, for 4 cornered slide for test
  38. Nitrate reduction test
    • Tests for enterics
    • Uses nitrate broth
    • Uses nitrate test reagents A&B
    • After inoculating overnight:
    • 1. observe for bubbles. If bubbles, don't need to add reagents. Is positive result. Took it past just reducing to nitrate = denitrification
    • 2. If no bubbles, add 5 drops each of reagent A&B
  39. Nitrate reduction test: after you add reagents A&B
    If the tube turns pink/red, it's positive. If nothing happens, you have to add zinc dust

    Zinc is added to see if nitrate is present, which will turn medium red. This will be a NEGATIVE result, as nitrate was not reduced

    If Zinc is added and there is no color change, it's a positive result. Indicates organism reduced the nitrate to some other nongaseous nitrogenous compound
  40. MSA
    • Mannitol salt agar
    • Used to differentiate Staph (which ferments mannitol) 
    • Is highly selective, as most bacteria can't tolerate high level of salt
    • If yellow halos occur around colonies, pos result
  41. Coagulase test
    Tests for Staph aureus

    • Coagulation in test tube (thickening) is positive result
    • Enzyme coagulase increases bacterial resistance to phagocytosis and antibodies by surrounding infecting organisms with a clot
  42. Blood Agar
    • Differential, Non-selective
    • tests for Streptococcus
    • Beta hemolytic - complete lysis of blood, becomes clear
    • Alpha hemolytic - some lysis, not clear, but olive green in color
    • Gamma hemolytic - doesn't do anything, NO HEMOLYSIS
  43. Bacitracin susceptibility test
    • Peptide antibiotic that inhibits cell wall synthesis (β-lactam)
    • Inhibits Streptococcus pyogenes

    Organism grown on blood agar plate w bacitracin, looking for zone of inhibition
  44. Bile Esculin test
    • Lite, "bile" gross color agar
    • Used to identify Group D strep (Strep faecalis)
    • Uses Bile Esculin plate
    • Pos result is black color around growth
    • Neg result is no black color or growth
  45. SIM tube
    • S-sulfide
    • I - indole
    • M - motility
  46. EMB
    • Eosin Methylene Blue agar
    • Selective and differential - used to Id enterics
    • Dark purplish color or agar
    • If lactose fermentor, will grow colored
    • colonies
    • If it is a non-lactose fermenter, no color
  47. McConkey agar
    • Selective and differential - used to Id enterics
    • Lite pink in color
    • Lactose fermentation turns it red, whereas non-lactose  remain normal color
    • Used to ID Enterics
  48. KIA
    • Kligler Iron Agar
    • Tests for enterics: all are rods
    • Uses KIA slant tubes
    • Used to differentiate on basis of glucose fermentation, lactose fermentation, and sulfur reduction

    • Yellow = Acid
    • Red = Base (Alkaline) 
    • If there is black, is + for H2S

    Lactose fermentation is yellow
  49. IMViC
    • Indole
    • Methyl red
    • Voges-Proskauer
    • Citrate
    • Tests are used to differentiate enterics
  50. IMViC: Indole test
    • Uses Sim medium
    • After 24 hours, add 5 drops of Kovacs reagent
    • Pos is red at surface
    • Neg is no change
  51. IMViC: Methyl red
    • Tests for mixed acids (ENTERICS)
    • Uses MR-VP tube
    • After 24 hours, add 5 drops of Methyl red
    • Pos result, medium turns red or pink
    • Neg result, medium has no change or goes orange, then fades to brown/yellow
  52. IMViC: VP
    • Voges-Proskauer test (ENTERICS)
    • Detects acetoin production
    • After 24 hrs, add 5 drops of VP-A and 5 drops of VP-B
    • Wait 10-15 mins
    • Pos result - med turns read near surface
    • Neg result - no change
  53. IMViC: Citrate agar slant
    • Uses Citrate agar slant, is green color
    • detects utilization of citrate
    • No reagents
    • Positive test, medium turns blue
    • Neg test, no change
  54. EnteroPluri Tube
    only works on enterics!
Card Set
Microlab Final
micro lab
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