MicroBio LabMidterm

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MicroBio LabMidterm
2015-04-14 16:05:29
MicroBio LabMidterm

MicroBio LabMidterm
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  1. How many lenses does a simple microscope have?
    One lens
  2. How many lenses does a compound microscope have? Explain the piece(s).
    • Two lens
    • 1. Ocular-eyepiece lens (usually 10x)
    • 2. Objective-nosepiece lenses (commonly 4x, 10x, 45x, 100x)
  3. At what objective magnification do you use oil immersion for a compound microscope?
  4. Magnification (total) = ?
    Mag(objective) x Mag(ocular)
  5. Explain the following piece of a microscope: Framework
    It is the arm, base, and structural parts of the microscope which support the basic frame
  6. Explain the following piece of a microscope: Stage
    Holds the slide. The mechanical stage clamps the slide and moves the slide around the stage
  7. Explain the following piece of a microscope:
    Lens system - Oculars
    Eyepiece lenses (usually 10x magnification)
  8. Explain the following piece of a microscope:
    Lens system - Objectives
    Lenses attached to rotatable nosepiece (common magnifications are 4x, 10x, 45x, 100x).
  9. _________ microscope focusing adjustments do not need to be made when changing objective lenses
    Parfocalized microscope focusing adjustments do not need to be made when changing objective lenses
  10. Detail why oil immersion is used to see higher magnification.
    Oil immersion lens uses oil with approximately the same refractive index as glass to prevent light loss due to diffraction (the bending of light rays) which would occur if light traveled from one refractive index to another (ex. glass to air)
  11. As magnification of the objective lens increase, ________ decreases.
    As magnification of the objective lens increase, Working Distance (distance between the object on the slide and the objective lens, when in focus) decreases.
  12. Explain the following piece of a microscope:
    Directs light towards the objective lens in bright field microscopy (in dark field microscopy, the condenser directs light at oblique angles away from the objective lens in a manner that allows only objects in the field of view to redirect or scatter light into the objective lens. This causes objects to appear white on a dark field)
  13. What are the effects that occur when you close down the condenser?
    • The light intensity decreases
    • Contrast improves
    • Depth of field increases
    • Limit Resolution (with oil immersion lens)
  14. Explain the following piece of a microscope:
    Iris Diaphragm
    Lever located in the condenser that adjusts the diameter of the cone of light so that it just fills the objective lens
  15. Give the two possible definition for resolution (d=?)
    • d = the smallest distance between two objects which can be seen as separated
    • d = the diameter of the smallest resolvable object
  16. What is the equation for resolution (d)?
    d = λ / 2NA
  17. How can you improve resolution (d)?
    d can be decreased by lowering λ or increasing the NA (numerical aperture)
  18. What three principle forms of microbiology does Light Microscopy reveal?
    • Cocci (more or less spherical)
    • Bacilli (rod shaped)
    • Helicoidal (spiral shaped)
  19. What are the 5 separate patterns in which Cocci may appear?
    Depending on the plane in which they divide and how they remain attached, cocci can appear as follows:

    • Diplococci (pairs) - divided in one plane
    • Streptococci (chains) - divide in one plane
    • Tetracocci (tetrads) - divide in two planes
    • Staphylococci (clusters) - divide in three planes irregularly
    • Sarcinae (cuboidal packets) - divide in three planes regularly
  20. Describe the 3 cylindrical patterns in which Bacilli can appear
    • Coccobacillus - very short and appears spherical, but they are just slightly longer in one direction than the other
    • Fusiform Bacilli - tapered at the ends, appearing as football like in shape
    • Filamentous Bacillary Forms - grow in long threads
  21. Most microorganisms are difficult to see using light microscopy due to their _________.
    Most microorganisms are difficult to see using light microscopy due to their size and lack of contrast between the cell and the environment.
  22. List the 4 types of dyes, and give examples of each
    • Cationic dyes (basic dye, positively charged chromophore) -  Methylene Blue, Crystal Violet
    • Anionic dyes (acidic dyes, negatively charged) - Acid Fuchsin, Congo Red, Nigrosin
    • Fat Soluble (no charge) - Sudan black (stains granuals of Poly-B-OH-butyric acid)
    • Insoluble Dyes (water insoluble) - India Ink (colloid suspension of carbon particles)
  23. Explain what the Negative Staining procedure stains, and which dye(s) is used?
    Stains background, not the cell in brightfield microscopy.

    • Two dyes used:
    • 1. Nigrosin - a black anionic(-) dye. The negatively charged dye is repelled by the negatively charged surface of the bacterial cell wall.
    • 2. India Ink - an insoluble dye (a colloidal suspension of carbon particles) which does not penetrate the cell surface
  24. What can a Simple Stain tell you about a cell? Which dye(s) is used?
    • A simple staining procedure can be used to tell morphology (shape) and size (although negative staining is better for size). 
    • Only one type of dye is used, a cationic dye (positively charged and are attracted by ionic forces to the negatively charged surface of the bacterial cell wall) - Methylene Blue, Crystal Violet
  25. Describe a Gram Stain (purpose and procedure)
    A differential staining procedure that causes cells to stain differently based on characteristics of their cell wall. Gm(+) bacteria have a higher peptidoglycan and lower lipid content than Gm(-) bacteria.

    • Procedure:
    • 1. Heat fix bacteria
    • 2. Cells are stained with Crystal Violet (primary stain) for 1min
    • 3. Cells are stained with Gram's Iodine (mordant) for 1min
    • 4. 95% Ethanol is then added as a decolorizer for 5sec
    • 5. Cells are counterstained with Safranin for 1min6. Rinse with DI water

    Gm(+) cells appear purple, Gm(-) cells appear redish-pink

  26. What effect does the decolorizer have on Gm(+) and Gm(-) bacteria during a Gram Staining procedure?
    Gm(+) cells resist decolorization from Ethanol due to the difference in cell wall consistency retaining the crystal violet-iodine complex.

    Gm(-) cells are easily decolorized because the ethanol dissolves the high lipid cell wall allowing the crystal violet-iodine complex to readily exit the cell wall.
  27. What are two important structural differences between Gm(+) and Gm(-) bacteria?
    Gm(+) bacteria have higher peptidoglycan content in their cell wall, but have lower lipid content than Gm(-) bacteria.
  28. Describe an Acid Fast Stain (purpose and procedure)
    A differential staining procedure that causes cells to stain differently based on characteristics of their cell wall. Acid Fast(+) microorganisms have a high wax content in their walls, which requires the use of steam to allow dye to penetrate the cell. Cells are steamed in the presence of carbol fuchsin and decolorized with acid alcohol. Cells which are Acid Fast(+) (microorganisms have a high wax content in their walls) will not decolorize and remain red, while Acid Fast(-) organism will readily lose their stain and become colorless.

    • Procedure:
    • 1. Heat fix bacteria
    • 2. Cells are steamed (mordant) in the presence of Carbol Fuchsin (primary stain) for a few minutes
    • 3. Cells are decolorized with Acid Alcohol for 5sec
    • 4. Cells are then counterstained with Methylene Blue for 1min

    • Acid Fast(+) cells appear red, Acid Fast(-) cells appear blue
  29. What is the alternate name for the Acid Fast Stain?
    The Ziehl-Neelsen Stain
  30. What is the specific type of wax that plays a factor in an Acid Fast Stain?
    Mycolic Wax
  31. List the two genera of Acid Fast Organisms (FYI: all other else are non-Acid Fast):
    • 1. Mycobacterium - do not gram stain well if mature because of high wax content within walls, if young they will appear as Gm(+) tapered rods that sometimes fragment.
    • Two important species: 
    • M. tuberculosis - causes tuberculosis
    • M. leprae- causes leprosy

    2. Nocardia - some species stain partially Acid Fast, but a lass harsh decolorizer than Acid Alcohol must be used
  32. Describe an Spore Stain (purpose + procedure)
    Some microorganisms produce heat and chemical resistance structures called endospores. To stain the spores, the cells must be steamed to allow for the dye to enter the spores. Once the spores are stained, all other organisms or vegetative cells can easily be decolorized, while free spores and endospores remain green. 

    • Procedure:
    • 1. Heat fix bacteria
    • 2. Cells are steamed (mordant) in the presence of Malachite Green (primary stain) for 1min
    • 3. Cells are decolorized with DI water
    • 4. Cells are counterstained with Safranin for 1min

  33. What is the alternate name for a Spore-Stain procedure?
    The Schaeffer-Fulton stain
  34. In a Spore Staining procedure, endospores will appear as _____, while free spores will appear as _______.
    In a Spore Staining procedure, endospores will appear as a green center within a pink sporangium, while free spores will appear as a small green oval body.
  35. List the three genera of Spore forming bacteria
    1. Bacillus - aerobic, Gm(+) rod

    2. Clostridium - Anaerobic, Gm(+) rod

    3. Sporosarcina - Cocci
  36. List the 5 Methods of Tube Media Preparation:
    • Pour = 15-20mL of liquid agar used to pour into plate
    • Broth = 5-7mL of liquid media
    • Deep = 5-7mL of media which has solidified in an upright position
    • Slant = 5-7mL of media which has solidified in an angled position
    • Fermentation Broth = broth with Durham Tube added
  37. Define "Natural Media":
    Media composed of complex raw materials whose actual chemical composition is unknown (ex: Nutrient Agar)
  38. Define "Synthetic Media":
    Media whose exact chemical composition is known and in many instances is designed for isolation, selection, or differentiation of specific types or microorganisms
  39. Describe the two types of Synthetic Media
    1. Selective Media: favors the growth of one type or microorganism over another. This is accomplished by either inhibiting unwanted microorganisms or enriching (providing conditions which are preferential to the desired microorganism)

    2. Differential Media: differentiates or distinguishes between different types of microorganisms based on differences in appearance of growth or color changes
  40. Describe the Selective and/or Differential Media:
    Phenylethyl Alcohol Agar (PEA)
    Type: Selective

    Objective: Selects for Gm(+)

    Significant Components: Phenylethyl Alcohol is an inhibitor of Gm(-)

    Indicator: Ability to grow with PEA

    Results: Growth= (+), No growth = (-)

  41. Describe the Selective and/or Differential Media:

    Desoxycholate Agar (DES)
    Type: Selective & Differential 

    Objective: Selects for Gm(-) mircoorganisms, differentiates for lactose fermenters (Desoxycholate Agar is inhibitory for the growth of Gm(+) microorganisms)

    Significant Components: Lactose, Desoxycholate (detergent)

    Indicator: Red Dye is absorbed into acid producing colony during lactose ferm.

    Results: Red Colony=Lactose (+), Non-Red Colony=Lactose (-)

    Extra Notes:Lactose fermenters produce acid, which preceipitates the bile salts in the media and absorbs the neutral red dye, therefore appearing red. Non-fermenters do not do this and do not appear red.

  42. Describe the Selective and/or Differential Media:

    Eosin Methylene Blue (EMB)
    Type: Selective & Differential 

    Objective: Selects for Gm(-) mircoorganisms, differentiates for lactose fermenters 

    Significant Components: Lactose, Eosin, & Methylene Blue Dye

    Indicator: Colonies absorb dyes by the low pH caused from lactose ferm.

    • Results
    • -Dark Colony=Lactose ferm (+) (absorbs MB dye by high acid production),
    • -Pink Colony=Lactose ferm (+) (absorbs Eosin by low acid production),
    • -No Dye Absorb=Lactose(-)

  43. How can you differentiate Lactose(+) fermenters in an EMB plate?
    You can further differentiate Lactose(+) fermenters based on the amounts of acid produced during lactose fermenters, and thus the different changes in color
  44. What is in EMB Agar that inhibits Gm(-) growth?
  45. Describe mixed acid fermenters
    Mixed Acid Fermenters produce more acid and produce colonies with dark blue-black centers (center is almost the size of the whole colony)
  46. Describe Butanediol Fermenters
    Butanediol Fermenters, like Enterobacter, produce less acid so that the colonies have pale pink to lavender centers. The centers are only a small part of the colony (i.e. bull's-eye colonies), and will not have a metallic green sheen.
  47. Describe the Biochemical Test: Blood Agar
    Type: Differential 

    Objective: Observe hemolysis 

    Significant Components: 5% Sheep Red Blood Cells (RBC)

    Indicator: Action of RBC

    • Results:
    • -Alpha (α) Hemolysis - Partial blood hemolysis and partial clearing around colony. Partial clearing sometimes appears green due to partial reduction of hemoglobin in blood
    • -Beta (β) Hemolysis - Complete blood hemolysis and complete clearing around the colony
    • -Gamma (γ) Hemolysis - No blood hemolysis, no zone of clearing around the colony

  48. Define Biochemical Tests
    Tests used to determine physiological characteristics of microorganism, particularly in terms of bacterial exo-enzymes and the chemistry of bio-oxidation.
  49. Describe the Biochemical Test: Starch Agar
    Type: Differential 

    Objective: Detect Amylase (enzyme that hydrolyzes starch into simple sugars)

    Significant Components: Soluble Starch

    Indicator: Grams Iodine(added after growth, thus a reagent) (Iodine + Starch = Purple)

    • Results
    • -Colorless area around colony = (+) 
    • -Purple all round colony = (-)

    Extra Notes:If amylase is present starch will be hydrolyzed and the blue/black color will not be observed around the amylase positive colonies.

  50. Describe the Biochemical Test: Milk Agar 
    Type: Differential 

    Objective: Detect Caseinase (enzyme that hydrolyzes casein, a predominant protein in milk, into amino acid products)

    Significant Components: Milk (containing opaque casein)

    Indicator: Loss or retention of opacity

    • Results:
    • -Clearing of opaque Milk=(+) 
    • -No Clearing of Opaque Milk=(-)

    Extra Notes:Casein gives milk its white color, so a breakdown in casein causes the milk plate to lose its white color and become clear around the Caseinase positive colonies.

  51. Describe the Biochemical Test: Lipase Plate
    Type: Differential

    Objective: Detect Lipase (enzyme that hydrolyzes lipids to fatty acids and glycerol)

    Significant Components: Corn Oil Lipid

    Indicator: Spirit Blue Dye

    • Results:
    • -Fatty Acid release lowers pH and Blue dye intensifies= (+)
    • -No Blue intensification= (-)

    Extra Notes:The production of the fatty acids lowers the pH just enough to produce a dark blue precipitate when a microorganism is Lipase positive.

  52. Describe the Biochemical Test:

    Sugar Fermentation Tubes
    Type: Differential 

    Objective: Detects fermentation of selected sugar, detects production of gas

    Significant Components: Sugar (variable, can be glucose, lactose, or mannitol), Durham Tube

    • Indicator: Phenol Red (pH indicator):...
    • -Yellow=acid 
    • -Red=neutral 
    • -Cerise=alkaline

    • Results:
    • -Yellow = Acid (ferm+)
    • -Gas in tube= Gas production
    • -Yellow + Gas = Acid, Gas
    • -Red/Dark Red or Cerise = Negative or Alkaline (ferm-)

    Extra Notes:If a microorganism is able to ferment the sugar being tested the result of the fermentation will result in the production of acid, therefore lowering the pH of the solution and causing the liquid to turn yellow from its original red color. Some microorganisms also produce gas during fermentation, which is important to know when identifying unknown bacteria. This gas will collect in the Durham tube and appear as a void or bubble in the inverted tube. An alkaline reaction can also occur, which is due to the utilization of the peptone in the broth and not the testing sugar. An alkaline reaction is indicated by the darkening of the red pH indicator color

  53. What causes an alkaline reaction with Phenol Red during the Sugar Fermentation Tube tests?
    An alkaline reaction can occur, which is due to the utilization of the peptone in the broth and not the testing sugar.
  54. Describe the Biochemical Test: Methyl Red (MR)

    Give an example of a MR(+) bacteria
    Type: Differential (tests for Mixed acid fermenters) 

    Objective: Detect Mixed Acid Ferm (Heavy Acid)

    Significant Components: Glucose

    Indicator: Methyl Red (added after growth) (3-5 drops), if pH is below 5.0 or 5.1=Red

    • Results:
    • -Dye stays Red=(+) 
    • -Dye turns Yellow=(-)

    • Extra Notes:
    • HCOOH --> CO2 + H2 (know this reaction)

    E. Coli is MR(+)

  55. Describe Mixed Acid Fermenters (what they produce+what it causes during an MR test).
    Mixed Acid Fermenters produce drastic amounts of acid from the fermentation of sugars. This acid ultimately results in the lowing of the pH below 5.1, so when theindicator (Methyl Red) is added to the culture, the Methyl Red remains red
  56. Describe the Biochemical Test:Voges-Proskauer (VP)
    Name a VP(+) bacteria
    Type: Differential

    Objective: Detect 2,3 Butanediol fermenters 

    Significant Components: Glucose

    Indicator: Barrit's reagents, also known as VPI (alpha-naphthol) and VPII (KOH), both added after growth (10 drops each)

    • Results:
    • -Brick Red ring on top=(+) 
    • -No ring on top=(-)

    • Extra Notes:
    • HCOOH --> acetyl methyl carbinol (AMC, or acetoin) (this is what you are actually testing for) --> 2,3 butanediol

    When oxygen is present, KOH will react with Acetoin to produce a brick red color and indicate that the microorganism is a 2,3 butanediol producer. Alpha-naphthol is used to intensify the red color.

    Enterobacter aerogenes = VP(+)

  57. What are the reagents VPI and VPII, and what are they collectively know as?
    • VPI (alpha-naphthol) 
    • VPII (KOH)

    They are collectively know as Barrit's Reagents
  58. Describe the difference(s) between 2,3 Butanediol Fermenters and Mixed Acid Fermenters.
    2,3 Butanediol Fermenters produce less acid and more neutral products than Mixed Acid Fermenters.
  59. Why is Acetyl Methyl Carbinol (Acetoin) used in Voges-Proskauer (VP) plates?
    Acetyl methyl carbinol (acetoin) is easier to detect than 2,3 butanediol, and that is the reason why it is what is tested for when determining if a microorganism is a 2,3 butanediol producer.
  60. Describe the Biochemical Test: Catalase 
    Type: Differential 

    Objective: Detect Catalase (enzyme which converts hydrogen peroxide to water and oxygen)

    Indicator: 3% Hydrogen Peroxide (added after growth)

    • Results:
    • -Bubbles =(+) 
    • -No Bubbles =(-)

    • Extra Notes:
    • 2 H2O2 --> 2 H2O + O2 (this is the balanced equation, know this!)

    Hydrogen Peroxide is produced during oxygen utilization and must therefore be eliminated since hydrogen peroxide is toxic. Catalase is an enzyme which converts hydrogen peroxide to water and oxygen, and can be tested for by merely adding H2O2 to the culture of question and look for the production of oxygen bubbles being produced.

    You cannot run Catalase test on Blood Agar plate, as blood naturally has catalase in it.

  61. Describe the Biochemical Test: Oxidase
    Name the only Oxidase(+) bacteria
    Type: Differential

    Objective: Detect Oxidase (enzyme which removes hydrogen from different substrates; can oxidize aromatic amines to form colored products )

    Indicator: Dimethyl-p-phenylenediamine hydrochloride (Oxidase Reagent- Added after growth; is an aromatic amine), which turns a dark blue/purple-black color when oxidized

    • Results
    • -Bacteria turn Dark Red/Purple to Black = (+) 
    • -Remains Colorless = (-)

    Pseudomonas aeruginosa is the only Oxidase(+) bacteria

  62. Describe the Biochemical Test: Nitrate
    Type: Differential 

    Objective: Detect Nitrate Reductase (Nitrase) (enzyme which converts/reduces nitrate(NO3-) to nitrite(NO2-) )

    Significant Components: Sodium Nitrate

    Indicator: Add Nitrate I (Sulfanilic Acid) & Nitrate II (Dimethyl-α- naphthylamine), and Zinc Dust (only after growth; is a CATALYST) (10 drops each), then look for Red complex to form

    • Results:
    • -Red (after N1 and N2)=(+) 
    • -No color change (after N1 and N2) = (-)
    • -Red (after N1, N2, and Zinc)=(-) 
    • -No color change (after N1, N2, and Zinc)=(+)

    • Extra Notes:
    • NO3- + 2e +2H+ --> NO2 + H2ONO2 --> --> N2 + NH3 other products

  63. What are NI and NII in a Nitrate Test?
    • Nitrate I = Sulfanilic Acid
    • Nitrate II = Dimethyl-α- naphthylamine
  64. Describe the Biochemical Test:"Tryptophan (Indole)"
    Type: Differential 

    Objective: Detect Tryptophanase (enzyme which hydrolyses tryptone to indole and pyruvic acid) (Indole is what is tested for)

    Significant Components: Tryptone polypeptide containing tryptophan

    Indicator: Kovac's Reagent (added after growth) (3-5 drops)

    • Results:
    • -Cherry Red ring on top=(+) 
    • -Yellow ring on top=(-)

    • Extra Notes:
    • Tryptophan --> Pyruvic Acid and Indole

    Indole can easily be tested for by adding Kovac's Reagent (p- dimethylaminobenzaldehyde, amyl or butyl alcohol (which extracts the non-polar product), and HCl), which will appear red in the presence of the product indole.

  65. What makes up Kovac's Reagent?
    • p- dimethylaminobenzaldehyde
    • Amyl or Butyl alcohol
    • HCL
  66. Describe the Biochemical Test: Urea

    Give an example of a Urea(+) bacteria
    Type: Differential

    Objective: Detect Urease (enzyme which degrades Urea to Ammonia and Carbon Dioxide)

    Significant Components: Urea

    • Indicator: Phenol Red (a pH indicator):
    • -Yellow=acid 
    • -Red=neutral 
    • -Cerise=alkaline

    • Reults:
    • -Red / Cerise(pink color)=(+) 
    • -Yellow to Light Orange=(-)

    • Extra Notes:
    • Urea --> 2 NH3 + CO2

    When ammonia is released the pH of the solution increases and once the pH is above 8.1 the phenol red indicator will appear Red. The red color indicates a positive test for Urease.

    Proteus vulgaris= Urease(+) (this bacteria also smells really bad) (also swarms on an NA plate, hazy/runny look)

  67. Describe the Biochemical Test: Hydrogen Sulfide Production (H2S)

    Name an H2S(+) bacteria
    Objective: Detects Cystein Desulfurase (enzyme which removes the sulfur side chain from cystein to produce H2S)

    • Results:
    • -Black Precipitate = (+)
    • -Unchanged = (-)

    • Extra Notes:
    • Cystein --> H2S + Amino Acrylic Acid --> Imino Acid --> Pyruvic Acid + NH3

    Tests for the enzyme cystein desulfurase which removes the sulfur side chain from cystein to produce H2S, which when in the presence of iron salts (contained in Klinger's Iron Agar and SIM medium) forms a black precipitate. This Black Precipitate is a positive test for cystein desulfurase (aka H2S positive)

    Proteus = H2S(+)

  68. Describe the Biochemical Test: SIM

    Give an example of a motility(+) bacteria
    Objective: Tests for Sulfur (H2S production), Indole, and Motility

    • Results:
    • -Black Precipitate= H2S(+)
    • -Kovacs Reagent turns red (cerise) after addition = Indole(+)
    • -Growth away from inoculation line (appears as cloudiness in tube) = Motility(+)

    • Extra Notes: 
    • Iron in the media reacts with the H2S to make the black precipitate (Ferric Sulfide)

    Enterobacter aerogenes = Motility(+)

  69. Describe the Biochemical Test:"Simmons Citrate"
    Type: Differential

    Objective: Detect use of Citrate as sole Carbon Source

    Significant Components: Citrate

    • Indicator: Bromothymol Blue: 
    • -Yellow=Acid 
    • -Green=Neutral 
    • -Blue=Alkaline

    • Results:
    • -Growth on slant AND Prussian Blue Color=(+) 
    • -No growth =(-)

    • Extra Notes:
    • If a microorganism can use citrate as the sole carbon source the microorganism will grow on the bacterial medium and the media will turn a deep Prussian Blue color. Growth AND the appearance of the Prussian Blue color are indications of a Citrate(+) microorganism.

  70. Describe the Biochemical Test: Phenylalanine (PPA) 
    Objective: Detect Phenylalanase (phenylalanine deaminase) (enzyme which converts phenylananine to phenylpyruvic acid (PPA) and NH3)

    Significant Components: Amino Acid Phenylalanine

    Indicator: 10% Feric Chloride (added after growth) (5-10 drops)

    • Results:
    • -Green = (+)
    • -No color change w/ addition of Ferric Chloride = (-)

    • Extra Notes:
    • Phenylalanine --> Phenylpyruvic Acid (PPA) + NH3

    To test for the presence of PPA ferric chloride is added to the media. Ferric Chloride in the presence of PPA will appear a deep green color. This green color is indicative of a positive test for phenylalanase.

  71. Describe the Biochemical Test: Litmus Milk 
    Objective: Detect Moderate/Heavy Acid from Lactose fermenters, Detect Caseinase, Detect Reductase 

    Significant Components: Lactose, Litmus, Casein from Milk

    • Indicator: Azolitmin Dye (Litmus-oxidized form): 
    • -Lavender=neutral
    • -Pink=Acid 
    • -Blue=Alkaline 
    • -No Color=Reduced form

    Results: 5 Rxns can be read in Litmus Milk* (separate slide)

    • Extra Notes: 
    • Tests for Lactose fermentation, reduction of litmus, presence of caseinase, and the deamination of amino acids to produce NH3

  72. Describe all 5 results that a Litmus Milk can show:
    • 1. Acid Reaction - Pink Liquid due to drop in pH from the fermentation of lactose
    • 2. Acid Curd Reaction - Pink Solid due to acid production and coagulation of proteins causing the solid formation
    • 3. Reduction - Litmus is reduced and is caused to be colorless and the tube appears white since only the Milk remains.
    • 4. Alkaline Reaction - Blue liquid that is usually caused when protein breakdown produces amino acids that are deaminated and release ammonia.
    • 5. Peptonization/Proteolysis- Clearing of medium (may be brown or amber) caused by enzyme caseinase, which breaks down the white protein casein inmilk.

    Multiple reactions can also be observed: ex Acid-Curd-Reduction (looks like Acid Curd but the tube turns white except for a small region at the top where oxygen reoxidizes the litmus to the colored form, which is red)
  73. Describe the Biochemical Test: Gelatin
    Objective: Detect Gelatinase (enzyme which hydrolyzes gelatin protein into amino acids)

    Significant Components: Gelatin

    Indicator: Re-solidification of gelatin at lower temperature

    • Results:
    • -Gelatinase(+) = LIQUID after being put on ice (gelatin was hydrolyzed) 
    • -Gelatinase(-) = SOLID after being put on ice (gelatin still present)

    • Extra Notes:
    • Gelatin is a protein that solidifies at lower temperatures. This tube is stab-inoculated and then placed on ice for several minutes after incubation. An organism that is positive for the enzyme gelatinase will be able to hydrolyze gelatin to amino acids.

  74. Describe the Biochemical Test: IMViC
    Set of four tests that are used to differentiate between Escherichia coli and Enterobacter aerogenes

    Indol, Methyl Red, Voges-Proskauer, and Citrate

    • E. Coli = I(+), MR(+), Vi(-), C(-)
    • E. aerogenes = I(-), MR(-), Vi(+), C(+)
  75. Describe the Biochemical Test: Motility Media 
    Objective: Determine if Bacteria are Motile

    Significant Components: Growth Indicator

    Indicator: Tetrazolium Chloride (Growth indicator), turns red in the presence of growing bacteria

    • Results:
    • -Red Throughout tube=Motile 
    • -Red only near Stab=Non Motile

    Extra Notes:Contains Tetrazolium chloride, a growth indicator that turns red in the presence of growing bacteria. Therefore Red color away from the inoculation line is an indicatorof growth.

    Red color is only a growth indicator and indicates that tetrazolium chloride is reduced; the red color does not mean motility. Where the red color appears is an indication of motility, not the appearance of red.

  76. Describe the Biochemical Test: Russel's Double Sugar (RDS)
    Objective: Test for Glucose and/or Lactose fermentation; Can also be used to test for gas production 

    Significant Components: contains 1% lactose and 0.1% glucose

    Indicator: pH indicator (phenol red)

    • Results: 
    • - Whole tube red = Glucose(-) and Lactose (-)
    • - Slant red+Butt Yellow = Glucose(+) and Lactose (-)
    • - Whole tube yellow = Glucose(+) and Lactose(+)

    • Extra Notes: RDS is read slant over butt, meaning that the results of the slant should be read first, and the results from the butt should be read second. 
    • RDS should ideally be read within 24-48 hours after inoculation. After 48 hours, the organism could begin to break down the peptone in the  media, which will raise the pH and cause the slant to turn back to red (leading to a false negative for lactose fermentation).

  77. Describe the Biochemical Test: Kligler's Iron Agar (KIA)
    Objective: Test for Glucose and/or Lactose fermentation, and H2S porduction; Can also be used to test for gas production 

    Significant Components: Iron Salts

    Indicator: Phenol Red + Iron Salts

    • Results:
    • - Whole tube red = Glucose(-), Lactose(-), H2S(-)
    • - Slant red + butt yellow = Glucose(+), Lactose(-), H2S(-)
    • - Whole tube yellow = Glucose(+), Lactose(+), H2S(-)
    • - Black precipitate + red slant = Glucose(+), Lactose(-), H2S(+)
    • - Black precipitate + yellow slant = Glucose(-), Lactose(-), H2S(+)

    Other Notes: KIA is ideally read after 18 hours. The lactose reaction should be read from the bottom of the slant as the tip of the slant may revert back to red as the inoculation ages beyond 18-24 hours

  78. Describe the Biochemical Test: Oxidative Fermentation (OF) Glucose
    Objective: Test for use of glucose in an oxidative (aerobic) or fermentative (anaerobic) state 

    Indicator: Brom Thymol Blue (pH indicator; turns yellow if glucose is utilized and acids are produced)

    • Results:
    • - Open Tube: Yellow
    • - Closed Tube: Uninoculated color
    • = Incompletely Oxidative (O)

    • - Open Tube: Yellow
    • - Closed Tube: Yellow
    • = Strictly Fermentation (F)

    • - Open Tube: Uninoculated color
    • - Closed Tube: Uninoculated color
    • = Strictly Oxidative

    • - Open Tube: Uninoculated color
    • - Closed Tube: Yellow
    • = Facultative 

    Extra Notes: Mineral Oil is used to cover one tube 

  79. Describe the Biochemical Test: Bisumuth Sulfide Agar (BSA)
    Objective: Isolation of Salmonella species


    • Results: 
    • - Salmonella typhi produces a black/very dark brown color

    Other Notes: It uses glucose as a primary source of carbon. Brilliant Green is inhibitory towards Gm(+) growth.

  80. Describe the Biochemical Test: Brilliant Green Agar (BGA)
    Objective: Differential for lactose/sucrose fermentation

    Indicator: Phenol Red

    • Results:
    • 1) Lactose/sucrose fermenting organisms produce yellow/ green or yellow colonies and turn the surrounding media yellow/green
    • 2) Non-lactose/ sucrose fermenting organisms produce opaque red/ pink/ white colonies and turn the surrounding media red

  81. Describe the Biochemical Test: SS Agar
    On this medium Salmonella usually produces a black colony, Shigella a colorless colony & all lactose positive colonies appear red 

    Indicator is Neutral Red Dye

  82. Describe the Biochemical Test: Desoxycholate Citrate
    Objective: used for the isolation and differentiation of Gm(-) enteric bacilli; Selects for Gm(-), Lactose(-); inhibitory towards Gm(+) growth

    Some Lactose(+) colonies do grow, but they will appear red

  83. Describe the Biochemical Test: Coagulase
    Incubate bacteria in small tube of plasma overnight

    If plasma becomes clumpy and or solidifies, then bacteria are coagulase positive

    Test is only valid on gram + staphylococcus like bacteria since gram negative bacteria are able to provide false positive reactions from a non coagulase like mechanism.

    Staph aureus is the only Coagulase(+) staph species

  84. Describe the Biochemical Test: Phenol Red Mannitol Sat Agar (MSA)
    Selects for Staphylococcus due to high salt concentration 7.5%

    Medium is red, but plate and colonies will turn yellow if organisms are mannitol positive

  85. Describe the Biochemical Test: Staphylococcus 110 Medium (SM110)
    Contains Mannitol and 7.5% NaCl, but lacks Phenol Red as in MSA plate

    Selects for Staphylococcus and allows for development of natural colony pigment formation unlike in MSA

  86. Describe the Biochemical Test: DNase
    Tests for exoenzyme DNase which is able to hydrolyze DNA

    Zones of clear around streaks either before or after addition of 0.1N HCl is a positive result for the presence of DNase

    Methyl Green is the indicator 

  87. Describe the Biochemical Test: M-staphylococcus broth
    Enriched media containing 10% NaCl, which selects for Staphylococcus since Staphylococcus prefer the higher salt concentration, which inhibits most other organisms
  88. Describe the Biochemical Test: Endo Agar
    Selects for Gm(-)

    Differential for lactose, lactose(+) = red colonies and surrounding medium

    Coliforms (such as E coli) produce a golden metallic golden sheen

  89. Staphylococcus:

    Where is it found?
    Is it penicillin resistant?
    How does it divide?
    What are some important species?
    Found in nasal membranes, the hair follicles, the skin, and perineum

    Most strains are penicillin resistant, which can cause epidemiology problems since 90% of healthcare workers carry Staphylococcus

    Divide in multiple planes and there appears as irregular clusters microscopically

    • Three important Staphylococcus species are:
    • 1) S. aureus (only one of 3 that has coagulase) 
    • 2) S. epidermidis
    • 3) S. saprophyticus
  90. Streptococcus:

    Where is it found?
    How does it divide?
    What are some important species?
    Found in pharynx, on surfaces of the teeth, saliva, shin, colon, rectum, and vagina

    Divide in only one plain and therefore appear as chains of cocci

    • Streptococci of greatest medical significance are:
    • 1) S. pyogenes
    • 2) S.pneumoniae
    • 3) S. agalactiae
  91. Describe the process of Isolation and Identification of Staphylococcus and Streptococcus
    • Day One:
    • 1) Swab Nose, Throat, and a Fomite
    • 2) Streak swab onto Blood Agar Plate as shown
    • 3) Place swabs into m-Staphylococcus broth

    • Day 2: 
    • Staphylococcus:
    • 1) Use the three m-Staphylococcus broths to inoculate two Staphylococcus medium 110 (SM110) and two Mannitol Salt Agar (MSA) Plates
    • 2) Select a Beta hemolytic staphylococcus from the Blood Agar plate and inoculate third SM 110 and MSA plate

    • Streptococcus: 
    • 1) Identify an Alpha and a Beta hemolytic Streptococcus and a Beta hemolytic Staphylococcus and streak each onto a new blood agar plate

    • Day 3: 
    • Staphylococcus:
    • 1) Using the SM110 and MSA plates from last period to identify 3 presumptive Staphylococcus colonies by their growth on the SM110 and MSA plates as well as the cluster formation of the gram + cocci.
    • 2) Inoculate each of these 3 colonies onto/into Coagulase, DNase, and Nitrate Broth and perform a catalase test on each (record these results, as well as any Mannitol + results)
    • 3) Into the 4th Coagulase, DNase, and Nitrate Broth inoculate with the provided Staphylococcus culture.

    • Streptococcus: 
    • 1) Using the Blood Agar plates from last period identify 3 Alpha or Beta hemolytic Streptococcus by identifying the chain formation of the gram + cocci
    • 2) Inoculate each of these 3 colonies into a Nitrate broth and perform a catalase test on each (record these results as well as the hemolytic results from the Blood Agar)
    • 3) Into the 4th Nitrate Broth inoculate with the provided Streptococcus culture.

    • Day 4:
    • Staphylococcus: 
    • 1) Examine the results of the Nitrate Broth, DNase, and Coagulase inoculations made last period and identify the species of Staphylococcus.

    • Streptococcus:
    • 1) Examine the results of the Nitrate Broth inoculations made last period and identify the species of Streptococcus.
  92. What two Gm(-) intestinal pathogens are a major concern for public health, and why?
    • Salmonella and Shigella
    • have the ability to cause enteric fevers, food poisoning, dysentery, and even typhoid fever
  93. Describe the process of identifying and differentiating Gram Negative Intestinal Pathogens:
    • Day One:
    • Perform Isolation streaks of the Salmonella containing mixture onto each of the selective/differential media provided.

    • Day Two:
    • 1) From the five selective/differential plates select 7 isolated colonies that are presumptive for being Salmonella based on their appearance and inoculate each into a SIM, Urea, and KIA media 

    • Colonies to be selected:
    • 1) EMB: Lactose (-) bacteria (colonies do not change color)
    • 2) DES citrate: Lactose (-) bacteria (colonies do not change color)
    • 3) BGA: Lactose (-) (colonies appear pink/white surrounded by red media)
    • 4) SS agar: Black colonies
    • 5) BSA: Black colonies

    • Day 3:
    • Determine the identity of each of the selected colonies using flow chart