Common and Hard Biochemical Questions

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Created by
Lee Carlos Boniel

Cards (24)

  • General chacracteristics of Staphylococcus and Micrococcus spp
    • Gram (+)
    • Catalase Test (+)
    • Effervesence or bubbling is produced when a colony is exposed to 3% H2O2
    • Do NOT use colonies from BAP; false positive reaction
    • Appearing in "clusters" or "grape-like" under a microscope
    • Beta-hemolytic on BAP
    • Resistant to Bacitracin
    • Steptococcus pyogenes is also resistant to bacitracin
    • Salt-tolerant = able to grow on 7.5-10% NaCl (Ex. MSA)
  • What is the definitive biochemical test for S. aureus?

    Coagulase Test
    • Slide Coagulase Test
    • Detects "Clumping Factor" aka "Bound Coagulase"
    • Tube Coagulase Test
    • Detects "Free Coagulase"
    • Requires incubation at 37 degC; it takes 20 hrs to do
    • 1' = 37 degC for 4 hours
    • 2' = RT for 16 hours
    • Reagent: Rabbit RBCs collected with EDTA
    • using Citrated RBCs will cause "false positive" results
  • In S. aureus biochemical test, define MSA and their respective characteristics?
    Mannitol Salt Agar (MSA) Components
    • Mannitol = Sugar; fermented by S. aureus
    • Phenol Red = pH indicator; Sugar fermentation turns the pH acidic which then turns the Phenol red agar into a yellow agar when there is growth of S. aureus colonies.
  • What organisms are generally known to be DNAse (+)?

    • Serratia marcesens
    • Moraxella catarrhalis
    • Staphylococcus aureus
    Mnemonic = "S.M.S"
  • S. aureus Biochem test

    S. aureus
    • Colony = yellow
    • Catalase = positive
    • (+) bubbling
    • Coagulase = positive
    • (+) formation of clot
    • DNAse = positive
    • DNA + Methyl Green = (+) Clear zone around the colony
    • DNA + 0.1 Toluidine Blue = (+) Pink zone; Cell is dead
    • Mannitol Fermentation = positive
    • (+) Yellow color on MSA
    • Hemolysis Pattern on BAP = beta hemolysis
    • Yellow discoloration on BAP
    • Novobiocin Susceptibility = Sensitive
    • Inhibitied by Novobiocin
    • Gelatinase = positive
    • (+): @ Ref Temp, gel will liquefy
  • S. epidermidis VS S. saprophyticus
    Shared Traits of the two
    • Colony = positive
    • Catalase = positive
    • Coagulase = Negative
    • DNAse = Positive
    • Mannitol Fermentation = Negative
    • Hemolytic Pattern = Gamma
    • Gelatinase = Positive
    S. epidermidis |VS| S. saprophyticus
    • Novobiocin sensitivity
    • S. epidermidis = Sensitive
    • S. saprophyticus = Resistant
    • Patholgies Associated
    • S. epidermidis = common contaminant of blood cultures, production of biofilms, prosethetic head valve infections
    • S. saprophyticus = common cause of UTI in young sexually active women; cystitis and pyelonephritis in px with catheters
  • What is the presumptive test for micrococci?
    • Appearance under microscope = tetrads or by clumps of 4
    • Modified oxidase Test
    • Reagent: Tetramethyl para-phenylenediamine dihyrochloride dimethylsulfoxide (DMSO)
    • (+): Blue to Purple;
    • BE-RECALL! *Purple* is the preferred answer!
  • What is the difference between oxidase and modified oxidase test?
    • Modified Oxidase aka "Microdase Test"
    • generally used for Gram positive bacteria
    • uses DMSO
    • differentiate Staphylococci from Micrococci
    • Micrococci = (+)
    • Staphylococci = (-)
    • Oxidase test aka "Cytochrome Oxidase Test"
    • generally used for Gram negative bacteria
    • does not use DMSO
  • Micrococci VS Staphylococci
    Micrococci:
    • Aerobic Culture = positive
    • Anaerobic Culture = negative
    • Micrococci are "strict aerobes"
    • Microdase Test = positive
    • Bacitracin Susceptibility = resistant
    • Glucose Utilization = Oxidizer
    Staphylococci
    • Aerobic Culture = positive
    • Anaerobic Culture = positive
    • Staphylococci are "facultative anaerobes"
    • Microdase Test = negative
    • Bacitracin Susceptibility = susceptible
    • Glucose Utilization = fermenter
  • What is MRSA or ORSA?
    MRSA "Methicillin Resistant S. aureus" aka ORSA "Oxacillin-resistant S. aureus" are strains of S. aureus that has gained resistance to penicilinase-resistant drugs. (Ex. Methicillin, Oxacillin, Nafacillin)

    Penicillinase-resistant drugs are given to S. aureus because we cannot use beta-lactam antibiotics (Ex. Penicillin, Carbapenem, Cephalosporin) since S. aureus has an innate production of beta-lactamase.

    MRSAs or ORSAs exist due to a gene called "MecA" which encodes for a protein called "PBP2A" or "Penicillin-binding protein 2A" that is found on the MRSAs' cell wall.
  • Tests used to Detect MRSA/ORSA

    1. Chrom Agar = selective and differential for MRSA
    2. MRSA = rose to mauve colored colonies
    3. Non-MRSA = blue colonies or no growth
    4. Oxacillin Screen Agar = infuses MHA with 6% oxacillin and 4% NaCl
    5. Presence of Growth = (+) Resistance to Oxacillin
    6. No growth = (-) Resistance to Oxacillin
    7. Cefoxitin Disk Diffusion Test = Induces expression of PBP2A
    8. PCR = Gold standard, detect the MecA gene
    Treaments used for MRSA/ORSA = Vancomycin, a glycopeptide cell-wall inhibitor
  • S. aureus || S. lugdunensis || S. intermedius
    Why we need to differentiate? Becuase the two other batceria resemble S. aureus, more biochemical tests are performed for proper identification.

    S. aureus || S. lugdunensis || S. intermedius

    PYR Test: (-) || (+) || (+)
    VP Test: (+) || (+) || (-)
    Slide Coag: (+) || (+) || (V)
    Tube Coag: (+) || (V/-) || (V)

    S. lugdunensis in positive for ornitihne decarboxylase.
  • What is wide zone hemolysis?

    • also called "alpha prime" hemolysis
    • it is the hemolytic pattern on BAP that shows a inner alpha-zone and a outer beta zone of hemolysis
    • not clinically significant and is usually caused by prolonged refrigeration
  • What is "Double Zone of Hemolysis"?
    • it also known as "Target hemolysis"
    • a hemolytic pattern that appears on BAP that is characterized by the appearance of an inner beta zone of hemolysis and an outer alpha zone of hemolysis
    • This pattern of hemolysis is clinically significant and must be reported ASAP
    • The bacteria that is known to cause this pattern of hemolysis is "Clostridium perfringens".
  • The classification of Streptococci based on the hemolytic patterns displayed on BAP is called as the "Smith and Brown" classification.
    • Alpha hemolysis = Viridans strep
    • Beta hemolysis = S. pyogenes, S. agalactiae
    • Gamma hemolysis = Enterococci and Non-enterococci


    The classification of Streptococci based on their surface carbohydrates is called as the "Lancefield" Classification
    The groups range from "A-K and H-U".
    • Group A = S. aureus
    • Group B = S. agalactiae
    • Group C = S. equisimilus
    • Group D = Enterococci
    • Non-groupable Streptococci
    • Viridans strep
    • S. pneumoniae
  • Quellung's Reaction - A test used to detect the presence of capsular antigens in bacteria such as S. Pneumoniae. It involves adding specific antibodies labeled with dye to the bacteria, resulting in swelling of the capsules if present.
  • What bacterial identification test is more specific to S. pyogenes?
    PYR test = Pyrrolidonil-arylamidase test
    • Substrate: Pyrrolidonil-B-naphthylamide
    • Reagent: P-dimethylamino-cinnamaldehyde
    • Result:
    • Group A Strep (S. pyogenes) = (+) Red
    • Group B Strep (S. agalactiae) = (-) Orange or no color change
  • Hippurate Hydrolysis test is
    • Purpose: a test that is primairly used in the identifcation S. agalactiae or Group B Beta hemolytic streptococci
    • Principle: Hippurate --[hippuric hydrolase]-> glycine + benzoic acid
    • Result: (+) Purple Color
  • CAMP test is ususalluy performed for ID of Group B Beta streptococci (S. agalactiae):
    • also known as "Christie, Atkins, Munch, Peterson"
    • Purpose: detects the ability of an organism to produce the CAMP factor. 
    • Principle: It's based on the observation that some weakly beta-hemolytic species cause intense beta hemolysis when grown with a strain of Staphylococcus.
    • Media: Blood Agar Plate
    • Known Organism: Staphyloccus aureus
    • (+) Result: Enhanced hemolysis showing an arrow-shaped Zone of Beta Hemolysis
    • (-) Result: No enhanced hemolysis
  • S. pyogenes (GAS) || S. agalactiae (GBS) || S. dysgalactiae (GCS)
    PYR: (+) || (-) || (-)
    Bacitracin: (S) || (R) || (R)
    Hippurate: (-) || (+) || (-)
    CAMP: (-) || (+) || (-)
    SXT*: (R) || (R) || (S)

    SXT = Trimethoprim sulfamethoxazole
  • Positive and Negative Controls of Bacterial ID tests
    1. Catalase
    2. (+) S. aureus
    3. (-) S. pyogenes
    4. Coagulase
    5. (+) S. aureus
    6. (-) S. epidermidis
    7. Bacitracin Disk Test
    8. (+) S. pyogenes / M. luteus
    9. (-) S. agalactiae / S. aureus
    10. CAMP Test
    11. (+) S. agalactiae
    12. (-) S. pyogenes
  • Bile Esculin Test
    • commonly used for Group D Streptococcus ID
    • Non-enterococci = (+) Bile Esculin
    • Enterococci = (+) Bile Esculin
    • Non-Group D Streptococci = (-) Bile esculin
    • Media = Bile Esculin Media (Slant)
    • Indicator = Ferric Ammonium Citrate
    • Results:
    • (+) Result = Blackening of media
    • (-) Result = no color change seen in the media
    • Controls:
    • (+) E. faecalis
    • (-) E. coli ; S. pyogenes
  • Group D Streptococci || Enterococci
    PYR: (-) || (+)
    Bile Esculin: (+) || (-)
    Growth in 6.5% NaCl: (-) || (+)
  • Between C. perfringens, C. botulinum, C. tetani, and C. difficile, who is the only one that is:

    1. Non-motile = C. Perfringens
    2. Lecithinase (+) = C. Perfringens
    3. Lactose (+) = C. perfringens
    4. Lipase (+) = C. botulinum
    5. Glucose (-) = C. tetani
    6. Indole (+) = C. tetani
    7. Gelatinase (-) = None, all are Positive