Mod 6

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Cards (133)

  • Enterobacterales

    New scientific order name adopted in 2020 for what was previously called Enterobacteriaceae
  • Enterobacteriaceae are now a family within the Enterobacterales order
  • Gram staining is not significant for the presumptive identification of Enterobacterales
  • Isolation of gram-negative organisms from a sterile site, including CSF, blood, and other body fluids, is critical and may assist the physician in prescribing appropriate therapy
  • Haemophilus influenzae

    Small, pleomorphic, gram-negative bacilli or coccobacilli with random arrangements
  • Type of specimen for Haemophilus influenzae
    • Blood
    • Cerebrospinal Fluid (CSF)
    • Sputum
    • Eye Discharge
    • Other Sterile Body Fluids
    • Middle Fluid
    • Direct Sinus Aspirate (Maxillary)
    • Bronchoalveolar Lavage (BAL)
    • Endotracheal Aspirate (ETA)
    • Tracheal Aspirate
  • Specimen transport and storage for Haemophilus influenzae
    1. Should be transported and processed as soon as possible to preserve viability
    2. Do not refrigerate specimens
    3. Deep body fluids other than blood are to be transported in plain sterile bottle
    4. For blood cultures, use appropriate blood culture media
  • Delay in transport for Haemophilus influenzae
    1. Should be transported in AMIES transport medium with or without charcoal
    2. Direct plating of specimen on CAP and transportation in candle jar should be considered
  • Biochemical tests for Haemophilus influenzae
    1. SIM: Stab once through the center of the agar depth
    2. TSI: Stab through the center of the butt up to the bottom and draw out and make vertical streak then fishtail over slant
    3. LIA: Double stab, draw out and make vertical streak then fishtail over slant
    4. SCA, UREA AGAR, NA: Vertical streak then fishtail over slant
    5. VP: Emulsify 1-2 isolated colonies in the medium
  • Serodiagnostic techniques are used for only two members of Enterobacterales: Salmonella serotype Typhi and Y. pestis
  • Indole test

    Principle: Detects the ability of an organism to split indole from the amino acid tryptophan
  • Conventional tube method for indole test
    Inoculate tryptophan broth, incubate, add Ehrlich's or Kovac's reagent, observe color change
  • Methyl red test

    Principle: Detects the ability of an organism to produce and maintain stable acid end products from glucose fermentation
  • Procedure for methyl red test
    Inoculate MR-VP broth, incubate, add methyl red indicator, observe color change
  • Voges-Proskauer test

    Principle: Detects the production of acetoin from glucose fermentation
  • Procedure for Voges-Proskauer test
    Inoculate MR-VP broth, incubate, add alpha-naphthol and KOH, observe color change
  • Voges-Proskauer test
    1. Inoculate MR/VP broth
    2. Incubate for 24 hours at 35°C
    3. Aliquot 1 mL of broth
    4. Add 0.6mL of 5% α-naphthol and 0.2 mL of 40% KOH
    5. Shake the tube and allow to remain undisturbed for 10 to 15 minutes
  • Voges-Proskauer test principle
    2pyruvate = acetoin + 2CO2<|>acetoin + NADH + H+ = 2,3-butanediol + NAD+
  • Production of acetoin from glucose. In alkaline pH, acetoin is oxidized to diacetyl. Diacetyl will react with guanidino compounds in medium to produce a cherry red color.
  • VP-positive

    Shows red coloration on top of the culture
  • VP-negative
    Has a yellowish color
  • Citrate utilization test

    1. Inoculate Simmons citrate agar lightly on the slant
    2. Place cap loosely on the tube
    3. Incubate aerobically at 35°C to 37°C for 18 to 24 hours
    4. Observe the development of blue color along the slant
  • Citrate utilization test principle
    When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromothymol blue indicator in the medium from green to blue above pH 7.6.
  • Citrate negative

    Trace or no growth will be visible. No color change will occur; the medium will remain the deep forest green color of the uninoculated agar.
  • Citrate positive

    Growth will be visible on the slant surface and the medium will be an intense Prussian blue.
  • Triple iron sugar test (TSI)
    1. Touch the top of a well-isolated colony with a straight inoculation needle
    2. Inoculate TSI by first stabbing through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant
    3. Leave the cap on loosely and incubate the tube at 35°C in ambient air for 18 to 24 hours
  • Triple sugar iron agar test principle
    The test is designed to differentiate among organisms based on the differences in carbohydrate fermentation patterns and hydrogen sulfide production. Carbohydrate fermentation is indicated by the production of gas and a change in the color of the pH indicator from red to yellow.
  • Triple sugar iron agar composition
    • CHON
    • Lactose (1%)
    • Sucrose (1%)
    • Glucose (0.1%)
    • Phenol red pH indicator
    • Ferrous sulphate H2S indicator
    • Na thiosulfate sulfur source
  • Slant reaction
    LF/SF = yellow
    Non LF/SF = red
  • Butt reaction
    GF = yellow
    Non GF = red
  • Other TSI reactions
    Gas = cracks or displacement of media
    H2S = blackening of medium
  • Formation of H2S requires an ACIDIC environment; even though a yellow butt cannot be seen because of the black precipitate, the butt is acidic
  • Lysine iron agar test (LIA)

    Inoculate LIA by twice stabbing through the center of the medium to the bottom of the tube and then streaking the slant
    Cap the tube tightly and incubate at 35°C in ambient air for 18 to 24 hours
  • Lysine iron agar test principle
    Lysine deamination is an aerobic process which occurs on the slant of the media.
    Lysine decarboxylation is an anaerobic process which occurs in the butt of the media.
  • Lysine iron agar composition
    • Small amount of CHON
    0.1% glucose
    1% lysine
    Bromcresol purple pH indicator
    Ferric ammonium citrate H2S indicator
  • Lysine decarboxylation

    If the organism produces lysine decarboxylase, cadaverine is formed. Cadaverine neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts to the alkaline state (purple).
  • Lysine deamination

    If oxidative deamination of lysine occurs, a compound is formed that, in the presence of ferric ammonium citrate and a coenzyme, flavin mononucleotide, forms a burgundy color on the slant.
  • LIA result interpretation
    Alkaline slant/alkaline butt = Lysine deaminase negative, Lysine decarboxylase positive
    Alkaline slant/acid butt = Lysine deaminase negative, Lysine decarboxylase negative, Glucose fermentation
    Red slant/acid butt = Lysine deaminase positive, Lysine decarboxylase negative, Glucose fermentation
  • The production of H2S can mask the purple color in the butt of the tube. Because H2S production in LIA occurs only in an ALKALINE environment.
  • Phenylalanine deaminase test (PAD)

    Streak the surface of a phenylalanine agar slant with a portion of a well-isolated colony
    Incubate the inoculated slant for 18-24 hours at 35°-37°C in ambient air with cap loose
    Add 4-5 drops of a 10% Ferric Chloride solution directly to the slant and observe for the development of a green color within 1-5 minutes