Enterobacteriaceae Identification

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lily

Cards (10)

  • GRAM STAIN
    Enterobacteriaceae are gram negative (pink or red)
  • MEDIA PREPARATION
    • MacConkey Agar
    • Eosin Methylene Blue
    • Use Quadrant Streak Method B
    • Incubate @35-37°C for 18-24 hours
  • BIOCHEMICAL TESTING
    1. CARBOHYDRATE UTILIZATION ON TSI
    2. METHYL RED/VOGES-PROSKAUER TEST
    3. SULFIDE INDOLE MOTILITY (SIM)
    4. LYSINE IRON AGAR [LIA] – GLUCOSE DETECTION
    5. UREASE TEST
  • CARBOHYDRATE UTILIZATION ON TRIPLE SUGAR IRON AGAR [TSI]
    1. Inoculate colonies
    2. Stab then streak
    3. Incubate @ 35 - 37 C for 18-24 hours
    Results: color change
    • A/A = Yellow/Yellow = Acid/Acid
    • K/A = Red/Yellow = Alkaline/Acid
    • K/K = Red/Red = Alkaline/Alkaline
    • Gas Formation = bubbles
    • Ferric Sulfide Production = Black color
  • METHYL RED/VOGES-PROSKAUER TEST
    [DO NOT EMULSIFY]
    1. Add 1 drop of bacterial suspension
    2. Incubate @35-37°C for 48 hours
    3. Observe for results
    Results:
    • Methyl Red – add MR reagent [3-5 drops] [after 48 hours]
    • Voges-Proskauer – add -naphthol after 48 hours [3-5 drops]
    • (+) – red color
  • CITRATE UTILIZATION ON SIMMONS CITRATE AGAR
    1. Inoculate colonies
    2. Streak
    3. Incubate @35-37°C for up to 7 days
    Results:
    • (+) Growth on media with or without color change
    • (-) No growth
  • SULFIDE INDOLE MOTILITY (SIM) [SEMI-SOLID AGAR]
    1. Inoculate colonies
    2. Stab halfway
    3. Incubate @35-37°C for 18-24 hours
    Results:
    • Sulfide – check for black color
    • Indole – Add Erlich’s reagent (5 drops)
    • (+) Pink to red color
    • Motility – Turbidity [growth will be form from the center]
  • LYSINE IRON AGAR [LIA] – GLUCOSE DETECTION
    1. Inoculate colonies
    2. Stab then Streak
    3. Incubate @35-37°C for 18-24 hours
    Results: color change
    • K/A – Purple/Yellow – Alkaline/Acid
    • K/K – Purple/Purple – Alkaline/Alkaline
    • R/A – Red/Yellow – Lysine Deamination
  • UREASE TEST
    • Can be from a broth [used in the laboratory] or slant agar [Christensen’s Urea Agar]
    1. Inoculate colonies (emulsify)
    2. Incubate @35-37°C for 24 hours
    3. Observe for growth
    Results:
    • (+) Color change – Light pink to orange
  • ANTIBIOTIC SUSCEPTIBILITY TESTING
    1. Make a bacterial suspension (emulsify colonies) in sterile NSS (1mL)
    2. Standardize turbidity. Compare with the McFarland Standard
    3. Swab in Mueller Hinton Agar
    4. Place Antibiotics
    Results:
    • (+) – Sensitive; Zone of inhibition
    • (-) – Resistant; No Zone of inhibition