MICR 206 Exam 2 Study Guide

Oxidase Test

  • Reagent Used: Oxidase
  • Purpose: Identify bacteria containing cytochrome c oxidase.
  • Procedure:
    • Transfer bacteria to sterile filter paper.
    • Saturate with oxidase reagent to test for color change.
  • Important Timing: Read results within 30 seconds (color change occurs in 20 seconds).
  • Interpretation:
    • Positive Reaction: Color change indicates presence of cytochrome c.
    • Negative Reaction: No color change indicates absence of cytochrome c.

Catalase Test

  • Reagent Used: Hydrogen peroxide
  • Purpose: Identify organisms that produce enzyme catalase; commonly differentiates between catalase-positive Staphylococceae/Micrococcaceae and catalase-negative Streptococcaeae.
  • Procedure:
    • Transfer bacteria to a slide.
    • Add 1-2 drops of hydrogen peroxide and watch for immediate bubble formation.
  • Interpretation:
    • Positive Reaction: Bubble formation indicates catalase is present.
    • Negative Reaction: No bubble formation indicates catalase is not present.

MacConkey Agar (MAC)

  • Purpose: Isolates or differentiates Enterobacteriaceae based on lactose fermentation ability.
  • Media Characteristics:
    • Supports growth of gram-negative bacteria, inhibits gram-positive.
    • Gram Negative:
      • Growth on MAC:
      • Colorless/orange if no lactose fermentation.
      • Pink if positive for lactose fermentation.
    • Gram Positive: No growth is visible.
  • Test Type: Both selective (only gram-negative grow) and differential (differentiates lactose fermenters).

Malonate Test

  • Interpretation:
    • Blue = positive; the organism can use malonate.
    • Green/slightly yellow = negative; organism cannot use malonate.
  • Purpose: Differentiate between Escherichia and Enterobacter.
  • Chemical Reaction: Malonate replaces succinate in the Krebs cycle, causing competitive inhibition without malonate utilization.
  • Color Change: Caused by bromothymol blue, indicates alkaline reaction from malonate utilization.

Simmon’s Citrate Test

  • Interpretation:
    • Blue = positive; organism can use citrate.
    • Green = negative; organism cannot use citrate.
  • Purpose: Determine if an organism utilizes citrate as its sole carbon source.
  • Chemical Reaction: Citrate transported into cell, converted to pyruvate via citrate-permease.
  • Color Change: Bromothymol blue turns blue due to increased pH from citrate conversion.

Urea Broth Test

  • Interpretation:
    • Pink = positive for urease production.
    • Orange/yellow = negative for urease production.
  • Purpose: Differentiate rapid urease-positive bacteria from slower urease-positive and urease-negative.
  • Chemical Reaction: Urea hydrolysis produces ammonia and carbon dioxide, increasing pH which turns the test pink.

MIO Test (Motility/Indole/Ornithine)

  • Parameters Tested: Motility, Indole, Ornithine decarboxylation.
  • Motility Test:
    • Medium becomes cloudy/no stab lines = positive (motile).
    • Medium clear/stab lines visible = negative (non-motile).
  • Indole Test: Add 5 drops of Kovac’s reagent.
    • Red/pink = positive (indole present).
    • No color change = negative (indole not present).
  • Ornithine Test:
    • Purple/grayish purple = positive (ornithine decarboxylation present).
    • Yellow = negative (no ornithine decarboxylation).
  • Color Changes: Normal reactions are affected by pH indicators; glucose fermentation lowers pH, while ornithine decarboxylation raises it.

LIA (Lysine Iron Agar) Test

  • Interpretation:
    • Purple = alkaline reaction;
    • Yellow = acidic;
    • Red = deamination.
    • Purple slant/purple butt = positive decarboxylase (K/K).
    • Red slant/yellow butt = positive deamination (R/A).
  • Purpose: Differentiate enterics based on decarboxylation/deamination of lysine and sulfur reduction.
  • Chemical Reactions:
    • Decarboxylation leads to alkaline conditions (purple).
    • Deamination produces compounds that turn red.

Carbohydrate Fermentation Test

  • Carbohydrates Included: Glucose, Lactose, Sucrose, etc.
  • Procedure with Durham Tube: Detect gas production (bubbles/air pocket).
  • Chemical Reactions: Fermentation lowers pH, turning medium yellow; deamination raises pH, turning it red/pink.
  • Interpretation:
    • Yellow = positive for carbohydrate fermentation.
    • Red = negative for carbohydrate fermentation.

Triple Sugar Iron Agar (TSIA)

  • Purpose: Differentiate bacteria based on sugar fermentation (glucose, sucrose, lactose), sulfur reduction, and gas production.
  • Color Codes:
    • K/A = Red slant/yellow butt (glucose fermenter).
    • A/A = Yellow/yellow (glucose and lactose/sucrose fermenter).
    • K/K = red/red (no fermentation).
  • Gas Production Indicators: Bubbles/cracks in the tube; H2S production indicated by black precipitate.
  • Common Observations: Yellow butt for all enterics; K/A reaction indicates improper inoculation.

Gram Stain Test

  • Purpose: Differentiate gram-positive and gram-negative bacteria.
  • Staining Process:
    1. Crystal Violet: Primary stain; penetrates thick peptidoglycan layer in gram-positive bacteria.
    2. Iodine: Mordant that fixes dye.
    3. Alcohol: Decolorizer for gram-negative bacteria; washes away crystal violet.
    4. Safranin: Counterstain that persists in gram-negative cells.
  • Final Outcomes:
    • Gram Positive: Purple;
    • Gram Negative: Pink.
  • Staining Steps:
    1. Prepare slide with DI water and bacteria.
    2. Allow to dry and heat fix.
    3. Sequentially cover with stains; rinse between steps.
    4. Observe under microscope for results.