Microbiology Lecture Review - Flashcards

Extracellular Enzymes Tests

• DNase Test

  • Purpose: Detects DNase enzyme activity, which breaks down DNA into nucleotides.
  • Medium: Plate.
  • Indicator: Methyl green dye (greenish/Listerine color).
  • Positive Result: Clear halo around growth, indicating DNase activity.
  • Molecule Broken Down: DNA is broken down into Nucleotides (DNA \rightarrow Nucleotides).

• Starch Test

  • Purpose: Detects amylase enzyme activity, which breaks down starch.
  • Medium: Plate.
  • Indicator: Iodine (added post-incubation; reacts with starch to produce a dark blue/black color).
  • Positive Result: Clear halo, indicating starch has been broken down.
  • Molecule Broken Down: Starch is broken down into Monosaccharides (Starch \rightarrow Monosaccharides).

• Gelatin Test

  • Purpose: Detects gelatinase enzyme activity, which breaks down gelatin.
  • Medium: Tube.
  • Positive Result: Liquefaction, where gelatin remains liquid following refrigeration.
  • Molecule Broken Down: Gelatin (protein) is broken down into Amino acids (Gelatin \rightarrow Amino acids).

• Urease Test

  • Purpose: Detects urease enzyme activity, which breaks down urea.
  • Medium: Slant or broth.
  • Indicator: Phenol red.
  • Positive Result: Hot pink color indicates an alkaline pH due to ammonia production.

Amino Acid-Targeting Enzyme Tests

• Phenylalanine Deaminase Test

  • Purpose: Detects deaminase enzyme, which removes the amino group from phenylalanine.
  • Medium: Slant.
  • Reagent Added: Ferric chloride (FeCl_3).
  • Positive Result: Green color indicates the presence of phenylpyruvic acid.

• Lysine Decarboxylase Test

  • Purpose: Detects the enzyme that removes the carboxyl group from lysine.
  • Medium: Broth.
  • Conditions: Anaerobic (achieved by adding mineral oil on top).
  • Indicator: Bromocresol purple.
  • Positive Result: Purple color, indicating an alkaline result from cadaverine production.

• Lysine Iron Agar (LIA) Slant

  • Purpose: Combines tests for lysine decarboxylase (anaerobic) and lysine deaminase (aerobic).
  • Medium: Slant with stab & fishtail inoculation.
  • Indicators: Bromocresol purple (pH) and iron salts (deaminase reaction).
  • Results:
    • Purple butt: Positive for decarboxylation.
    • Rusty red slant: Positive for deamination.

Coliform Detection Labs

• MacConkey Agar

  • Selective: Gram-negative bacteria.
  • Differential: Lactose fermentation.
  • Positive for Coliforms: Pink/red colonies indicate acidic pH from lactose fermentation.

• EMB Plate (Eosin Methylene Blue)

  • Selective: Gram-negative bacteria.
  • Differential: Lactose/sucrose fermentation.
  • Positive for Coliforms:
    • Dark colonies: Lactose fermentation.
    • Green metallic sheen: E. coli.

• (Unspecified 3rd Test)

  • There was mention of a third coliform test, but its name was not specified.

Coliform and Selective Media Overview

• Coliform Identification: All coliforms ferment lactose.

• Selective Media (MacConkey, EMB, Hektoen, BGLB):

  • All are selective for Gram-negative bacteria; they inhibit Gram-positive growth.
  • Growth = Gram-negative, regardless of colony color.
  • All include bile salts to suppress Gram-positive bacteria.

BGLB (Brilliant Green Lactose Bile Broth)

• Structure: Tube, contains a Durham tube for gas detection.
• Ingredients: Brilliant green (inhibitory dye), lactose (fermentable sugar), bile (selective).
• Positive result: Turbid broth with gas bubble indicates a coliform is present.
• Negative result: No gas, even if cloudy, indicates no lactose fermentation.

MacConkey, EMB, Hektoen Enteric (HE)

• MacConkey & EMB:

  • Contain lactose and bile salts.
  • Coliforms = growth + color change (e.g., E. coli = metallic green on EMB).

• Hektoen:

  • Selective for non-coliforms.
  • Also contains bile salts and lactose, plus extra sugars.
  • Color indicators:
    • Orange/yellow colonies = lactose fermentation (coliform, negative test).
    • Green colonies with/without black centers = non-lactose fermenters (Shigella = green; Salmonella = green + black).
  • Black precipitate = H_2S gas (hydrogen sulfide), indicates sulfur metabolism.

Hydrogen Sulfide (H₂S) Production

• Black coloration in media = H_2S gas production.
• Occurs if bacteria metabolize:
  • Thiosulfate or
  • Cysteine (a sulfur-containing amino acid).

Fermentation Labs

• Phenol Red Broths

  • Indicator: Phenol red.
  • Starts red, turns yellow if acid produced (positive fermentation).
  • Hot pink = alkaline, negative (uses peptone instead of sugar).

• Mannitol Salt Agar (MSA)

  • Selective: High salt for Staphylococcus spp.
  • Differential: Mannitol fermentation turns phenol red → yellow.

• OF Glucose Test

  • Tests for oxidation vs fermentation.
  • Two tubes: one aerobic, one anaerobic (covered with oil).
  • Indicator: Bromothymol blue (starts green).
  • Yellow/orange color = acid.
    • Both tubes yellow = facultative anaerobe.
    • Only aerobic tube yellow = oxidative metabolism.
    • Only anaerobic yellow = rare, possibly anaerobe.
  • Enterobacteriaceae expected to be facultative anaerobes (both tubes yellow).

Citrate Test

• Medium: Simmon’s citrate agar.
• Starts green, turns blue if citrate is used as sole carbon source.
• Tests for citrate permease enzyme.
• Blue = positive, green = negative.

Staining Techniques

• Gram Stain: Identify Gram-positive (purple) vs Gram-negative (pink).

• Negative Stain:

  • Uses negatively charged dye (e.g., nigrosin).
  • Dye is repelled by negatively charged bacterial cell walls.
  • Background stains, not the cells.

Indole Test (SIM Media)

• Tests for tryptophanase enzyme → indole production.
• Reagent added after incubation (e.g., Kovac’s).
• Positive = cherry red layer on top.

Methyl Red and Voges-Proskauer (MR-VP)

• Both test for glucose metabolism pathways.

• Methyl Red: Tests for mixed acid fermentation.

  • Positive = red.

• VP Test: Tests for 2,3-butanediol fermentation (neutral products).

  • Positive = red after adding reagents.

Final Notes

• Gas production = key sign of fermentation (esp. lactose).
• Turbidity + gas = positive in BGLB.
• Some organisms grow but don’t ferment → turbid but negative (no gas).
• Practice interpreting color changes across media for:
  • Acid (yellow)
  • Alkaline (pink)
  • H_2S (black precipitate)
  • Growth (selectivity confirmation)