Microbiology Lecture Notes Review

Gram Stain

• Test Name: Gram Stain
• Media: None (used directly on bacterial smear)
• Metabolic Activity: Differentiates bacteria based on cell wall structure (Gram-positive vs. Gram-negative).
• Key Ingredients: Crystal violet, iodine, alcohol, safranin.
• Chemicals Added: Crystal violet, iodine, alcohol (decolorizer), safranin.
• Interpretation:
  • Purple cells = Gram-positive (thick cell wall).
  • Pink/red cells = Gram-negative (thin cell wall).

Catalase Test

• Test Name: Catalase Test
• Media: Nutrient Agar or Blood Agar Plate
• Metabolic Activity: Tests for the presence of the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen.
• Key Ingredients: None in the media.
• Chemicals Added: Hydrogen Peroxide.
• Interpretation:
  • Bubbles form = Catalase positive (enzyme present).
  • No bubbles = Catalase negative (enzyme absent).

Oxidase Test

• Test Name: Oxidase Test
• Media: Nutrient Agar or Blood Agar Plate
• Metabolic Activity: Tests for the presence of cytochrome c oxidase, involved in the electron transport chain.
• Key Ingredients: None in the media.
• Chemicals Added: Oxidase reagent.
• Interpretation:
  • Purple color = Oxidase positive.
  • No color change = Oxidase negative.

Dextrose (Glucose) Fermentation

• Test Name: Dextrose (Glucose) Fermentation
• Media: Phenol Red Broth with glucose
• Metabolic Activity: Tests for the ability to ferment glucose, producing acid and/or gas.
• Key Ingredients: Glucose, phenol red (pH indicator).
• Chemicals Added: None.
• Interpretation:
  • Yellow broth with gas = Glucose fermented (acid and gas).
  • Yellow broth, no gas = Glucose fermented (only acid).
  • Red broth = No fermentation (neutral pH).
  • Pink broth = No fermentation (alkaline).

Lactose Fermentation

• Test Name: Lactose Fermentation
• Media: Phenol Red Broth with lactose
• Metabolic Activity: Tests for the ability to ferment lactose, producing acid and/or gas.
• Key Ingredients: Lactose, phenol red (pH indicator).
• Chemicals Added: None.
• Interpretation:
  • Yellow broth with gas = Lactose fermented (acid and gas).
  • Yellow broth, no gas = Lactose fermented (only acid).
  • Red broth = No fermentation (neutral).
  • Pink broth = No fermentation (alkaline).

Mannitol Fermentation

• Test Name: Mannitol Fermentation
• Media: Phenol Red Broth with mannitol
• Metabolic Activity: Tests for the ability to ferment mannitol, producing acid and/or gas.
• Key Ingredients: Mannitol, phenol red.
• Chemicals Added: None.
• Interpretation:
  • Yellow broth with gas = Mannitol fermented (acid and gas).
  • Yellow broth, no gas = Mannitol fermented (only acid).
  • Red broth = No fermentation (neutral).
  • Pink broth = No fermentation (alkaline).

Methyl Red Test

• Test Name: Methyl Red Test
• Media: MR-VP Broth
• Metabolic Activity: Tests for mixed acid fermentation (producing stable acids like lactic acid).
• Key Ingredients: Glucose, buffer, pH indicator (methyl red).
• Chemicals Added: Methyl Red (after incubation).
• Interpretation:
  • Red color = Positive (acidic pH, mixed acid fermentation).
  • Yellow color = Negative (no acid production).

Voges-Proskauer Test

• Test Name: Voges-Proskauer Test
• Media: MR-VP Broth
• Metabolic Activity: Tests for the production of acetoin from fermentation (a neutral product).
• Key Ingredients: Glucose, buffer.
• Chemicals Added: Barritt’s Reagents (A and B) (after incubation).
• Interpretation:
  • Red color = Positive (acetoin produced).
  • No color change = Negative (acetoin not produced).

Hydrogen Sulfide (H2S) Production

• Test Name: Hydrogen Sulfide Production
• Media: SIM Agar or TSI Agar
• Metabolic Activity: Tests for the ability to reduce sulfur (to produce H2S gas).
• Key Ingredients: Cysteine, iron salts.
• Chemicals Added: None.
• Interpretation:
  • Black precipitate = H2S production (positive).
  • No color change = No H2S production (negative).

Citrate Utilization

• Test Name: Citrate Utilization
• Media: Citrate Agar
• Metabolic Activity: Tests for the ability to use citrate as the sole carbon source, which produces an alkaline pH.
• Key Ingredients: Citrate, bromthymol blue (pH indicator).
• Chemicals Added: None.
• Interpretation:
  • Blue color = Positive (citrate utilized).
  • Green color = Negative (citrate not utilized).

Litmus Milk

• Test Name: Litmus Milk Test
• Media: Litmus Milk Agar
• Metabolic Activity: Tests for the fermentation of lactose, reduction of litmus, and production of acid or gas.
• Key Ingredients: Milk, litmus (pH indicator).
• Chemicals Added: None.
• Interpretation:
  • Pink (acid) = Lactose fermentation (acid production).
  • Blue (alkaline) = No lactose fermentation (alkaline production).
  • White (reduction) = Litmus reduction.
  • Curdling = Coagulation of proteins (acid production).
  • Gas bubble = Gas production.

Starch Hydrolysis

• Test Name: Starch Hydrolysis
• Media: Starch Agar Plate
• Metabolic Activity: Tests for the ability to break down starch using the enzyme amylase.
• Key Ingredients: Starch.
• Chemicals Added: Iodine (after incubation).
• Interpretation:
  • Clear zone around growth = Starch hydrolyzed (positive).
  • No clear zone = Starch not hydrolyzed (negative).

Phenylethyl Alcohol (PEA) Agar

• Test Name: Phenylethyl Alcohol (PEA) Agar Test
• Media: PEA Agar
• Metabolic Activity: Selective medium to inhibit the growth of Gram-negative bacteria.
• Key Ingredients: Phenylethyl alcohol.
• Chemicals Added: None.
• Interpretation:
  • Growth = Gram-positive bacteria.
  • No growth = Gram-negative bacteria.

Mannitol Salt Agar (MSA)

• Test Name: Mannitol Salt Agar (MSA) Test
• Media: MSA Agar
• Metabolic Activity: Selective and differential; tests for the ability to ferment mannitol and tolerate high salt concentrations.
• Key Ingredients: Mannitol, salt (7.5%), phenol red.
• Chemicals Added: None.
• Interpretation:
  • Yellow agar around growth = Mannitol fermentation (acid production).
  • Red or no change in color = No fermentation.
  • Growth = Salt tolerance (positive for salt tolerance).
  • No growth = Inability to tolerate salt (negative).

MacConkey's Agar

• Test Name: MacConkey's Agar Test
• Media: MacConkey Agar
• Metabolic Activity: Selective and differential; tests for the ability to ferment lactose and tolerate bile salts.
• Key Ingredients: Lactose, bile salts, crystal violet, neutral red.
• Chemicals Added: None.
• Interpretation:
  • Pink colonies = Lactose fermentation (acid production).
  • Colorless colonies = No lactose fermentation.
  • Growth = Tolerance to bile salts.
  • No growth = Inability to tolerate bile salts.

Dichotomous Key

• Tool used to identify bacteria based on test results.
• Series of "yes/no" questions.
• Steps:
  1. Start at the first question.
  2. Answer the question based on test results.
  3. Move to the next question based on the answer.
  4. Continue answering questions.
  5. Arrive at the identification.
• Key Points:
  • Understand question structure and test result interpretation.
  • Each question has two options: "yes" or "no."

ELISA Test

• Purpose of Each Step:
  1. Sample: Contains the substance you're testing for.
  2. Primary Antibody: Binds to the target molecule.
  3. Secondary Antibody: Binds to the primary antibody and is linked to an enzyme.
  4. Enzyme Substrate: Reacts with the enzyme, producing a color change or signal.
  5. Wash Buffer: Removes unbound antibodies or proteins.
• Interpreting an ELISA Test:
  • Positive Result: Color change occurs.
  • Negative Result: No color change.