Gram Positive Cocci and Rods (1)

Gram Positive Cocci and Rods

Learning Objectives

• Understand the purpose of selective and differential media in the isolation and identification of Gram positive bacteria.

• Understand the purpose of hydrolytic and degradative reactions in the identification of Gram positive bacteria.

• Understand the purpose of fermentative reactions in the identification of Gram positive bacteria.

Selective and Differential Media

• Selective Medium:

  • Designed to allow growth of specific microorganisms while inhibiting others.

  • Contains antibiotics, antifungals, dyes, and other inhibitory compounds.

  • Example: Mannitol Salt Agar (MSA) - selects for Staphylococcus genus due to high sodium chloride content.

• Differential Medium:

  • Contains substances that lead to a visible differentiation of microorganisms based on appearance.

  • Example: MSA allows for identification of Staphylococcus aureus, which ferments mannitol, changing the agar from red to yellow, while other non-fermenters keep it red.

  • Sheep Blood Agar (SBA):

    • Used to identify hemolytic activity.

    • Beta hemolysis: complete lysis of red blood cells (clear zone).

    • Alpha hemolysis: partial lysis (greenish discoloration).

    • Gamma hemolysis: no lysis (no effect).

Oxidative and Fermentative Reactions

• Aerobic Bacteria: Use oxygen for respiration, producing hydrogen peroxide (H2O2) as a by-product.

  • Catalase: Enzyme produced by aerobes to degrade H2O2 into water and oxygen.

• Strict Anaerobes and Aerotolerant Bacteria: Lack catalase, cannot detoxify H2O2, important for differentiation.

  • Example of differentiation using a catalase test:

    • Aerobic Staphylococcus (catalase positive) vs. Aerotolerant Streptococcus and Enterococcus (catalase negative).

• Fermentation: Production of acid indicated by pH change; pH indicator phenol red changes from red to yellow in acidic conditions.

Hydrolytic Reactions

• Starch Macromolecule: Composed of amylose and amylopectin, too large to enter bacteria directly.

  • Bacteria secrete exoenzymes (amylases) to hydrolyze starch into maltose, glucose, and dextrins.

  • Starch hydrolysis detection: Using Gram's iodine—changes color based on hydrolysis.

    • Dark agar indicates starch present; clear zone indicates hydrolysis.

• Bile Esculin Agar:

  • Selectively inhibits growth of non-Enterococcus, differentiates based on esculin hydrolysis.

  • Positive result: dark brown/black color formation indicates hydrolysis.

Coagulase Test

• Purpose: Confirms identification of Staphylococcus aureus through its ability to produce coagulase, causing plasma to clot.

• Procedure: Inoculate blood plasma, incubate, observe for clotting.

  • Valid only for Gram positive staphylococcus-like organisms.

Practice Unknown #1 - Gram Positives

Lab Objectives

• Identify Gram positive cocci or rods through biochemical testing.

• Use primary tests to determine unknown number, perform Gram stain, catalase, oxidase, KOH tests.

• Identify bacterial family: Streptococcaceae, Staphylococcaceae, Bacillaceae.

Primary Test Results

Table I: Primary Tests by Family

• Streptococcaceae: Cocci, Catalase -, Oxidase -

• Staphylococcaceae: Cocci, Catalase +, Oxidase -

• Bacillaceae: Rods, Catalase +, Oxidase +/-

Secondary Tests

• Perform different secondary tests based on Gram positive family identified.

Table II: Streptococcaceae Results

Table III: Staphyloccocaceae Results

Table IV: Bacillaceae Results

Lab Steps

• Record unknown number, perform tests, interpret results to identify unknown bacterium.

Biochemical Tests

BHI with 6.5% NaCl

• Differentiates based on ability to grow in high salt.

  • Positive: Growth (turbidity)

  • Negative: No growth

Bile Esculin Hydrolysis

• Differentiates based on ability to hydrolyze esculin.

  • Positive: Darkening of the medium

  • Negative: No color change or < 1/2 dark

Catalase Test

• Differentiates based on catalase enzyme production.

  • Positive: Bubbles with H2O2

  • Negative: No bubbles

Coagulase Test

• Detects Staphylococcus aureus coagulase.

  • Positive: Blood plasma coagulates

  • Negative: No coagulation

Hemolysis on SBA

• Differentiates based on hemolytic activity.

  • Gamma: No hemolysis

  • Alpha: Partial hemolysis

  • Beta: Complete hemolysis

KOH Test

• Confirms Gram stain by lysing bacterial cell wall.

  • Positive: Strings present (Gram-negative)

  • Negative: No strings (Gram-positive)

MSA (Mannitol Salt Agar)

• Purpose: Selects for organisms with high salt tolerance and differentiates on mannitol fermentation.

  • Positive: Yellow colonies (acid produced)

  • Negative: Colorless colonies or no growth

Nitrate Reduction

• Differentiates based on ability to reduce nitrate.

  • Positive: Red color after reagents A & B

  • Negative: Red after zinc or no color change

Oxidase Test

• Differentiates based on presence of cytochrome C oxidase.

  • Positive: Blue to purple color change

  • Negative: No color change

Phenol Red Carbohydrate Tubes

• Tests for carbohydrate fermentation.

  • Positive: Yellow color (fermentation)

  • Negative: Red color

Starch Hydrolysis

• Tests hydrolytic ability on starch.

  • Positive: Clear zone around streak

  • Negative: Dark agar up to streak line