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Microbiology Laboratory Techniques: Media, Biochemical Tests, and Staining

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Selective and Differential Media

Introduction to Media Types

Microbiologists use various types of culture media to isolate, identify, and differentiate microorganisms. Selective media favor the growth of specific microbes, while differential media distinguish between organisms based on metabolic traits.

  • Selective Media: Contain agents that inhibit the growth of some microbes while allowing others to grow.

  • Differential Media: Contain indicators that reveal differences between organisms, often through color changes.

EMB (Eosin Methylene Blue) Agar

  • Purpose: Selective for Gram-negative bacteria; differential for lactose fermentation.

  • Key Features:

    • Inhibits Gram-positive bacteria (e.g., Staphylococcus aureus shows no growth).

    • Escherichia coli produces a green metallic sheen (strong lactose fermenter).

    • Enterobacter aerogenes forms pink to purple colonies (weaker fermenter).

    • Proteus vulgaris and Salmonella typhimurium do not ferment lactose and appear colorless.

MSA (Mannitol Salt Agar)

  • Purpose: Selective for Staphylococcus species; differential for mannitol fermentation.

  • Key Features:

    • High salt concentration inhibits most bacteria except Staphylococcus.

    • Staphylococcus aureus ferments mannitol, turning the medium yellow.

    • Staphylococcus epidermidis grows but does not ferment mannitol (medium remains red).

    • Micrococcus luteus does not grow.

Blood Agar

  • Purpose: Enrichment and differential medium for hemolysis patterns.

  • Key Features:

    • Beta-hemolysis: Complete lysis of red blood cells (clear zone, e.g., Streptococcus pyogenes).

    • Alpha-hemolysis: Partial lysis (greenish zone, e.g., Escherichia coli).

    • Gamma-hemolysis: No lysis (no change, e.g., Staphylococcus epidermidis).

    • "Fish eye" colonies (e.g., Enterobacter) show distinctive morphology.

Antibiotic Sensitivity Testing

Principle and Procedure

Antibiotic sensitivity tests determine the susceptibility of bacteria to various antibiotics. The Kirby-Bauer disk diffusion method is commonly used.

  • Antibiotic disks are placed on an agar plate inoculated with the test organism.

  • After incubation, zones of inhibition (clear areas) around disks indicate sensitivity.

  • Zone diameters are measured and compared to standard tables to classify bacteria as sensitive, intermediate, or resistant.

Antibiotic

Sensitive (mm)

Intermediate (mm)

Resistant

Carbenicillin (100 μg)

≥23

18-22

<18

Kanamycin (30 μg)

≥18

14-17

<14

Other antibiotics

Refer to specific standards

Example: A 25 mm zone for carbenicillin indicates sensitivity; no zone indicates resistance.

Biochemical Tests for Bacterial Identification

The Catalase Test

  • Principle: Catalase enzyme converts hydrogen peroxide () into water and oxygen:

  • Purpose: Differentiates Staphylococcus (catalase +) from Streptococcus (catalase -).

  • Procedure:

    • Place a colony on a slide, add 3% hydrogen peroxide.

    • Positive: rapid bubbling; Negative: no bubbles.

    • False positives may occur if blood agar is used or procedure is reversed.

The Oxidase Test

  • Principle: Detects cytochrome c oxidase enzyme (present in Micrococcus, absent in Staphylococcus).

  • Procedure:

    • Moisten test strip, apply bacteria, observe for color change.

    • Positive: blue to dark purple; Negative: no color change.

Durham Sugar Tube Fermentation

  • Purpose: Tests ability to ferment carbohydrates (glucose, lactose, sucrose) to acid and/or gas.

  • Indicators: Phenol red (red at alkaline pH, yellow at acidic pH); Durham tube collects gas.

  • Results:

    • Red: negative for fermentation.

    • Yellow: positive for acid production.

    • Yellow with bubble: positive for acid and gas production.

IMViC Tests

  • Series of four tests used to differentiate Enterobacteriaceae:

  • Indole test

  • Methyl red test

  • Voges-Proskauer test

  • Citrate utilization test

Sulfur Indole Motility (SIM) Medium

  • Purpose: Tests for hydrogen sulfide production, indole production, and motility.

  • Key Reactions:

    • Sulfur reduction: black precipitate indicates H2S production.

    • Indole production: red ring after adding Kovac's reagent indicates positive.

    • Motility: diffuse growth from stab line indicates motility.

Methyl Red Test

  • Purpose: Detects stable acid production from glucose fermentation (mixed acid pathway).

  • Indicator: Methyl red (red at pH < 4.4, yellow at pH > 6.2).

  • Results: Red = positive; Yellow = negative.

Voges-Proskauer Test

  • Purpose: Detects acetoin production (precursor to 2,3-butanediol) from glucose fermentation (butylene glycol pathway).

  • Reagents: Barritt's A (alpha-naphthol) and Barritt's B (KOH).

  • Results: Red = positive; Yellow/Orange = negative.

Simmons Citrate Agar

  • Purpose: Tests ability to use citrate as sole carbon source and ammonium salts as sole nitrogen source.

  • Indicator: Bromthymol blue (green at neutral pH, blue at alkaline pH).

  • Results: Prussian blue = positive; Green = negative.

Starch Hydrolysis Test

  • Purpose: Detects production of exoenzymes (amylase, oligo-1,6-glucosidase) that hydrolyze starch.

  • Procedure: After incubation, add iodine. Clearing around growth indicates starch hydrolysis.

  • Results:

    • Escherichia coli: negative

    • Bacillus subtilis: positive

    • Proteus vulgaris: variable

Gelatinase Test

  • Purpose: Tests for production of gelatinase, which hydrolyzes gelatin.

  • Results: Liquefied medium after refrigeration = positive.

  • General Results:

    • Serratia marcescens: positive

    • Escherichia coli: negative

    • Proteus vulgaris: positive

Casein Hydrolysis Test

  • Purpose: Tests for production of caseinase, which hydrolyzes milk protein casein.

  • General Results:

    • Escherichia coli: negative

    • Bacillus subtilis: positive

    • Pseudomonas aeruginosa: positive

Urease Test

  • Purpose: Detects urease enzyme, which hydrolyzes urea to ammonia and carbon dioxide.

  • Reaction:

    • Urea + H2O → 2NH3 + CO2

  • Indicator: Phenol red (orange/yellow at acidic pH, bright pink at alkaline pH).

  • Results:

    • Rapid positive: Proteus spp. (within 1-2 hours)

    • Slow positive: Klebsiella spp. (may require 3+ days)

    • Negative: Escherichia coli

Staining Techniques in Microbiology

Principle of Staining

Bacterial cells are nearly colorless and transparent, making them difficult to observe under a microscope. Staining techniques are used to enhance contrast and reveal cell morphology.

  • Stains: Chemical dyes that bind to cellular components.

  • Basic dyes: Colored cation + colorless anion (e.g., methylene blue: MB+ + Cl-).

  • Acidic dyes: Colored anion + colorless cation (e.g., eosin: Na+ + eosin-).

  • Bacterial cells are slightly negatively charged (due to nucleic acids), so they bind basic dyes.

  • Acidic dyes stain the background, not the cell (negative staining).

Types of Staining Techniques

  • Simple Staining: Uses a single dye to reveal cell shape and arrangement.

  • Differential Staining: Uses two contrasting dyes separated by a decolorizing agent to distinguish cell types or structures (e.g., Gram stain, acid-fast stain, spore stain, capsule stain).

Smear Preparation and Fixation

  • Objective: Kill microorganisms and fix them to the slide to prevent loss during staining.

  • Steps: Spread bacteria on slide, air dry, heat fix.

Gram Stain

  • Purpose: Most important differential stain; classifies bacteria as Gram-positive or Gram-negative.

  • Procedure:

    1. Crystal violet (primary stain)

    2. Gram's iodine (mordant)

    3. Alcohol or acetone-alcohol (decolorizer)

    4. Safranin (counterstain)

  • Results:

    • Gram-positive: thick peptidoglycan, retains crystal violet (purple)

    • Gram-negative: thin peptidoglycan, loses crystal violet, stained by safranin (red/pink)

  • Examples:

    • Staphylococcus aureus: Gram-positive cocci, violet

    • Candida albicans: Gram-positive oval, violet

    • Bacillus subtilis: Gram-positive bacilli in chains, violet

    • Gram-negative bacilli: rods, single, red

Endospore Staining (Schaeffer-Fulton Method)

  • Purpose: Identifies endospore-forming bacteria (e.g., Bacillus, Clostridium).

  • Procedure:

    1. Prepare smear, air dry, heat fix.

    2. Flood with malachite green, steam for 10 minutes (keep moist).

    3. Cool, rinse with water.

    4. Counterstain with safranin (1 min), rinse, blot dry.

  • Results: Endospores appear green; vegetative cells appear red/pink.

  • Significance: Endospores are highly resistant to environmental stress; only certain Gram-positive genera form them.

Examples of spore-forming pathogens: Clostridium botulinum (botulism), C. perfringens (gas gangrene), C. tetani (tetanus), Bacillus anthracis (anthrax).

*Additional info: The notes above cover laboratory techniques relevant to Ch. 3 (Observing Microorganisms Through a Microscope), Ch. 4 (Functional Anatomy of Prokaryotic and Eukaryotic Cells), Ch. 6 (Microbial Growth), Ch. 7 (The Control of Microbial Growth), and Ch. 20 (Antimicrobial Drugs) as outlined in standard microbiology curricula.*

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