Microbiology Lab Techniques: Media, Biochemical Tests, and Staining

Selective and Differential Media

Introduction to Selective and Differential Media

Selective and differential media are essential tools in microbiology for isolating and identifying microorganisms based on their growth characteristics and biochemical properties. These media help distinguish between different types of bacteria by promoting or inhibiting growth and by revealing metabolic differences.

• Selective media: Contain ingredients that inhibit the growth of some organisms while allowing others to grow.

• Differential media: Contain substances that cause some bacteria to take on an appearance that distinguishes them from other bacteria.

EMB (Eosin Methylene Blue) Agar

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

• Key Ingredients: Eosin Y and methylene blue dyes inhibit Gram-positive bacteria and react with lactose fermenters.

• Results:

  • Escherichia coli: Green metallic sheen (strong lactose fermenter).

  • Enterobacter aerogenes: Pink to purple colonies (moderate lactose fermenter).

  • Proteus vulgaris, Salmonella typhimurium: Colorless or pale colonies (non-lactose fermenters).

  • Staphylococcus aureus: No growth (Gram-positive inhibited).

MSA (Mannitol Salt Agar)

• Purpose: Selective for Staphylococcus species due to high salt concentration; differential for mannitol fermentation.

• Key Ingredients: 7.5% NaCl (selective), mannitol (differential), phenol red (pH indicator).

• Results:

  • Staphylococcus aureus: Growth with yellow color (ferments mannitol, acid production turns indicator yellow).

  • Staphylococcus epidermidis: Growth with no color change (does not ferment mannitol).

  • Micrococcus luteus: No growth (not salt-tolerant).

Blood Agar

• Purpose: Enrichment and differential medium for hemolysis patterns.

• Key Feature: Contains 5% sheep blood to detect hemolytic activity.

• Types of Hemolysis:

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

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

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

• Special Note: 'Fish eye' colonies of Enterobacter may be observed.

Antibiotic Sensitivity Testing

Principle and Procedure

Antibiotic sensitivity testing determines the susceptibility of bacteria to various antibiotics using the disk diffusion method (Kirby-Bauer test).

• Procedure: Antibiotic-impregnated disks are placed on an agar plate inoculated with the test organism. After incubation, zones of inhibition (clear areas) around the disks indicate sensitivity.

• Interpretation: Measure the diameter of the zone of inhibition and compare to standard tables to classify 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 tables

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

Biochemical Tests for Bacterial Identification

The Catalase Test

• Principle: Detects the enzyme catalase, which converts hydrogen peroxide to water and oxygen.

• Purpose: Differentiates Staphylococcus (catalase positive) from Streptococcus (catalase negative).

• Procedure:

  1. Place a small amount of bacterial colony on a glass slide.

  2. Add 1-2 drops of 3% hydrogen peroxide.

  3. Observe for rapid bubble formation (positive) or no bubbles (negative).

• Possible False Positives: Reversed procedure order or contamination with red blood cells.

The Oxidase Test

• Principle: Detects cytochrome c oxidase enzyme, present in Micrococcus but not Staphylococcus.

• Purpose: Differentiates Micrococcus (oxidase positive) from Staphylococcus (oxidase negative).

• Procedure:

  1. Moisten test strip with distilled water.

  2. Rub a small amount of bacteria onto the strip.

  3. Observe for blue to dark purple color (positive) or no color change (negative).

Durham Sugar Tube Fermentation

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

• Key Components: Peptone broth, single carbohydrate, phenol red indicator, Durham tube for gas collection.

• Results:

  • Red: No fermentation (negative).

  • Yellow: Acid production (positive).

  • Yellow with bubble: Acid and gas production (positive).

IMViC Tests

• Components: Indole, Methyl Red, Voges-Proskauer, Citrate utilization tests.

• Purpose: Used to differentiate members of the family Enterobacteriaceae.

Indole Test (part of SIM medium)

• Principle: Detects tryptophanase enzyme that converts tryptophan to indole.

• Positive Result: Red ring after adding Kovac's reagent.

Methyl Red Test

• Principle: Detects stable acid production from glucose fermentation.

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

• Results: Red = positive (mixed acid fermentation); yellow = negative.

Voges-Proskauer Test

• Principle: Detects acetoin, a neutral end product of glucose fermentation (butanediol pathway).

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

• Results: Red = positive for acetoin; yellow/orange = negative.

Citrate Utilization Test (Simmon's Citrate Agar)

• Principle: 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.

Sulfur Indole Motility (SIM) Medium

• Purpose: Differential medium for sulfur reduction, indole production, and motility.

• Key Reactions:

  • Sulfur reduction: Black precipitate indicates H2S production.

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

  • Motility: Diffuse growth radiating from stab line indicates motility.

Starch Hydrolysis Test

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

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

• Results:

  • Bacillus subtilis: Positive (clearing).

  • Escherichia coli: Negative (no clearing).

  • Proteus vulgaris: Variable.

Gelatinase Test

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

• Procedure: Inoculate nutrient gelatin, incubate, refrigerate. Liquid after refrigeration indicates positive.

• Results:

  • Serratia marcescens: Positive.

  • Escherichia coli: Negative.

  • Proteus vulgaris: Positive.

Casein Hydrolysis Test

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

• Results:

  • Bacillus subtilis: Positive.

  • Pseudomonas aeruginosa: Positive.

  • Escherichia coli: Negative.

Urease Test

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

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

• Results:

  • Bright pink: Positive (alkaline, rapid hydrolysis).

  • No color change/yellow: Negative.

  • Klebsiella pneumoniae, Proteus vulgaris: Positive.

  • Escherichia coli: Negative.

Staining Techniques in Microbiology

Principle of Staining

Bacterial cells are typically colorless and transparent, making them difficult to observe under a microscope. Staining techniques are used to color cells, enhancing visibility and allowing for the determination of cell shape, size, and arrangement.

• Stains: Chemical compounds that bind to cellular components.

• Types of Dyes:

  • Basic dyes: Colored cation + colorless anion (e.g., methylene blue, crystal violet).

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

• Bacterial cells: 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 visualize cell morphology and arrangement.

• Differential Staining: Uses two contrasting stains separated by a decolorizing agent to distinguish between cell types or structures.

  • Examples: Gram stain, acid-fast stain, spore stain, capsule stain.

Smear Preparation and Fixation

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

• Steps: Spread a thin layer of bacteria, air dry, and 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, appears purple.

  • Gram-negative: Thin peptidoglycan, loses crystal violet, takes up safranin, appears red/pink.

• Examples:

  • Staphylococcus aureus: Gram-positive cocci, violet.

  • Candida albicans: Gram-positive oval, violet.

  • Bacillus subtilis: Gram-positive bacilli, violet.

  • Gram-negative bacilli: Single rods, 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, cover with filter paper, steam for 10 minutes.

  3. Rinse, counterstain with safranin for 1 minute.

  4. Rinse, blot dry, observe under oil immersion.

• Results: Spores appear green; vegetative cells appear red or pink.

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

Table: Examples of Endospore-Forming Pathogens

Genus	Disease
Clostridium botulinum	Botulism
Clostridium perfringens	Gas gangrene
Clostridium tetani	Tetanus
Bacillus anthracis	Anthrax (Woolsorter's Disease)

Additional info: The location (central, subterminal, terminal) and shape (swollen, not swollen) of the spore are useful for identification.