Microbiology Laboratory Media and Organism Identification Guide

PEA Media (Phenylethyl Alcohol Agar)

• Definition and Classification: Phenylethyl Alcohol Agar (PEA) is a selective medium, not differential, used in microbiology to isolate Gram-positive bacteria, especially within clinical samples.
• Key Selective Ingredient:     * Contains Phenylethyl Alcohol (PEA).     * Mechanism of Action: It inhibits the growth of Gram-negative organisms by disrupting their cell membrane integrity and interfering with their DNA synthesis.
• Primary Application: Used to isolate Gram-positive cocci, specifically Staphylococcus and Streptococcus species.
• Growth Patterns:     * Gram-positive bacteria thrive on PEA.     * Gram-negative bacteria (such as Escherichia coli or Proteus species) are inhibited or exhibit extremely limited growth, facilitating the focus on Gram-positive organisms in mixed cultures.
• Interpretation of Results:     * Growth Present: Significant growth suggests the organism is likely a Gram-positive bacterium (e.g., Gram-positive cocci like Staphylococcus and Streptococcus, or some Gram-positive rods).     * No Growth or Inhibited Growth: Minimal or absent growth suggests the organism is a Gram-negative bacterium. Examples include Escherichia coli, Proteus, and Pseudomonas species. These may show very sparse or inhibited colonies if any growth occurs at all.

Mannitol Salt Agar (MSA)

• Definition and Classification: Both a selective and differential medium utilized for the isolation and identification of Staphylococcus species, particularly Staphylococcus aureus.
• Key Characteristics:     * Selective Ingredient: Contains $7.5\%$ Sodium Chloride ($NaCl$). This high-salt environment inhibits most bacteria while allowing halotolerant organisms, specifically Staphylococcus species, to grow.     * Differential Ingredient: Contains Mannitol (a sugar alcohol) and Phenol Red (a pH indicator).
• Interpretation of Results (Mannitol Fermentation):     * Positive Result: If an organism ferments mannitol, acidic byproducts are produced, lowering the pH. This causes the medium color to change from red to yellow. This is typical for Staphylococcus aureus.     * Negative Result: Non-fermenters, such as Staphylococcus epidermidis, do not produce acid; consequently, the medium remains red or pink around the colonies.
• Salt Tolerance: Only halotolerant organisms grow on MSA. It is specifically useful for distinguishing S. aureus (yellow colonies) from other Staphylococcus species that do not ferment mannitol.

Eosin Methylene Blue (EMB) Agar

• Definition and Classification: A selective and differential medium used to isolate and differentiate Gram-negative enteric bacteria, particularly those in the family Enterobacteriaceae.
• Key Characteristics:     * Selective Ingredients: Eosin Y and Methylene Blue dyes. These inhibit Gram-positive growth, permitting only Gram-negative organisms to grow.     * Differential Ingredients: Lactose is the primary carbohydrate. Eosin and Methylene Blue serve as pH indicators that respond to acidic byproducts of lactose fermentation.
• Interpretation of Results:     * Strong Lactose Fermenters: Organisms like Escherichia coli produce high levels of acid, resulting in dark purple colonies with a characteristic metallic green sheen.     * Moderate Lactose Fermenters: Organisms like Enterobacter aerogenes produce pink to purple colonies without the metallic green sheen, indicating less acid production than E. coli.     * Non-Lactose Fermenters: Organisms such as Salmonella and Shigella species produce colorless or translucent colonies. They may appear the same color as the medium or slightly amber due to the lack of acid.     * Inhibited Growth: Gram-positive organisms are typically inhibited by the dyes. Any growth that occurs will be limited or weak.

Thioglycolate Broth

• Definition and Classification: An enriched, differential medium designed to support a wide range of bacteria and determine an organism's specific oxygen ($O_2$) requirements.
• Key Characteristics:     * Oxygen Gradient: Contains Sodium Thioglycolate, a reducing agent that lowers oxygen content. This creates a gradient from aerobic ($O_2$ rich) at the top to anaerobic (no $O_2$) at the bottom.     * Indicator: Resazurin, an oxygen-sensitive dye. It turns pink in the presence of $O_2$ (top of the tube) and remains colorless in anaerobic conditions (deeper in the tube).     * Broad Support: Supports both fastidious and non-fastidious organisms across all oxygen tolerance levels.
• Interpretation of Results (Oxygen Requirements):     * Obligate Aerobes: Grow only at the top of the broth. Examples: Pseudomonas aeruginosa, Mycobacterium tuberculosis.     * Obligate Anaerobes: Grow only at the bottom of the tube. Examples: Clostridium perfringens, Bacteroides fragilis.     * Facultative Anaerobes: Grow throughout the tube, but more growth occurs at the top because they prefer oxygen for respiration. Examples: Escherichia coli, Staphylococcus aureus.     * Microaerophiles: Grow just below the surface where $O_2$ levels are lower than atmospheric but still present. Examples: Helicobacter pylori, Campylobacter jejuni.     * Aerotolerant Anaerobes: Grow evenly throughout the broth; they do not require $O_2$ and are unaffected by its presence. Examples: Lactobacillus spp., Streptococcus spp.

Phenol Red Broths

• Description: Includes Phenol Red Glucose, Phenol Red Lactose, and Phenol Red Sucrose. These are differential media used to test ability to ferment specific carbohydrates and produce gas.
• Key Characteristics:     * Carbohydrate Specificity: Each tube contains a single sugar (glucose, lactose, or sucrose).     * pH Indicator: Phenol Red. It is yellow at acidic pH ($pH < 6.8$) and red to pink at neutral or alkaline pH ($pH > 6.8$).     * Durham Tube: A small inverted vial inside the tube to capture gas produced during fermentation.
• Interpretation of Results:     * Acid Production (Positive Fermentation): Medium turns yellow, indicating fermentation of the specific carbohydrate.     * Gas Production: A bubble is present in the Durham tube, indicating gas was a byproduct of fermentation.     * No Fermentation (Negative): The medium remains red or turns pink.     * Alkaline Reaction: Medium turns deep pink, meaning the organism metabolized peptones instead of the carbohydrate, producing alkaline byproducts.

SIM (Sulfide, Indole, Motility) Medium

• Definition: A multi-purpose differential medium used to detect three bacterial traits. It is especially useful for distinguishing members of Enterobacteriaceae (e.g., Escherichia coli vs. Proteus vulgaris).
• Key Characteristics:     * Hydrogen Sulfide ($H_2S$) Production: Medium contains sodium thiosulfate and iron salts. Sulfur reduction produces $H_2S$ gas, which reacts with iron to form black precipitates (iron sulfide).     * Indole Production: Contains tryptophan. Some bacteria break this down into indole. Detection requires the addition of Kovacs' reagent.     * Motility: The medium is semi-solid, allowing motile bacteria to move away from the initial stab line.
• Interpretation of Results:     * H2S Production: Positive result is indicated by blackening of the medium; negative is no blackening.     * Indole Production: Positive result is a red or pink layer appearing at the top after adding Kovacs' reagent; negative is no color change.     * Motility: Positive is diffuse, cloudy growth extending from the stab line; negative is growth restricted to the stab line.

Nitrate Broth

• Definition: A differential medium used to detect nitrate reduction ($NO_3$ to $NO_2$ or $N_2$ gas) during anaerobic respiration. Used primarily for identifying Enterobacteriaceae and anaerobic bacteria.
• Key Characteristics:     * Nitrate Source: Potassium nitrate ($KNO_3$).     * Reagents: Reagent A (sulfanilic acid) and Reagent B (alpha-naphthylamine).
• Interpretation of Results:     * Step 1 (Add Reagents A and B): If the broth turns red, it is positive for nitrate reduction to nitrite. If no color change, proceed to Step 2.     * Step 2 (Add Zinc Dust):         * Red Color After Zinc: Negative for nitrate reduction. Zinc reduced the remaining nitrate to nitrite artificially.         * No Color Change After Zinc: Positive for complete nitrate reduction. The organism reduced nitrate beyond nitrite to nitrogen gas ($N_2$) or other compounds, leaving no nitrate for the zinc to react with.

Simmons Citrate Agar

• Definition: A selective and differential medium (part of the IMViC series) used to determine if an organism can use citrate as its sole carbon source.
• Key Characteristics:     * Sole Carbon Source: Sodium citrate.     * Sole Nitrogen Source: Ammonium dihydrogen phosphate.     * pH Indicator: Bromothymol blue. It is green at neutral pH ($pH \approx 6.9$) and blue at alkaline pH ($pH \ge 7.6$).
• Interpretation of Results:     * Positive: The medium changes to blue. Any visible growth, even without clear color change, is considered a positive result.     * Negative: No growth and the medium remains green.

MRVP Broth (Methyl Red and Voges-Proskauer)

• Definition: A differential medium used to identify glucose fermentation pathways. It differentiates between mixed-acid fermentation and the butylene glycol fermentation pathway.
• Methyl Red (MR) Test:     * Detects stable acid end products.     * Positive: Red color after adding methyl red indicator (indicates mixed-acid fermentation, e.g., E. coli).     * Negative: Yellow or no color change.
• Voges-Proskauer (VP) Test:     * Detects acetoin (neutral end product).     * Procedure: Add Reagent A (alpha-naphthol) and Reagent B (KOH); wait $10\text{--}30$ minutes.     * Positive: Red color (indicates butylene glycol pathway, e.g., Enterobacter aerogenes).     * Negative: No color change or a copper color.

Urea Broth

• Definition: A differential medium to detect the production of the enzyme urease, which hydrolyzes urea into ammonia ($NH_3$) and carbon dioxide ($CO_2$).
• Mechanism: Urease-positive organisms produce ammonia, raising the pH.
• pH Indicator: Phenol Red. Yellow/orange at neutral ($pH \approx 6.8$); bright pink/fuchsia at alkaline (pH > 8.1).
• Interpretation of Results:     * Positive: Bright pink/fuchsia (e.g., Proteus mirabilis, Helicobacter pylori).     * Negative: Yellow or orange (e.g., Escherichia coli).     * Weakly Positive: Pale pink.

Phenylalanine Agar Slant

• Definition: Also known as the Phenylalanine Deaminase Test; identifies bacteria that produce phenylalanine deaminase.
• Mechanism: The enzyme catalyzes the deamination of phenylalanine to produce phenylpyruvic acid.
• Reagent: Ferric chloride ($FeCl_3$) is added after incubation.
• Interpretation of Results:     * Positive: Green color on the slant after adding reagent (e.g., Proteus, Providencia, Morganella).     * Negative: No color change or a yellowish color (e.g., E. coli, Klebsiella pneumoniae).

Litmus Milk

• Definition: A complex differential medium used to assess metabolism of milk components (lactose and casein).
• Indicators: Litmus serves as a pH and redox indicator.     * pH: Pink (Acidic, $pH < 4.5$); Blue (Alkaline, $pH > 8.3$); Purple (Neutral, $pH \approx 6.8$).     * Redox: White at the bottom indicates a reduced environment.
• Metabolic Reactions:     * Lactose Fermentation: Pink color; may include an acid clot (firm) and gas (cracks/fissures called "stormy fermentation").     * Alkaline Reaction: Blue color from protein digestion (peptone utilization) and ammonia release.     * Reduction: Litmus turns white due to enzymatic activity in anaerobic conditions.     * Coagulation: Acid Clot (firm) or Rennet Clot (soft, enzymatic).     * Peptization: Clearing of the medium as casein is digested.

Spirit Blue Plates

• Definition: A differential medium used to detect lipase production, which hydrolyzes lipids into glycerol and fatty acids.
• Key Characteristics: Lipid substrate (e.g., tributyrin) and Spirit blue dye indicator.
• Interpretation of Results:     * Positive: A clear halo/zone around growth and sometimes a darker blue intensification around colonies (e.g., Bacillus subtilis, S. aureus).     * Negative: No clear zone; medium remains uniform (e.g., E. coli).

Lysine Decarboxylase Test

• Definition: Detects the enzyme lysine decarboxylase, which converts lysine to cadaverine (alkaline).
• Conditions: Must be anaerobic; the tube is overlaid with mineral oil.
• Indicator: Bromocresol purple: yellow (Acidic, $pH < 5.2$); purple (Alkaline, $pH > 6.8$).
• Process: Initial glucose fermentation creates an acidic environment (yellow) that activates the decarboxylase enzyme, which then raises the pH (back to purple).
• Interpretation:     * Positive: Purple (e.g., Salmonella spp., Klebsiella pneumoniae).     * Negative: Yellow (acid produced from glucose but no decarboxylation).

Catalase and Oxidase Tests

• Catalase Test: Determines the presence of catalase enzyme which breaks down hydrogen peroxide ($H_2O_2$) into water ($H_2O$) and oxygen ($O_2$).     * Positive: Immediate bubbling (e.g., Staphylococcus).     * Negative: No bubbles (e.g., Streptococcus).
• Oxidase Test: Detects cytochrome c oxidase in the electron transport chain.     * Reagent: Tetramethyl-p-phenylenediamine dihydrochloride.     * Interpretation: Must be read before $30$ seconds.     * Positive: Purple or dark blue (e.g., Pseudomonas aeruginosa, Neisseria).     * Negative: No color change (e.g., Escherichia coli).

Triple Sugar Iron (TSI) Test

• Composition: Glucose ($0.1\%$), Lactose ($1.0\%$), Sucrose ($1.0\%$), Phenol Red, Sodium Thiosulfate, and Ferrous Sulfate.
• Slant/Butt Interpretations:     * Yellow Slant/Yellow Butt (A/A): Fermentation of glucose and lactose/sucrose (e.g., E. coli).     * Red Slant/Yellow Butt (K/A): Glucose fermentation only (e.g., Shigella, Salmonella).     * Red Slant/Red Butt (K/K): No sugar fermentation; peptones used (e.g., P. aeruginosa).     * Black Precipitate: $H_2S$ production (e.g., Salmonella, Proteus).     * Cracks/Bubbles: Gas production.

Profiles of Specific Microorganisms

Proteus vulgaris

• PEA: Growth (weakly tolerates PEA).
• MSA: No growth (salt intolerant, non-mannitol fermenter).
• EMB: Growth; colonies colorless/pink (non-lactose fermenter).
• Thioglycolate: Facultative anaerobic.
• SIM: Positive for Sulfide ($H_2S$), Indole, and Motility.
• Nitrate: Positive.
• Citrate: Negative.
• Urea/Phenylalanine: Positive (rapid urease and deaminase producer).
• Spirit Blue: Positive.
• Other: Glucose/Sucrose Fermenter; Catalase Positive; Oxidase Negative.

Serratia marcescens

• PEA/MSA: Poor or no growth.
• EMB: Growth; colorless/light pink.
• Thioglycolate: Facultative anaerobic.
• Nitrate/Citrate/Lysine: Positive.
• MR/VP: Both Positive (mixed-acid and acetoin production).
• SIM: Motility positive; H2S/Indole negative.
• Urea: Variable (weak or absent).

Staphylococcus aureus

• PEA: Growth (Gram-positive).
• MSA: Growth; Yellow colonies (Halotolerant, mannitol fermenter).
• EMB: No growth (inhibited).
• SIM: Negative for H2S, Indole, and Motility.
• MRVP: MR Positive; VP Negative.
• Litmus Milk: Acidic reaction with coagulation.
• Catalase: Positive (characteristic for Staphylococci).

Escherichia coli

• EMB: Growth with metallic green sheen (vigorous lactose fermentation).
• SIM: Indole Positive; Motility Positive; H2S Negative.
• Nitrate/Lysine: Positive.
• Citrate/Urea/Phenylalanine/Lipase: Negative.
• MRVP: MR Positive; VP Negative.
• Fermentation: Glucose/Lactose Positive with gas; Sucrose Negative.

Enterobacter aerogenes

• EMB: Growth; pink to purple colonies.
• MRVP: MR Negative; VP Positive (produces acetoin).
• Citrate/Lysine: Positive.
• Urea: Negative.
• Fermentation: Glucose/Lactose/Sucrose Positive.

Citrobacter freundii

• EMB: Growth; dark colonies, variable green sheen.
• SIM: H2S Positive; Motility Positive; Indole variable.
• MRVP: MR Positive; VP variable.
• Urea: Positive (slower than Proteus).
• Citrate: Positive.
• Nitrate: Positive.