After isolating a pure culture, the next step is identification. Traditional biochemical tests assess metabolic capabilities, while modern automated systems accelerate and standardize the process.

Core Biochemical Tests

• Catalase: detects the enzyme catalase, which breaks down hydrogen peroxide. A drop of 3% H₂O₂ on a colony produces bubbles (positive) within seconds. Staphylococci are catalase-positive; streptococci are catalase-negative.
• Oxidase: detects cytochrome c oxidase. A colony smeared on a filter paper impregn with oxidase reagent (tetramethyl-p-phenylenediamine) turns dark purple within 10–30 seconds (positive). Neisseria and Pseudomonas are oxidase-positive; Enterobacteriaceae are oxidase-negative.
• Coagulase: distinguishes Staphylococcus aureus (positive) from other staphylococci. Free coagulase is detected by mixing a colony with rabbit plasma and checking for clot formation at 4–24 hours.
• Indole: some bacteria (E. coli among others) produce indole from tryptophan. Indole reacts with Kovac’s reagent (p-dimethylaminobenzaldehyde) to produce a red layer.
• Methyl red / Voges–Proskauer (MR/VP): MR detects mixed acid fermentation (red with methyl red indicator). VP detects the butanediol fermentation pathway (pink/red with α-naphthol and KOH). Classic for E. coli (MR⁺, VP⁻) vs. Enterobacter / Klebsiella (MR⁻, VP⁺).
• Citrate utilization: bacteria that use citrate as the sole carbon source produce ammonia, raising pH and turning bromothymol blue from green to blue. Klebsiella pneumoniae is positive; E. coli is negative.
• TSI (Triple Sugar Iron) agar: a slant that detects glucose, lactose, and sucrose fermentation, gas production, and H₂S production. The pattern of acid/alkaline reactions and black precipitate is distinctive for enteric bacteria.

Miniaturized Systems (API Strips)

The API (Analytical Profile Index) system miniaturizes 20–50 biochemical tests in a plastic strip. Each test is a dehydrated substrate in a cupule. The strip is inoculated with a standardized bacterial suspension, incubated for 18–24 hours, and reactions are read based on color changes. The resulting 7- or 9-digit profile number is looked up in a database to identify the organism.

Automated Systems

• VITEK (bioMérieux): uses plastic cards containing 30–60 biochemical reactions. A bacterial suspension is loaded into the card, which is sealed and read automatically every 15 minutes. Identification is generated in 2–8 hours. VITEK 2 also provides antimicrobial susceptibility testing (AST).
• MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry): the most rapid identification method available. A bacterial colony is spotted onto a target plate, overlaid with a matrix (α-cyano-4-hydroxycinnamic acid), and analyzed by mass spectrometry. The resulting protein spectrum (mainly ribosomal proteins) is compared to a database, providing species-level identification in under a minute per sample.

MALDI-TOF has largely replaced biochemical testing in clinical microbiology laboratories because of its speed, accuracy, and low consumable cost after instrument acquisition. It identifies bacteria, yeast, and fungi from a single colony without requiring prior knowledge of the organism.