Overview
Once bacteria have been isolated in pure culture, the next essential step in the diagnostic microbiology workflow is identification down to the genus and species level. Correct identification allows the clinician to select the appropriate antibiotic and is also vital for epidemiological surveillance and outbreak tracing.
This lesson walks through the full identification ladder used in a clinical microbiology laboratory — from simple staining reactions, through the classic biochemical tests (catalase, coagulase, oxidase, indole, citrate, urease), to serology, phage typing, and the modern automated and molecular identification systems.
Learning Objectives
After this lesson you will be able to…- Describe the processes involved in the identification of bacteria.
- Explain the significance of microscopy in the process of identification of bacteria.
- Explain the significance of biochemical tests in the process of identification of bacteria.
- Describe the significance of serology in the process of identification of bacteria.
- Describe the significance of phage typing in the identification of bacteria.
- Explain the significance of antimicrobial susceptibility testing in bacterial identification.
Clinical Story
Why This MattersA pus swab from an infected surgical wound grows creamy, golden colonies on blood agar with a wide zone of clear hemolysis. Before the physician can prescribe the right antibiotic, the lab technologist must confirm whether this is Staphylococcus aureus or a look-alike coagulase-negative species — a distinction made in minutes with a simple catalase and coagulase test.
Core Concepts
Staining forms the foremost and most important step in bacterial identification.
- Gram staining — differentiates bacteria into Gram positive (staphylococci, streptococci, pneumococci) and Gram negative (E.coli, Klebsiella, Salmonella, Shigella) groups.
- Albert staining — used when Corynebacterium spp. is suspected (shows metachromatic granules).
- Acid-fast staining — used for suspected mycobacterial infections such as tuberculosis and leprosy.
- Special staining — required for spirochetes and other unusual organisms.
Motility is assessed by preparing a wet mount and observing under the microscope, or by inoculating the organism into a semisolid motility medium and observing for spreading growth away from the stab line.
A battery of biochemical reactions narrows down bacterial identity. The commonly used tests are: catalase, coagulase, oxidase, sugar fermentation, indole, citrate, and urease. Each is described in detail in the Laboratory Principle and Procedure sections below.
Serology detects antigens by enzyme or fluorescence immunoassay and is used to confirm identification obtained by other methods (e.g. slide agglutination typing of Salmonella and Vibrio cholerae).
Phage typing detects single strains of bacteria using strain-specific bacteriophages, and is invaluable for tracing the source of infection outbreaks.
Identification discs — the Kirby-Bauer disc diffusion method is primarily used for antibiotic susceptibility but can also help identify organisms such as Micrococci, Streptococci and Moraxella spp.
Semiautomated and automated identification systems (Microscan Walkaway, Vitek, Sensititre, Phoenix) identify bacteria and simultaneously perform antibiotic susceptibility testing. The Bactec AFB system and MGIT / MGIT 960 are used for mycobacterial identification.
Molecular methods include G+C% content, DNA-DNA hybridisation, DNA base sequencing, and amplification techniques such as PCR, ligase chain reaction, strand displacement amplification, and nucleic acid sequence-based amplification — used for direct detection of organisms like Neisseria gonorrhoeae and Leptospira.
Laboratory Principle
Catalase test: detects the enzyme catalase, which splits hydrogen peroxide into water and oxygen (visible as bubbles). It separates catalase-positive Micrococcaceae (e.g. Staphylococcus) from catalase-negative Streptococcaceae (e.g. Streptococcus).
Coagulase test: detects staphylocoagulase, which clots plasma by activating prothrombin (bound coagulase causes direct clumping in the slide test; free coagulase clots plasma in the tube test) — the definitive marker of Staphylococcus aureus.
Oxidase test: detects cytochrome oxidase, which oxidises a colourless reagent (Kovács reagent) to a dark purple coloured product within seconds in positive organisms such as Pseudomonas and Vibrio cholerae.
Indole test: detects the enzyme tryptophanase, which degrades tryptophan to indole; indole reacts with Kovác's reagent to form a pink "cherry-red ring".
Citrate test: detects the ability of an organism to use citrate as its sole carbon source, raising the pH of Simmons citrate medium and turning the bromothymol blue indicator from green to Prussian blue.
Urease test: detects urease, which hydrolyses urea to ammonia and CO₂, raising the pH of Christensen's urea agar and turning it bright pink (fuchsia).
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Hydrogen peroxide | 3% H₂O₂ | Catalase test | Cool, dark bottle, room temperature |
| Rabbit / human plasma | EDTA, oxalate, or heparin anticoagulated | Coagulase test (slide & tube) | Refrigerated (2–8°C) |
| Kovács oxidase reagent | 1% solution | Oxidase test (filter paper method) | Refrigerated, protect from light |
| Kovác's indole reagent | Standard formulation | Indole test | Room temperature, protect from light |
| Simmons citrate agar | Bromothymol blue indicator | Citrate utilisation test | Refrigerated slants, 2–8°C |
| Christensen's urea agar | Phenol red indicator | Urease test | Refrigerated slants, 2–8°C |
Step-by-Step Procedure
Place a drop of 3% H₂O₂ on a microscope slide inside a petri dish. Using a sterile loop, pick a well-isolated 18–24 hour colony (avoid picking up any agar or red blood cells) and touch it into the peroxide. Cover with the petri dish lid and observe immediately for bubble formation (O₂ + water). Immediate bubbling = catalase positive.
Emulsify the isolate in a drop of saline on a slide, check for absence of autoagglutination, then add a drop of rabbit/human plasma and mix. Prompt clumping within 10 seconds = positive for bound coagulase (may give false negatives — confirm with tube test).
Mix ~0.1 mL of an overnight broth culture with ~0.5 mL of plasma in a narrow test tube. Incubate at 37°C and examine at intervals up to 24 hours. Formation of any size clot that does not flow on tilting = positive (definitive test for free coagulase).
Soak filter paper in 1% Kovács oxidase reagent and let dry. Rub a fresh (18–24 hour) colony onto the treated paper using a loop. A colour change to dark purple within 5–10 seconds = oxidase positive; purple within 60–90 seconds = delayed positive; no change or >2 minutes = negative.
Inoculate tryptone broth (indole), Simmons citrate slant (citrate), and Christensen's urea agar slant (urease) each with a pure culture. Incubate at 35–37°C. Read indole after adding Kovác's reagent (cherry-red ring = positive); read citrate for growth and colour change to blue (positive); read urease for pink/fuchsia colour on the slant (positive).
Flow Diagram
Quality Control
Every batch of biochemical media/reagents must be tested with known positive and negative control strains (e.g. Staphylococcus aureus ATCC 25923 for coagulase-positive, Staphylococcus epidermidis for coagulase-negative) before use on patient isolates. Discard any H₂O₂ that fails to bubble with a known catalase-positive control.
Laboratories should enrol in an external quality assurance (proficiency testing) scheme in which blinded reference strains are periodically sent for identification, allowing inter-laboratory comparison of identification accuracy.
Reference Values
Typical Reactions⚠️ Reactions can vary between strains and species. Always confirm with positive/negative controls run in parallel.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Catalase positive + Coagulase positive | Suggests Staphylococcus aureus — a virulent pyogenic pathogen | Proceed to antibiotic susceptibility testing (see Lesson 12) |
| Catalase negative, chain-forming cocci | Suggests Streptococcus spp. | Perform hemolysis pattern and Lancefield grouping |
| Oxidase positive, Gram negative rod, non-fermenter | Suggests Pseudomonas aeruginosa — common nosocomial pathogen | Report urgently; guide targeted anti-pseudomonal therapy |
| Indole positive, citrate negative, lactose fermenter | Classic pattern of E. coli | Correlate with clinical picture (UTI, wound, GI) |
| Urease positive within 1–6 hours | Suggests Proteus spp. — associated with struvite renal calculi | Consider urease-related complications in UTI patients |
Common Errors & How to Avoid Them
Cause: Picking up red blood cells from blood agar along with the colony can cause a false bubble reaction.
Prevention: Pick colonies carefully avoiding underlying agar/blood cells; use a non-blood-containing medium colony when possible.
Cause: Some S. aureus strains produce free coagulase but lack bound coagulase, giving a negative slide test.
Prevention: Always confirm a negative slide coagulase with the tube test before ruling out S. aureus.
Cause: Reading the oxidase colour change too late (after 2 minutes) can give a false positive due to auto-oxidation of the reagent.
Prevention: Time the reaction precisely and read within the first 10–60 seconds; use a nichrome/platinum loop, not iron, since iron can cause false-positive reactions.
Laboratory Tips from the Bench
Always run known positive and negative control organisms alongside every batch of biochemical tests — this is the single most reliable way to catch a bad reagent lot before it leads to a misidentification.
Observe the catalase bubble reaction against a dark background — it dramatically improves readability of small bubble formation.
Remember "CCOICU" for the six classic biochemical tests: Catalase, Coagulase, Oxidase, Indole, Citrate, Urease — the core identification ladder for Gram positive cocci and Enterobacteriaceae.
Important Notes
When the slide and tube coagulase tests disagree, the tube test result is always the deciding factor since it detects free coagulase directly, while the slide test can occasionally miss it.
PCR-based and other molecular identification techniques are not used routinely in most hospital laboratories due to cost, but are invaluable for fastidious or slow-growing organisms such as Neisseria gonorrhoeae and Leptospira.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgePresents with a painful, red, swollen abscess on the forearm following a minor injury at a construction site, three days ago. Fever 38.9°C. Pus is draining from the lesion.
Gram positive cocci in clusters that are catalase-positive, coagulase-positive and mannitol-fermenting are diagnostic of Staphylococcus aureus — the classic cause of localized pyogenic abscesses.
- →Coagulase positivity is the single most reliable marker distinguishing S. aureus from other staphylococci.
- →Antibiotic susceptibility testing should always follow identification (see Lesson 12) as MRSA is common.
- →Localized abscess formation is a hallmark of staphylococcal infection, unlike the spreading nature of streptococcal disease.
Frequently Asked Questions
The slide test is rapid (results in 10 seconds) but can give false negatives because it only detects bound coagulase. The tube test detects free coagulase and is considered the gold standard, though it can take up to 24 hours.
Bubble formation can be subtle, especially with weak catalase producers. A dark background improves contrast, making tiny bubbles easier to visualize and reducing the chance of a false-negative reading.
Not entirely. Molecular techniques (PCR, sequencing) are increasingly used, especially for fastidious or slow-growing organisms, but classic biochemical tests remain the routine backbone of most hospital laboratories due to their low cost and simplicity.
Quick Revision
10-Minute ReviewKey Takeaways
- Bacterial identification proceeds from staining, through motility and biochemical testing, to serology and molecular methods where needed.
- Gram, Albert, and acid-fast staining each target different organism groups.
- The catalase, coagulase, oxidase, indole, citrate and urease tests form the core biochemical identification ladder.
- Serology and phage typing confirm identification and support epidemiological investigation.
- Automated and molecular systems are increasingly used alongside classic biochemical methods.
- Antibiotic susceptibility testing always follows successful identification.
Competency Checklist
Track Your MasteryReferences
- Collee JG, Fraser AG, Marmion BP, Simmons A. Mackie & McCartney Practical Medical Microbiology. 14th ed. Churchill Livingstone.
- Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott's Diagnostic Microbiology. 13th ed. Mosby Elsevier.
- National Institute of Open Schooling (NIOS). Microbiology — Bacterial Identification Tests, Module Notes.