Unit 2: Biochemical identification tests
Systemic Bacteriology notes · PTU syllabus (BMLS201-18)
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Unit summary
Biochemical tests identify bacteria by what they metabolise. This unit covers the principle, procedure and interpretation of catalase, coagulase, indole, methyl red, Voges–Proskauer, urease, citrate, oxidase, triple sugar iron, nitrate reduction, carbohydrate fermentation, Hugh and Leifson, bile solubility, H₂S production and motility tests.
After this unit you can
- Perform catalase, coagulase and oxidase tests
- Perform the IMViC tests
- Perform urease, TSI, nitrate, sugar fermentation and O-F tests
- Perform bile solubility, H₂S and motility tests
PTU syllabus topics
- Principle
- procedure and interpretation of catalase
- coagulase
- indole
- methyl red
- Voges-Proskauer
- urease
- citrate
- oxidase
- TSI
- nitrate reduction
- carbohydrate fermentation
- Hugh and Leifson
- bile solubility
- H2S production and motility tests
Catalase
Bubbles with H2O2
Staphylococcus (Streptococcus is negative)
Coagulase
Plasma clots
Staphylococcus aureus
Indole
Red ring with Kovac's reagent
E. coli
Urease
Pink colour
Proteus
Oxidase
Purple colour
Pseudomonas, Vibrio
Topic 1
Catalase test
- Principle: catalase breaks hydrogen peroxide into water and oxygen. Procedure: touch a colony to a drop of 3% H₂O₂ on a slide. Positive: brisk bubbles — Staphylococcus (positive) vs Streptococcus (negative).
Exam tip
Do not take colonies from blood agar if possible — red cells contain catalase and give false positives.
Topic 2
Coagulase test
Detects
Bound coagulase (clumping factor)
Free coagulase
Procedure
Emulsify colony in saline, add a drop of plasma
0.5 mL plasma + colony; incubate at 37 °C, read at 4 and 24 hours
Positive
Clumping in 10 seconds
Clot formation
Significance
Staphylococcus aureus is positive; coagulase-negative staphylococci (S. epidermidis) are negative
Confirms doubtful slide tests
Topic 3
Oxidase test
- Principle: organisms with cytochrome c oxidase oxidise tetramethyl-p-phenylenediamine to a purple compound. Procedure: rub a colony on oxidase reagent paper with a platinum loop or stick (not nichrome). Positive: deep purple within 10 seconds — Pseudomonas, Vibrio, Neisseria; Enterobacteriaceae are negative.
Topic 4
The IMViC tests
Indole
Tryptophanase splits tryptophan to indole; Kovac's reagent after 48 hours in peptone water
Red ring
Methyl red (MR)
Mixed acid fermentation keeps pH below 4.4; methyl red indicator
Red colour
Voges–Proskauer (VP)
Acetoin from butanediol fermentation; α-naphthol and 40% KOH (Barritt's)
Pink–red in 15 minutes
Citrate
Uses citrate as sole carbon source; Simmons citrate agar with bromothymol blue
Growth and blue colour
Example
E. coli: + + − − ; Klebsiella pneumoniae: − − + +.
Topic 5
Urease test
- Principle: urease splits urea into ammonia and CO₂; ammonia raises pH, turning phenol red pink. Medium: Christensen's urea agar slope. Positive: pink — Proteus (rapid, within 4 hours), Klebsiella (slow), Helicobacter pylori.
Topic 6
Triple sugar iron (TSI) agar
- Contents: glucose (0.1%), lactose and sucrose (1% each), ferrous sulphate and phenol red; slant and butt.
Acid slant / acid butt (A/A)
Ferments glucose and lactose or sucrose
E. coli, Klebsiella
Alkaline slant / acid butt (K/A)
Glucose only
Shigella; Salmonella (with H₂S)
K/A with black butt
Glucose only + H₂S
Salmonella Typhimurium, Proteus
Alkaline / alkaline (K/K)
Non-fermenter
Pseudomonas
Cracks or lifted agar
Gas production
E. coli
Topic 7
Nitrate reduction test
- Principle: nitrate reductase converts nitrate to nitrite. After incubation in nitrate broth, add sulphanilic acid and α-naphthylamine — red means nitrite present (positive). If no colour, add zinc dust: red means nitrate still present (true negative); no colour means nitrate was reduced beyond nitrite (positive). All Enterobacteriaceae are positive.
Topic 8
Carbohydrate fermentation
- Procedure: peptone water with 1% sugar (glucose, lactose, sucrose, mannitol) and Andrade's indicator, with an inverted Durham's tube. Positive: pink (acid); gas in the Durham's tube. Uses: differentiating Enterobacteriaceae; mannitol fermentation by S. aureus.
Topic 9
Hugh and Leifson oxidation–fermentation test
- Two tubes of O-F glucose medium; one sealed with paraffin oil. Oxidative organisms produce acid (yellow) only in the open tube (Pseudomonas); fermentative organisms produce acid in both (Enterobacteriaceae); non-reactive organisms change neither.
Topic 10
Bile solubility test
- Principle: bile salts (10% sodium deoxycholate) activate autolysins and lyse Streptococcus pneumoniae. Positive: a broth culture clears (or colonies dissolve on the plate). Viridans streptococci are insoluble. Supported by optochin sensitivity.
Topic 11
H₂S production
- Organisms reducing sulphur compounds produce H₂S, which reacts with iron salts to form a black precipitate — seen in TSI, SIM medium or lead acetate paper. Positive: Salmonella (most), Proteus, Citrobacter.
Topic 12
Motility tests
Hanging drop
Young broth culture viewed under high power
Darting (Vibrio), tumbling (Listeria), sluggish or active movement
Semisolid (Craigie's tube or SIM) stab
Stab with a straight wire; incubate
Diffuse growth spreading from the stab = motile
Non-motile examples
—
Klebsiella, Shigella, Staphylococcus
Key terms
- Catalase
- Enzyme splitting hydrogen peroxide
- Coagulase
- Enzyme clotting plasma, marker of S. aureus
- IMViC
- Indole, methyl red, Voges–Proskauer and citrate tests
- TSI
- Triple sugar iron medium for fermentation and H₂S
- Durham's tube
- Small inverted tube trapping gas
Quick revision
- Catalase (bubbles), coagulase (clot or clump), oxidase (purple).
- IMViC: E. coli ++−−, Klebsiella −−++.
- Urease (pink — Proteus), TSI patterns, nitrate (zinc dust), sugars, O-F.
- Bile solubility for pneumococcus; H₂S black; hanging drop and semisolid motility.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.How is Staphylococcus distinguished from Streptococcus?
- Q2.What does a positive coagulase test indicate?
- Q3.Give the IMViC pattern of E. coli.
- Q4.What is the role of zinc dust in the nitrate test?
- Q5.Interpret K/A with H₂S in TSI.
- Q6.Which organism is bile soluble?
Long-answer questions
- Q1.Explain the catalase, coagulase and oxidase tests.
- Q2.Describe the IMViC tests with principles and interpretation.
- Q3.Explain the TSI agar test and its interpretation.
- Q4.Describe the urease, nitrate reduction, bile solubility and motility tests.
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