Biochemical identification
A drop of 3% hydrogen peroxide on a colony: brisk bubbling within seconds means catalase. The first split of Gram-positive cocci, staphylococci (positive) from streptococci and enterococci (negative).
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Catalase
A drop of 3% hydrogen peroxide on a colony: brisk bubbling within seconds means catalase. The first split of Gram-positive cocci, staphylococci (positive) from streptococci and enterococci (negative).
How it differs from the standard method
- Take growth from the top of the colony with a plastic loop or wooden stick; blood agar carried over with it contains red-cell catalase.
- Use fresh peroxide from a dark bottle; it decomposes in light and with age.
- Test 18–24 h growth; old cultures can lose activity.
- Never add peroxide to an open blood agar plate of an unknown organism: the bubbles make aerosols.
1From a colony to a name
A Gram stain and a colony on a differential plate narrow an unknown down to a group: Gram-positive cocci in clusters, an oxidase-negative Gram-negative rod that does not ferment lactose. Identification goes the rest of the way by asking what the organism can do (which enzymes it has, which sugars it ferments, which amino acids it breaks down) or, more recently, by weighing its most abundant proteins.
Catalase
Hydrogen peroxide on a colony: bubbles of oxygen mean catalase. Splits staphylococci (positive) from streptococci and enterococci (negative).
Oxidase
A colony on tetramethyl-p-phenylenediamine turns purple within about 10 s if it has cytochrome c oxidase. Splits Pseudomonas and other non-fermenters from the Enterobacterales.
Biochemical strips
About twenty miniature tests from one suspension, read as a profile number and looked up in a database for a species and a confidence.
MALDI-TOF
A mass spectrum of the colony's ribosomal and other abundant proteins, matched against reference spectra for a species-level score.
2Spot tests and their false positives
Catalase converts hydrogen peroxide into water and oxygen, so a catalase-positive colony fizzes in a drop of 3% peroxide. Red blood cells have catalase too, so a colony scraped off blood agar together with some agar can give weak bubbles from a streptococcus.
3Biochemical strips: probability, not a key
A strip such as the classic 20-test panel for Enterobacterales holds dehydrated substrates in a row of cupules. Each is filled with the same standardised suspension; some (decarboxylases, urease, H₂S) are sealed under mineral oil to keep oxygen out, and some (tryptophan deaminase, indole, acetoin) only show a result once a reagent is added at the end.
4MALDI-TOF: weighing the ribosome
In matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, a thin smear of colony is covered with a matrix (typically α-cyano-4-hydroxycinnamic acid) that absorbs the laser's energy and helps proteins ionise. The ions are accelerated and their flight time gives their mass.
- Profile number
- The code built from a strip's results, each group of three tests contributing one digit (1, 2 and 4 for positives).
- %ID
- The probability of the best candidate relative to all candidates in the database, given the observed profile.
- Score (MALDI)
- A log-scale measure of how well a spectrum matches a reference: share of peaks matched in both directions and agreement of intensities.
Key terms
Practise these as flashcards →Enzyme that converts hydrogen peroxide into water and oxygen; detected by bubbling in 3% H₂O₂.
The terminal respiratory enzyme detected by the oxidase test, present in Pseudomonas, Neisseria and Vibrio but not in Enterobacterales.
Detects removal of the carboxyl group from lysine, ornithine or arginine; the alkaline products turn the indicator red. Needs low oxygen, hence the oil overlay.
A turbidity reference used to standardise bacterial suspensions; 0.5 McFarland is roughly 1–2 × 10⁸ CFU/mL.
A small aromatic acid that co-crystallises with the sample, absorbs the laser energy and transfers protons to the proteins.
A MALDI spotting method in which the smear is overlaid with 70% formic acid before matrix, to release proteins from thick-walled cells.
Converting flight time to mass using a standard of known masses spotted on the same target; it drifts and is redone regularly.
When to use it
- Sorting a Gram-positive coccus (catalase) or a Gram-negative rod (oxidase) in seconds, to decide which identification route and which media come next.
- Identifying an isolate where a MALDI-TOF is not available, or confirming a MALDI result in a group it does not separate, with a biochemical strip.
- Routine species identification of bacteria and yeasts from colonies in minutes, when a validated MALDI-TOF library is available.
Compared with alternatives
Comes first: it decides whether catalase (Gram-positive cocci) or oxidase (Gram-negative rods) is the next test.
Lactose fermentation on MacConkey and haemolysis on blood agar are themselves biochemical clues, read from the plate before any strip is set up.
16S rRNA gene sequencing identifies organisms that biochemistry and MALDI cannot place, or slow and unusual ones.
Gives species, strain type and resistance genes at once; slower and costlier, but definitive where close relatives must be told apart.
Uses the same physics with chromatography and fragmentation to identify individual proteins and peptides rather than fingerprinting a whole cell.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A drop of 3% hydrogen peroxide on a colony: brisk bubbling within seconds means catalase. The first split of Gram-positive cocci, staphylococci (positive) from streptococci and enterococci (negative).
- Take growth from the top of the colony with a plastic loop or wooden stick; blood agar carried over with it contains red-cell catalase.
- Use fresh peroxide from a dark bottle; it decomposes in light and with age.
- Test 18–24 h growth; old cultures can lose activity.
- Never add peroxide to an open blood agar plate of an unknown organism: the bubbles make aerosols.