FOOD90023 · Food Microbiology
Microbial Identification
Identification answers which organism is this?, and there are two philosophies. Conventional (phenotypic) methods read what a microbe looks like and does — shape, Gram reaction, colony morphology, biochemistry — cheap and foundational but slow and sometimes ambiguous. Modern (molecular) methods read its genes or proteins — PCR, 16S rRNA sequencing, MALDI-TOF, immunoassays — fast, specific and able to detect the unculturable. Alongside both runs enumeration: not who but how many, reported in colony-forming units (CFU). You should be able to describe the conventional methods and the classification decision tree, explain the problems with conventional classification and the molecular fix, distinguish validation vs evaluation of a method, name selective vs differential media, and explain plate-count enumeration. Keep identification (the process) separate from classification (the scheme) — the exam penalises swapping them.
What this chapter covers
- 01Two philosophies — conventional (phenotypic) vs modern (molecular)
- 02Conventional methods — microscopy/staining, culture, biochemistry, serology
- 03Selective vs differential media
- 04The classification decision tree (Gram, catalase, oxidase, fermentation)
- 05Modern methods — PCR, 16S rRNA sequencing, MALDI-TOF, immunoassays
- 06Problems with conventional classification (and the molecular fix)
- 07Enumeration — plate counts and CFU; validation vs evaluation
Worked example: place an isolate on the classification tree
- +1(a) Gram + shape. Gram-positive cocci splits the field into a few genera (e.g. Staphylococcus, Streptococcus, Enterococcus, Micrococcus) — the Gram reaction and morphology are the first branch.
- +1(a) Catalase. Catalase-positive points toward Staphylococcus / Micrococcus and away from catalase-negative Streptococcus / Enterococcus — the next branch of the tree.
- +1(b) Further test. A coagulase test separates Staphylococcus aureus (coagulase-positive, a pathogen) from the coagulase-negative staphylococci; alternatively, growth on a selective/differential medium (e.g. mannitol-salt) narrows it further.
- +1(c) Limitation — overlap. Phenotypic traits overlap between species and depend on growth conditions, so the tree gives a genus or group, not always a confident species — it can misclassify.
- +1(c) Limitation — culturability/speed. It also misses unculturable / VBNC organisms and is slow (days), which is why molecular methods (16S rRNA, MALDI-TOF) are used for fast, strain-level confirmation.
Key terms
- Phenotypic (conventional) identification
- Identifying a microbe by what it looks like and does — shape and Gram reaction, colony morphology, biochemical tests (catalase, oxidase, sugar fermentation), motility. Cheap and foundational, but slow (days for culture) and only moderately specific because traits overlap.
- 16S rRNA sequencing
- A molecular identification method that sequences the highly conserved 16S ribosomal RNA gene, whose variable regions differ between species. It identifies bacteria (including unculturable ones) to species or strain level far faster and more specifically than phenotypic tests.
- Selective vs differential media
- Selective media contain agents that suppress unwanted organisms so a target can grow (e.g. high salt selects staphylococci). Differential media let many organisms grow but make the target visually distinct (e.g. a colour change from fermenting a sugar). Many media are both.
- Colony-forming unit (CFU)
- The unit of viable count: each colony on a plate is assumed to have grown from one viable cell (or clump), so the count is reported as CFU per gram or millilitre. It measures how many live, culturable cells are present, not the total cell count.
- Validation vs evaluation
- Validation establishes that a method actually does what it claims (fit for purpose) under defined conditions; evaluation compares a method's performance (e.g. sensitivity, specificity, speed, cost) against a reference or alternative. The exam asks you to distinguish the two for a given method.
Microbial Identification FAQ
What is the difference between identification and classification?
Classification is the scheme — the system that arranges organisms into groups by similarity or phylogeny. Identification is the process of working out that a particular new isolate belongs to one of those groups. The exam penalises swapping them: identification is what you do at the bench (run the tests), classification is the map you place the result onto. State that distinction explicitly when asked.
How do I answer 'the problems with conventional classification'?
Frame it as the limitations of phenotype-based ID, then give the molecular fix. The problems: phenotypic traits overlap between species and shift with growth conditions (so results can be ambiguous or wrong); culture is slow (days); and it misses unculturable / viable-but-non-culturable (VBNC) organisms entirely. The fix: molecular methods (16S rRNA sequencing, MALDI-TOF, PCR) read genes or proteins for fast, specific, strain-level identification that does not depend on culturing the organism.
When would you choose a molecular method over a conventional one?
When you need speed (hours not days), high specificity (strain-level, e.g. distinguishing pathogenic from harmless strains), or the ability to detect organisms you cannot culture. Conventional methods remain valuable because they are cheap, foundational and give useful information (a Gram stain and catalase test cost almost nothing), so in practice the two are used together — phenotype to narrow, molecular to confirm.
What does CFU actually measure, and why does it matter?
A colony-forming unit is a viable count — each colony is taken to have grown from one live, culturable cell or clump, so CFU/g or CFU/mL tells you how many cells could grow, not the total number present (which includes dead and VBNC cells). That matters for safety and shelf-life decisions, because it is the growing cells that spoil food or cause illness, and it is why a plate count can underestimate a population containing stressed or unculturable cells.
Exam move
Keep the two-philosophy framing at the centre: conventional/phenotypic (reads appearance and behaviour; cheap, slow, moderately specific) vs modern/molecular (reads genes/proteins; fast, specific, detects the unculturable). Be able to walk the classification decision tree (Gram → shape → catalase/oxidase → fermentation) and to name selective vs differential media. Pre-write the recurring 'problems with conventional classification' answer (overlap, slow, misses unculturable — plus the molecular fix) and the validation vs evaluation distinction, both of which recur as short-answer marks. Finally, be able to explain plate-count enumeration and CFU as a viable count, and never confuse identification (the process) with classification (the scheme).
