Diagnostic Bacteriology
The practical bacteriological workup follows the sequence a clinical specimen actually takes, from collection through to a reported, actionable result.
The specimen type must match the suspected site and clinical question:
- blood cultures for suspected bacteraemia or sepsis
- midstream urine for suspected urinary tract infection
- sputum or bronchoalveolar lavage for suspected pneumonia
- wound swab or, preferably, aspirated pus or tissue for suspected soft tissue infection
- CSF for suspected meningitis
- stool for suspected enteric infection
Specimens should ideally be collected before antimicrobial therapy is started. Even a single antibiotic dose can suppress growth and produce a false-negative culture.
This is of particular importance for blood cultures and CSF, given the severity of the conditions these specimens are used to diagnose.
Gram stain is the first and fastest piece of diagnostic information available for most bacterial specimens, providing preliminary organism morphology, cocci, bacilli, coccobacilli, and Gram reaction, positive or negative, within minutes, often directly informing initial empirical antibiotic choice before culture results are available.
Direct Gram stain of a normally sterile fluid, CSF, joint fluid, pleural fluid, showing organisms is considered a significant, actionable finding even before culture confirmation, given the clinical urgency such findings typically represent.
Solid, liquid, selective, differential, and enriched media each serve distinct purposes in bacterial isolation and identification.
Aerobic culture uses standard incubation conditions, ambient air, sometimes with added CO2 for capnophilic organisms such as Neisseria species, and is used for most routine specimens.
Anaerobic culture requires an oxygen-free environment, an anaerobic jar or chamber, and, critically, a specimen collected and transported in a manner that minimises oxygen exposure, aspirated fluid or deep tissue, not a superficial swab, transported in anaerobic-specific transport media. Anaerobic culture is generally set up in parallel with aerobic culture for specimens from sites where anaerobic infection is plausible, deep abscess, intra-abdominal infection, deep wound.
Blood culture is typically collected as paired sets, with aerobic and anaerobic bottles per set, from two separate venepuncture sites where possible.
This practice helps distinguish true bacteraemia from skin-flora contamination. In true bacteraemia the organism grows in multiple, separately collected bottles. In contamination it grows in only one of several bottles.
Continuous-monitoring automated blood culture systems detect growth, via CO2 production or other metabolic markers, typically within 12 to 48 hours for most common bacterial pathogens.
Slower-growing or fastidious organisms may require extended incubation. Examples are Brucella, nutritionally variant streptococci, and some HACEK group organisms. Notify the laboratory specifically if such an organism is clinically suspected.
Visual characteristics of bacterial growth on solid media, size, shape, colour, texture, mucoid, dry, glistening, and haemolysis pattern on blood agar, provide preliminary identification clues before formal biochemical or molecular confirmation. Several organism-specific examples recur across bacteriology: the mucoid, nonmotile colonies of Klebsiella pneumoniae, the swarming motility pattern of Proteus species, the pigmented, sweet-odoured colonies of Pseudomonas aeruginosa, and the double-zone haemolysis of Clostridium perfringens.
The catalase test distinguishes Staphylococcus, positive, from Streptococcus and Enterococcus, negative. The coagulase test distinguishes S. aureus, positive, from coagulase-negative staphylococci. The oxidase test distinguishes the Enterobacterales, negative, from Neisseria, Pseudomonas, and most other nonfermenting Gram negative bacilli, positive, with Acinetobacter as a notable oxidase-negative exception within the nonfermenter group.
The urease test identifies urease-positive organisms including Proteus, linked to struvite stone formation, Klebsiella, and Helicobacter pylori, the basis of the urea breath test. The indole test is positive for E. coli, distinguishing it from most other Enterobacterales, based on the organism's ability to convert tryptophan to indole.
Lactose fermentation is the primary initial split within the Enterobacterales and enteric pathogens, fermenters including E. coli, Klebsiella, Enterobacter, nonfermenters including Salmonella, Shigella, Proteus, Yersinia, historically performed on MacConkey agar.
Matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry is a rapid identification method. It analyses the characteristic protein mass spectrum, a fingerprint, of an organism grown from a colony on solid media.
The spectrum is compared against a reference database to give species-level identification, typically within minutes once a colony is available.
This is a dramatic improvement in turnaround time compared with traditional biochemical identification panels, which can take a day or more.
MALDI-TOF does not, on its own, provide antimicrobial susceptibility information. That is a separate process, still required after identification.
PCR and related molecular methods detect organism-specific nucleic acid sequences directly.
They can be used on a primary clinical specimen, bypassing the need for culture entirely and offering same-day results. This is particularly valuable for slow-growing or fastidious organisms such as Mycobacterium tuberculosis, Bordetella pertussis, and Neisseria gonorrhoeae or Chlamydia trachomatis.
They can also be used on an isolated colony, for rapid, targeted confirmation or resistance gene detection.
These methods are increasingly incorporated into rapid, syndrome-based molecular panels. An example is a single respiratory or gastrointestinal panel simultaneously testing for multiple potential pathogens.
This reflects a broader shift in diagnostic microbiology toward faster, panel-based molecular testing alongside, rather than entirely replacing, traditional culture.
Antigen detection identifies specific organism antigens directly in a clinical specimen without requiring culture or nucleic acid amplification, generally faster than culture but with variable sensitivity depending on the specific test and organism.
Recurring examples include:
- rapid streptococcal antigen testing for S. pyogenes pharyngitis
- urinary pneumococcal and Legionella antigen testing in suspected pneumonia
- Clostridioides difficile toxin antigen and enzyme immunoassay testing, typically combined with a GDH screen and/or PCR in a multistep algorithm
Disc diffusion places antibiotic-impregnated discs on an inoculated agar plate; zone of inhibition diameter indicates relative susceptibility, a qualitative or semiquantitative method.
Minimum inhibitory concentration, MIC, testing is a quantitative determination of the lowest antibiotic concentration inhibiting visible growth, used to guide precise dosing decisions and to detect intermediate or borderline resistance that disc diffusion alone might not clearly distinguish.
Specific resistance phenotype screening, recognising ESBL, AmpC, carbapenemase, MRSA, and VRE patterns, is typically incorporated into modern automated susceptibility testing platforms, flagging these clinically significant resistance patterns directly within the reported result rather than requiring separate, additional testing in most routine laboratory workflows.
