Introductory Laboratory Microbiology

This subsection introduces the core principles of specimen handling and basic laboratory technique. Detailed interpretation of laboratory results, and the full range of diagnostic modalities, is covered in the dedicated Diagnostic Microbiology section.

• Correct specimen: the specimen type must be appropriate to the suspected site and organism (for example, sputum for suspected pneumonia, midstream urine for suspected urinary tract infection); an inappropriate specimen produces a result that does not answer the clinical question.
• Correct timing: ideally collected before antimicrobial therapy is started, since even a single dose of antibiotic can suppress organism growth and produce a false-negative culture.
• Aseptic collection: technique must minimise contamination by normal microbiota or the environment, particularly critical for specimens from normally sterile sites (blood cultures, CSF), where contamination can generate a false impression of infection.
• Appropriate container: sterile, leak-proof, and matched to the test required (for example, specific transport media for anaerobic culture, or viral transport media for suspected viral infection).
• Rapid transport: delay between collection and laboratory processing allows overgrowth of contaminating or rapidly growing organisms and death of more fastidious pathogens, degrading the specimen's diagnostic value; some specimens require refrigeration or specific transport conditions to preserve organism viability during transit.

• Light microscopy: the basic tool for direct visualisation of organisms in a specimen, providing rapid preliminary information before culture results are available.
• Gram stain: differentiates Gram positive (purple) from Gram negative (pink/red) organisms and reveals basic morphology (cocci, bacilli, arrangement), often the first and fastest piece of diagnostic information available, frequently guiding initial empirical antibiotic choice.
• Acid fast staining: identifies organisms with a waxy, mycolic-acid-rich cell wall that resists standard Gram staining (Mycobacterium species, and to a lesser extent Nocardia), using a strong stain (carbol fuchsin, Ziehl-Neelsen method) that, once bound, resists decolourisation with acid-alcohol.
• Wet preparation: unstained or lightly stained specimen examined directly, used particularly for detecting motile organisms (Trichomonas vaginalis, motile bacteria) or parasitic forms (ova, cysts, trophozoites) in stool or other fluid specimens.

• Culture media: nutrient preparations designed to support microbial growth outside the host, the foundation of traditional diagnostic microbiology.
• Solid media: agar-based media allowing isolated colony formation, essential for obtaining a pure culture from a mixed specimen and for assessing colony morphology.
• Liquid media (broth): supports growth in suspension, used for blood cultures and for enrichment of low-density specimens before subculture to solid media.
• Selective media: contains agents that inhibit the growth of unwanted organisms while permitting growth of the target organism (for example, MacConkey agar, which inhibits Gram positive organisms, selecting for Gram negative growth).
• Differential media: contains an indicator that allows visual distinction between organisms based on a specific biochemical property, without necessarily being selective (for example, MacConkey agar is also differential, distinguishing lactose fermenters, which produce pink colonies, from non-fermenters).
• Enriched media: contains additional nutrients (blood, serum, specific growth factors) to support the growth of fastidious organisms that would not grow on standard media (for example, chocolate agar, heated blood agar providing factors X and V, required for Haemophilus influenzae).

• Colony morphology: visual characteristics of bacterial growth on solid media (size, shape, colour, haemolysis pattern on blood agar, odour) provide preliminary identification clues.
• Biochemical tests: assess specific metabolic capabilities (carbohydrate fermentation, enzyme production such as catalase, coagulase, oxidase, urease) to narrow or confirm organism identity.
• Antigen detection: identifies specific organism surface or secreted antigens directly in a specimen, without requiring culture, often faster than culture-based methods (for example, rapid streptococcal antigen testing, urinary pneumococcal and legionella antigen testing).
• Molecular testing: detects organism-specific nucleic acid sequences (PCR and related techniques), offering high sensitivity and rapid turnaround, increasingly central to modern diagnostic microbiology (full detail in Diagnostic Microbiology and Microbial Genetics and Molecular Microbiology).

• Disc diffusion: antibiotic-impregnated discs are placed on an agar plate inoculated with the isolated organism; the diameter of the resulting zone of inhibited growth around each disc indicates relative susceptibility.
• Minimum inhibitory concentration (MIC): the lowest concentration of an antibiotic that visibly inhibits growth of the organism in a standardised test, providing a quantitative measure of susceptibility used to guide dosing and to compare susceptibility between organisms or between drugs.