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Laboratory Procedures of Clinical Microbiology

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1. Specimen Collection and Transport

• Specimens must be collected early in the acute phase of infection, before the organism ceases to be shed (e.g., respiratory viruses shed for only 3-7 days; HSV/VZV may not be recoverable from lesions more than 5 days after onset).
• The shorter the interval between collection and laboratory delivery, the higher the diagnostic yield - many organisms are labile and samples are susceptible to bacterial/fungal overgrowth.
• Specimen type depends on the differential diagnosis (e.g., arboviral meningitis requires CSF + blood; enteroviral meningitis requires CSF + throat swab + stool).
• The most purulent or bloody portion of a specimen is selected for smear preparation.
• For sterile body fluids (e.g., CSF), the specimen should be centrifuged to concentrate organisms 10-100x before smear preparation.
• Viruses are best transported on ice in media containing antibiotics and proteins (serum albumin or gelatin); enveloped viruses (HSV, VZV, influenza) suffer significant loss at room temperature or when frozen at -20°C.

• Medical Microbiology 9e, Chapter 39 (Specimen Collection)

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2. Microscopy

2a. Brightfield (Light) Microscopy

• The standard method using a light source, condenser, and objective + ocular lens system.
• Three objective lenses: low power (10×) for scanning; high dry (40×) for parasites and filamentous fungi; oil immersion (100×) for bacteria and yeasts (total magnification 1000× with 10× ocular).
• Resolving power: ~0.2 µm (can see bacteria but not viruses).
• Limitation: similar refractive indices of organisms and background means organisms must be stained to be visible.

2b. Darkfield Microscopy

• Uses a special condenser that blocks transmitted light; only oblique scattered light reaches the specimen.
• Resolving power improved to ~0.02 µm - allows detection of extremely thin bacteria such as Treponema pallidum (syphilis) and Leptospira spp.
• Limitation: light passes around rather than through organisms, so internal structures are difficult to study.

2c. Phase-Contrast Microscopy

• Exploits differences in optical density between cellular structures. Beams of light passing through objects of different densities move out of phase; annular rings in the condenser and objective lens translate these phase differences into contrast variations.
• Allows examination of internal microbial details and is used to visualize unstained, living organisms.

2d. Fluorescent Microscopy

• Uses high-intensity light (UV or short-wavelength visible) to excite fluorescent dyes or antibody-fluorochrome conjugates bound to organisms.
• Very sensitive and specific; widely used for organisms like Mycobacterium (auramine-rhodamine), Pneumocystis (calcofluor white), Chlamydia, and Legionella (fluorescent antibody stains).

2e. Electron Microscopy

• Not a standard clinical technique but can detect and identify viruses if sufficient particles are present.
• Immunoelectron microscopy: addition of virus-specific antibody causes viral particles to clump, facilitating simultaneous detection and identification.

• Medical Microbiology 9e, p. 34-36

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3. Staining Procedures

3a. Direct (Simple) Stains

3b. Differential Stains

• 10^5 organisms/mL must be present to see one organism per oil immersion field.
• Report includes size, shape, Gram reaction, and arrangement (e.g., "gram-positive diplococci resembling S. pneumoniae" rather than just "gram-positive cocci").
• White cell and squamous epithelial cell quantitation assesses specimen quality.
• Acridine orange stain can be used as a more sensitive alternative for blood culture bottles, CSF, or buffy coat - bacteria/fungi fluoresce orange, mammalian cells stain green.

3c. Acid-Fast Stains

3d. Fluorescent Stains

• Medical Microbiology 9e, p. 36-37; Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 1298

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4. In Vitro Culture (Culture Media and Incubation)

1. Biology of the organism (aerobic, anaerobic, fastidious growth requirements)
2. Site of infection (determines specimen type)
3. Patient's immune response
4. Quality of culture media

4a. Nonselective Enriched Media

• Blood agar (sheep blood agar): supports most bacteria; hemolysis patterns (alpha, beta, gamma) aid identification.
• Chocolate agar: blood agar heated to lyse RBCs, releasing hemin and NAD; required for fastidious organisms like Haemophilus spp. and Neisseria gonorrhoeae.
• Brucella agar, Brain-Heart Infusion (BHI) agar: additional enriched nonselective media.

4b. Selective Media

• MacConkey agar: selects gram-negative enteric bacilli; bile salts and crystal violet inhibit gram-positive organisms; lactose fermenters produce pink/red colonies.
• Mannitol salt agar: selects staphylococci (7.5% NaCl inhibits most organisms).
• Thayer-Martin agar: selects pathogenic Neisseria (contains vancomycin, colistin, nystatin).
• TCBS agar: selects Vibrio cholerae.
• Hektoen enteric / XLD agar: selects Salmonella and Shigella.

4c. Selective-Differential Media

• MacConkey agar (also differential - lactose fermentation).
• CLED agar (cystine lactose electrolyte-deficient): urinary pathogen differentiation.

4d. Specialized / Enrichment Broth Media

• Selenite F broth / GN broth: enrichment for Salmonella and Shigella from stool.
• Thioglycollate broth: supports anaerobes.
• Legionella: requires charcoal yeast extract agar with iron and L-cysteine supplements (BCYE agar) - was not recovered until this nutritional requirement was recognized.

4e. Culture for Specific Organism Groups

• Mycobacteria: Lowenstein-Jensen (LJ) egg-based medium; Middlebrook 7H10/7H11 agar; BACTEC MGIT 960 liquid broth system.
• Fungi: Sabouraud dextrose agar (low pH and high glucose selective for fungi); inhibitory mold agar.
• Anaerobes: Brucella blood agar or CDC anaerobe blood agar incubated in anaerobic chambers.
• Viruses: cell culture lines showing characteristic cytopathic effects (CPE) - syncytia formation (paramyxoviruses, HSV, HIV), vacuolation, cell lysis, inclusion bodies.
  • Intranuclear owl's-eye inclusions: CMV
  • Cowdry type A inclusions: HSV, VZV
  • Cytoplasmic Negri bodies: rabies

• Medical Microbiology 9e, p. 37-40

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5. Biochemical Identification Tests

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6. Antimicrobial Susceptibility Testing (AST)

6a. Disk Diffusion (Kirby-Bauer)

• Antibiotic-impregnated disks placed on a lawn of the test organism on Mueller-Hinton agar.
• After 16-18 hours incubation, zone of inhibition (diameter in mm) is measured.
• Zone size interpreted as Susceptible (S), Intermediate (I), or Resistant (R) using CLSI/EUCAST breakpoints.

6b. Broth Microdilution (MIC)

• Serial 2-fold dilutions of antibiotic in broth wells are inoculated with a standardized bacterial suspension.
• Minimum Inhibitory Concentration (MIC): lowest concentration that visibly inhibits growth.
• MIC is the standard for precise quantitative AST and is required for organisms treated with concentration-dependent antibiotics (aminoglycosides, fluoroquinolones).

6c. Gradient Diffusion (E-test / MIC Test Strips)

• A plastic strip with an antibiotic gradient is placed on an agar plate.
• At the point where the inhibition ellipse intersects the strip, the MIC is read directly.

6d. Automated AST Systems

• VITEK 2, MicroScan, BD Phoenix: inoculate pre-formatted cards/panels; provide MIC results in 4-8 hours via turbidimetric or fluorometric monitoring.

6e. Specialized Resistance Testing

• Beta-lactamase detection: nitrocefin disk (chromogenic cephalosporin).
• MRSA detection: chromogenic MRSA agar; mecA gene PCR.
• ESBL/carbapenemase detection: combined disk tests; modified carbapenem inactivation method (mCIM); molecular testing for bla-KPC, bla-NDM, bla-OXA-48.

• Tietz Textbook of Laboratory Medicine, 7th Edition; Murray & Nadel's Textbook of Respiratory Medicine

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7. Molecular Diagnostic Procedures

7a. Nonamplified Nucleic Acid Probes

• Short DNA/RNA oligonucleotides labeled with reporter molecules hybridize to complementary microbial sequences.
• Limitation: require large numbers of target sequences; low sensitivity for direct detection in clinical specimens.
• Use: identification of organisms already grown in culture (mycobacteria, dimorphic fungi, viruses) where large numbers of organisms are present.

7b. Nucleic Acid Amplification Tests (NAATs)

• The dominant amplification method.
• Thermocycling with template denaturation (94°C), primer annealing, and DNA synthesis (72°C with Taq polymerase).
• Quantitative PCR (qPCR/Real-Time PCR): fluorescent probes (TaqMan, SYBR Green) allow simultaneous amplification and quantitation.
• Multiplex PCR: multiple primer sets detect multiple organisms in one reaction (e.g., respiratory multiplex panels, enteric panels, blood culture panels).

• Isothermal; amplifies RNA targets without thermocycling.
• Used for HIV viral load, Chlamydia/Gonorrhoeae testing.

• Isothermal DNA amplification using restriction enzyme + polymerase.

• Isothermal; uses 4-6 primer pairs; amplification monitored by turbidity of magnesium pyrophosphate precipitate.
• Rapid and does not require expensive instrumentation.

7c. Nucleic Acid Sequencing

• Targeted sequencing: 16S rRNA gene for bacteria; ITS regions for fungi - provides definitive genus/species identification.
• Whole Genome Sequencing (WGS): sequences entire microbial genome; gold standard for outbreak investigation and epidemiologic subtyping; tracks single nucleotide polymorphisms (SNPs).

7d. Restriction Fragment Length Polymorphism (RFLP) / Pulsed-Field Gel Electrophoresis (PFGE)

• Chromosomal DNA is cut with restriction endonucleases; fragments separated by PFGE create banding patterns unique to each strain.
• Used for epidemiologic strain typing (e.g., outbreak investigations of S. aureus, Salmonella, C. difficile).

7e. Matrix-Assisted Laser Desorption/Ionization - Time of Flight (MALDI-TOF MS)

• Protein analysis by mass spectrometry.
• Rapidly replacing traditional biochemical identification.
• A colony is spotted onto a target plate, ionized by laser, and the protein mass spectrum is compared to a reference database.
• Provides genus and species identification in minutes at low cost per test.

7f. Key Molecular Test Applications

• Medical Microbiology 9e, Chapter 5

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8. Serologic (Immunologic) Procedures

8a. Precipitation and Immunodiffusion

• In the equivalence zone, antigen-antibody complexes cross-link and precipitate out of solution.
• Single radial immunodiffusion (SRID): antigen diffuses from a well into antibody-containing agar; precipitation ring diameter correlates with antigen concentration.
• Ouchterlony double diffusion: antigen and antibody diffuse toward each other; lines of identity, non-identity, or partial identity are formed.

8b. Agglutination Tests

• Antibodies cross-link particulate antigens (bacteria, RBCs, latex beads) into visible clumps.
• Widal test (typhoid), VDRL/RPR (syphilis), latex agglutination for capsular antigens (Cryptococcus, H. influenzae, S. pneumoniae, N. meningitidis, Group B Streptococcus).
• Tube dilution: titer is the greatest dilution of serum retaining detectable agglutination.

8c. Complement Fixation

• If antigen-antibody complexes form, they fix complement; absence of hemolysis of indicator RBCs signals a positive test.
• Used for viral serology (e.g., influenza, arbovirus).

8d. Enzyme-Linked Immunosorbent Assay (ELISA)

• Antibody or antigen is immobilized on a solid phase; enzyme-conjugated secondary antibody produces colorimetric signal.
• Both antigen detection (e.g., HIV p24, HBsAg, C. difficile toxin) and antibody detection (HIV, hepatitis, Lyme disease, etc.) formats.
• High throughput, automatable, quantitative.

8e. Western Blot (Immunoblot)

• Proteins from organism are separated by electrophoresis, transferred to membrane, and probed with patient serum.
• Used to confirm ELISA results (e.g., HIV, Lyme disease).

8f. Immunofluorescence Assays (IFA)

• Direct IFA: labeled antibody applied directly to specimen.
• Indirect IFA (IIFA): patient serum applied first; labeled anti-human antibody is secondary probe.
• Used for ANA, ANCA, and detection of many intracellular pathogens.

8g. Lateral Flow / Rapid Antigen Tests

• Nitrocellulose membrane with test and control lines; results in 5-15 minutes.
• Examples: influenza rapid antigen tests, SARS-CoV-2 antigen tests, malaria RDTs, strep throat tests.

• Medical Microbiology 9e, Chapter 6

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9. Cytology and Histopathology

• Tissue biopsies or cytologic specimens (e.g., bronchial washings, skin scrapings) are examined for pathognomonic inclusion bodies, granulomas, and organism morphology.
• Cytopathic effects (CPE) in cell culture or tissue sections:
  • Syncytia (multinucleated giant cells): paramyxoviruses, HSV, HIV
  • Owl's-eye intranuclear inclusions: CMV
  • Cowdry type A inclusions (intranuclear): HSV, VZV
  • Negri bodies (cytoplasmic): Rabies virus
• Special stains for histopathology: PAS (fungi), Gomori methenamine silver (GMS, for Pneumocystis and fungi), Warthin-Starry (spirochetes, Bartonella), Ziehl-Neelsen (mycobacteria).
• In situ hybridization (ISH) or PCR can be applied directly to tissue sections for specific viral identification.

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10. Blood Culture Systems

• Automated continuous-monitoring systems (e.g., BACTEC FX, BacT/ALERT) detect CO2 produced by growing organisms via fluorescent or colorimetric sensors.
• Detected positive bottles are Gram stained first; then subcultured to solid media for isolation and identification.
• Rapid multiplex PCR panels (FilmArray Blood Culture ID) can identify organisms and common resistance genes directly from a positive bottle in ~1 hour.

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Summary Overview

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• Medical Microbiology 9e (Murray, Rosenthal & Pfaller), Chapters 4, 5, 6 - microscopy, molecular diagnosis, serology
• Henry's Clinical Diagnosis and Management by Laboratory Methods, Chapter 57 - Gram stain and bacteriology procedures
• Tietz Textbook of Laboratory Medicine, 7th Edition - antimicrobial susceptibility testing