Clinical Microbiology: Specimen Collection, Processing, and Identification

Specimen Collection and Processing in Clinical Microbiology

Overview

Accurate diagnosis of infectious diseases relies on the proper collection, transport, and processing of clinical specimens. This chapter outlines the main types of specimens, their collection methods, etiologic agents, and laboratory processing techniques, including media selection and preliminary identification tests.

Specimens from the Respiratory Tract

Throat Cultures

• Etiologic Agents: Common pathogens include viruses, Streptococcus pyogenes, and Candida albicans.

• Specimen Collection:

  • Swab the posterior pharynx, avoiding normal oropharyngeal flora.

  • Inoculate onto blood agar plates for culture.

  • Rapid antigen testing is available for Group A streptococci.

Nasopharyngeal Specimens

• Etiologic Agents: Bordetella pertussis (whooping cough).

• Carrier State: Staphylococcus aureus, Neisseria meningitidis.

• Collection: Use a flexible thin wire swab inserted through the nares to the posterior pharynx.

Upper Respiratory Tract Specimens

• Oral Cavity: Associated with gingivitis and dental caries, often caused by Streptococci and anaerobes.

• Middle Ear: Otitis media commonly caused by nontypeable Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis.

• Sinuses: Infections often involve staphylococci and streptococci.

Lower Respiratory Tract Specimens

• Etiologic Agents: Viruses, Streptococcus pneumoniae, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, anaerobes, and Mycoplasma pneumoniae.

• Specimen Collection: Expectorated sputum, endotracheal specimens, translaryngeal aspirates, and bronchoalveolar lavage.

Sputum and Mucus

• Sputum: Phlegm coughed up from the lower airways, often due to infection. Contains secretions from respiratory tract cells and may harbor bacteria.

• Evaluation: Gram stain and Bartlett’s classification (enumeration of neutrophils and squamous epithelial cells per low power field).

• Interpretation: High PMNs suggest infection; high squamous cells indicate contamination with oral flora.

• Mucus: Normal, slippery fluid produced by lining tissues; contains antibodies and bactericidal enzymes. Normal production is 1–1.5 L/day.

Gastrointestinal and Urinary Specimens

Fecal Specimens

• Composition: 75% water, 25% solid material (undigested food, bacteria).

• Diagnostic Use: Identification of parasites by detection of eggs in stool.

Urine Specimens

• Composition: 91–96% water, inorganic salts, urea, organic compounds, proteins, hormones, and metabolites.

• Etiologic Agents: Escherichia coli and other Enterobacteriaceae.

• Collection Methods:

  • Midstream clean catch (preferred).

  • Straight catheterization, suprapubic aspirates, or collection during cystoscopy.

  • Avoid random voided urine and Foley catheters due to contamination risk.

• Colony Count:

  • Use a 1 μL calibrated inoculating loop to plate urine.

  • Incubate for 24 hours, count colonies, and convert to CFU/mL by multiplying by 1000.

  • Counts > $1.0 \times 10^5$ CFU/mL indicate infection.

• Types of UTIs: Cystitis, urethritis, pyelonephritis, pyelitis, glomerulonephritis.

• Urine Color Interpretation:

Color	Possible Meaning
Fizzy	Normal, excess protein, kidney problems
Clear	Excess water
Pale Straw Yellow	Healthy
Transparent Yellow	Normal
Honey/Amber	Dehydrated
Dark Yellow	Normal but needs water
Orange	Dehydrated, liver/bile duct condition, food dye
Brown	Severely dehydrated, liver disease
Pink/Red	Blood in urine or certain foods
Blue/Green	Rare genetic disease, bacteria, medication, food dye

Blood and Cerebrospinal Fluid (CSF) Specimens

Blood Specimens

• Composition: 55% plasma, 44% RBCs, 1% WBCs and platelets.

• Etiologic Agents: Staphylococcus aureus, Enterococcus, E. coli, other Enterobacteriaceae.

• Bacteremia: Presence of bacteria in blood, confirmed by blood cultures. Can be transient, intermittent, or continuous.

• Septicemia: Acute bloodstream infection with bacteria or their toxins, causing severe clinical symptoms.

• Collection:

  • Proper skin preparation to avoid contamination (alcohol, iodophor, sterile water, etc.).

  • Collect from two sites at least one hour apart; at least 20 mL per set (10 mL per tube), with both aerobic and anaerobic bottles.

  • No more than three sets per 24 hours.

Cerebrospinal Fluid (CSF) Specimens

• Function: Cushions brain and spinal cord, provides nutrients, removes waste.

• Normal Appearance: Clear and transparent.

• Abnormal Appearance: Bloody (traumatic tap or hemorrhage), yellowish (possible pathology).

• Etiologic Agents: Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae.

• Collection: Needle aspirate from 3rd or 4th lumbar vertebrae; three tubes collected (third tube for microbiology).

• Fastidious Organisms: Require enriched media and incubation at 37°C (e.g., Neisseria species).

Genital Tract Specimens

• Etiologic Agents: Neisseria gonorrhoeae, Treponema pallidum, Herpes Simplex Virus type 2, Chlamydia trachomatis, Trichomonas vaginalis.

• Collection: Urethral exudate, uterine cervix, urethra. Use specialized media (e.g., Martin Lewis, Modified Thayer Martin) and proper transport media to maintain viability.

Wound and Abscess Specimens

• Etiologic Agents: Staphylococcus aureus, Enterococcus, anaerobes, fungi.

• Collection: Aspirates, irrigation fluids, purulent drainage, necrotic tissue. Needle aspirates preferred over swabs.

• Exudate: Fluid produced during wound healing; normal part of the process.

• Pressure Wounds: Common in immobile patients; require careful cleaning and management.

Specimen Transport and Laboratory Processing

Specimen Transport

• Maintain specimen integrity; transport within 2 hours if possible.

• Use transport media (e.g., Stuart's or Amies) to preserve viability.

• On receipt, log and evaluate specimen; notify provider of concerns.

Initial Microscopic Examination

• Gram Stain: Assesses specimen quality and provides preliminary identification.

• Interpretation: Neutrophils indicate infection; epithelial cells suggest contamination.

• Bacterial Morphology: Gram-positive or negative, cocci or bacilli, arrangement (chains, pairs, clusters), presence of anaerobes or fungi.

Culture Media and Isolation Techniques

Primary Media Selection

• Media provide nutrients, energy sources, and buffers for microbial growth.

• Types include broth and agar (1.5% agar for solid media).

• Isolated colonies are essential for further biochemical and serological testing.

Types of Culture Media

• Supportive Media: Nutrient agar, trypticase soy agar, nutrient broth.

• Nonselective/Enriched Media:

  • Blood Agar Plate (BAP): Trypticase soy agar with 5% defibrinated RBCs (sheep, horse, or rabbit).

  • Hemolysis Types:

    • Alpha: Partial (greenish color)

    • Beta: Complete (clear/yellow)

    • Non-hemolytic: No change

  • Chocolate Agar: Boiled blood; RBCs lysed to release NAD and hematin. Used for fastidious organisms (e.g., Haemophilus).

• Differential Media: Distinguish bacteria based on metabolic properties.

  • MacConkey Agar: Isolates gram-negative bacteria; lactose fermenters appear pink, non-fermenters are clear.

  • Eosin Methylene Blue (EMB): Isolates gram-negative bacteria; strong lactose fermenters (e.g., E. coli) show green metallic sheen, others are purple or clear.

• Enrichment Broth: Inhibits normal flora to enhance growth of pathogens (e.g., selenite broth, tetrathionate broth, thioglycollate broth).

• Selective Media: Inhibit all but target bacteria (e.g., Hektoen Enteric, Salmonella-Shigella, XLD for stool pathogens).

• Antibiotic Media: Contain antibiotics to select for or against certain bacteria (e.g., CNA for gram-positives, MTM for Neisseria gonorrhoeae).

Incubation Conditions

• Temperature: Most clinical bacteria are mesophilic; optimal growth at 35 ± 2°C.

• Oxygen Requirements: Strict aerobes, strict anaerobes, facultative anaerobes.

• Atmosphere: Capnophilic organisms require increased CO2 (candle jar or CO2 incubator); strict anaerobes require anaerobic chambers.

Colony Characteristics

• Hemolysis: Type and zone size on blood agar.

• Color: On differential/selective media (e.g., Micrococcus luteus is banana yellow).

• Other Features: Pigments, odors (e.g., Proteus vulgaris has a strong smell), swarming motility.

Preliminary Biochemical Identification Tests

Carbohydrate Utilization

• Tests for fermentation or oxidation of specific sugars.

• Indicators: pH changes (color change), gas production.

Catalase Test

• Detects enzyme catalase, which converts hydrogen peroxide to water and oxygen (bubbles = positive).

• Differentiates:

  • Staphylococcus (catalase +) from Streptococcus (catalase -)

  • Bacillus (catalase +) from Clostridium (catalase -)

Coagulase Test

• Detects conversion of fibrinogen to fibrin (clotting).

• Slide test (bound coagulase) and tube test (free coagulase).

• Differentiates: Staphylococcus aureus (coagulase +) from coagulase-negative staphylococci.

Cytochrome Oxidase Test

• Detects cytochrome oxidase enzyme; positive result is color change to indophenol blue.

• Oxidase Positive Genera: Neisseria, Pseudomonas, Campylobacter, Aeromonas, Pasteurella.

Spot Indole Test

• Detects tryptophanase enzyme, which breaks down tryptophan to indole.

• Positive: Pink/red color after adding Kovac's reagent.

• Differentiates:

  • E. coli (indole +) from other lactose fermenters

  • Proteus mirabilis (indole -) from Proteus vulgaris (indole +)

IMViC Testing

• Used to differentiate E. coli from Enterobacter species.

• IMViC stands for Indole, Methyl Red, Voges-Proskauer, and Citrate tests.

Summary Table: Common Media and Their Uses

Medium	Purpose	Key Features
Blood Agar Plate (BAP)	General isolation, hemolysis detection	Supports many bacteria, shows hemolysis
Chocolate Agar	Fastidious organisms	Lysed RBCs, enriched nutrients
MacConkey Agar	Gram-negative isolation, lactose fermentation	Lactose fermenters pink, non-fermenters clear
EMB Agar	Gram-negative isolation, lactose fermentation	E. coli green sheen, others purple/clear
CNA Agar	Gram-positive selection	Contains colistin and nalidixic acid
MTM/Martin Lewis	Neisseria gonorrhoeae selection	Chocolate base + antibiotics
Thioglycollate Broth	Aerobe/anaerobe differentiation	Oxygen gradient

Key Definitions

• Etiologic Agent: The microorganism responsible for causing a disease.

• Colony Forming Unit (CFU): A measure of viable bacterial or fungal cells.

• Hemolysis: The breakdown of red blood cells, observed as color changes on blood agar.

• Fastidious Organism: Microorganisms with complex nutritional requirements.

• Exudate: Fluid produced by tissue, often during inflammation or infection.

Additional info:

• Some details about rare urine colors and their causes are inferred from standard clinical knowledge.

• IMViC test details are expanded for clarity.

• Tables are reconstructed for clarity and completeness.