A Comprehensive Guide to Urine Culture Processing and Interpretation for Laboratory Technicians

Urine cultures are critical for diagnosing urinary tract infections (UTIs) and guiding effective treatment. This article synthesizes key protocols, challenges, and best practices for laboratory technicians and technologists, based on current guidelines and evidence-based methods.

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Specimen Collection and Handling:

Proper collection is paramount to avoid contamination and ensure accurate results:

• Clean-Catch Midstream Urine:
  • Instruct patients (especially women) to clean the periurethral area thoroughly and discard the initial urine stream.
  • Use sterile containers; avoid contact with skin or clothing.
• Pediatric Populations:
  • Bagged urine: Suitable for urinalysis but unreliable for culture due to contamination. Confirm positive results with suprapubic aspiration (SPA) or catheterization.
  • SPA or catheterization: Gold standards for infants <2 years. Colony count thresholds:
    • SPA: >100 CFU/mL (gram-negative rods)
    • Catheterization: >50,000 CFU/mL
• Indwelling Catheters:
  • Collect via port after ethanol disinfection. Discard initial 15–30 mL.
  • Reject catheter tips or bag urine for culture.

Transport & Storage:

• Process within 2–4 hours. Refrigerate (≤24 hours) or use boric acid preservative (avoid if leukocyte esterase testing is needed).

Term	Definition
Bacteriuria	Presence of bacteria in urine (>10⁵ CFU/mL in voided specimens).
Cystitis	Bladder inflammation, typically causing dysuria, urgency, and frequency.
Pyuria	>5 WBCs/hpf in urine; correlates with infection but may be absent in Proteus or neutropenia.
Urosepsis	Systemic infection originating from a UTI, often with bacteremia.
Asymptomatic Bacteriuria	≥10⁵ CFU/mL without symptoms; requires treatment in pregnancy.
Complicated UTI	UTI with structural/functional urinary tract abnormalities, diabetes, or immunosuppression.

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Culture Techniques and Media:

• Calibrated-Loop Method:
  • Use 0.01 mL (10 µL) or 0.001 mL (1 µL) loops for semiquantitative plating.
  • Key thresholds:
    • Significant bacteriuria: ≥10⁵ CFU/mL for voided urine; ≥10³ CFU/mL for catheterized specimens.
  • Calibrate loops monthly using Evans blue dye to ensure accuracy (±20% error tolerance).
• Media Selection:
  • Blood Agar (5% SBA): For total colony count and gram-positive organisms.
  • MacConkey/EMB: Isolate gram-negative rods (e.g., E. coli, Klebsiella).
  • CLED Agar: Prevents Proteus swarming and supports lactose fermenters.
  • CNA/PEA: Select for gram-positive cocci (e.g., Enterococcus, Staphylococcus).
• Automated Screening:
  • Bioluminescence (ATP detection) and photometry systems lack sensitivity for low-grade infections.
  • Particle filtration (e.g., Bac-T-Screen) is sensitive but prone to false positives.

Protocol for Workup of Urine Cultures:

Specimen Type	Inoculation	Workup Criteria	Reporting
Voided Midstream	0.001 mL loop on BAP, MAC/EMB	≥10⁵ CFU/mL: ID + AST Mixed flora: Report contamination	“Mixed microbiota; recollect if symptomatic.”
Catheterized	0.001 mL loop on BAP, CNA, MAC/EMB	≥10⁴ CFU/mL: ID + AST ≥3 isolates: Contamination	“Polymicrobial growth; clinical correlation advised.”
Suprapubic Aspiration	0.01 mL loop on BAP, CNA, MAC/EMB	≥100 CFU/mL (gram-negative rods): Report Any growth in symptomatic patients	“Pathogen identified; correlate clinically.”
Pediatric (SPA)	0.01 mL loop on BAP, CHOC for fastidious	≥100 CFU/mL (gram-negative rods): Urgent report	“Significant growth; recommend treatment.”

Key:

• BAP: Blood agar plate.
• MAC/EMB: MacConkey/Eosin Methylene Blue agar.
• CNA: Columbia colistin-nalidixic acid agar.
• CHOC: Chocolate agar.

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Interpretation Guidelines:

Key Criteria:

Collection Method	Significant CFU/mL	Probability of Infection
Suprapubic Aspiration (SPA)	≥100 (gram-negative rods)	>99%
Catheterization	≥50,000	95%
Clean-Catch (Women)	≥10⁵ (1 specimen)	80%

• Mixed Flora: Report as contamination if ≥3 organisms or skin/vaginal commensals dominate.
• Pyuria: >5 WBCs/hpf correlates with infection but may be absent in Proteus UTIs (alkaline pH lyses leukocytes).

Special Populations:

• Pregnancy: Screen for asymptomatic bacteriuria (≥10⁵ CFU/mL at 12–16 weeks). Treat Group B Streptococcus to prevent neonatal sepsis.
• Elderly: Higher asymptomatic bacteriuria rates; correlate with symptoms before reporting.
• Catheter-Associated UTIs: Treat only if symptomatic (≥10³ CFU/mL + symptoms).

Urinary Microbiota:

Category	Organisms	Clinical Relevance
Urogenital Microbiota	Lactobacillus spp., viridans streptococci, diphtheroids, Neisseria spp., anaerobes	Typically contaminants; report only if dominant (>10× other flora).
Skin Microbiota	Coagulase-negative staphylococci, Staphylococcus epidermidis, diphtheroids	Report as contaminants unless predominant in catheterized/immunocompromised patients.
Uropathogens	Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Proteus spp.	Always report and perform antimicrobial susceptibility testing (AST).
Yeasts	Candida albicans, Candida glabrata	Report species if pure; consider colonization vs. infection.
Fastidious Pathogens	Corynebacterium urealyticum, Aerococcus urinae, Gardnerella vaginalis	Require extended incubation (48–72 h) or special media.

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Pathogen Identification and Reporting

• Common Pathogens:
  • E. coli (80–90% of outpatient UTIs), Klebsiella, Enterococcus, Pseudomonas, Staphylococcus saprophyticus (young women).
• Contaminants:
  • Lactobacillus, Corynebacterium, coagulase-negative staphylococci (unless predominant in immunocompromised patients).
• Uncommon Pathogens:
  • Aerococcus urinae (elderly males), Corynebacterium urealyticum (urease-positive, alkaline urine), Candida (catheterized patients).

Reporting Tips:

• Add comments for ambiguous results:
  • “No urinalysis performed; clinical correlation advised.”
  • “Mixed skin flora; suggest recollection if clinically indicated.”
• For sterile pyuria: Consider Chlamydia, TB, or prior antibiotic use.

Bacteria Isolation in Urine Culture:

Bacteria Name	Agar Media	Key Identification Features	Minimal Testing Required
Escherichia coli	MAC, EMB, CLED, BAP	– Gram-negative rods – Lactose fermenter (pink colonies on MAC/EMB) – Indole-positive	– Gram stain – Lactose fermentation – Indole test
Klebsiella pneumoniae	MAC, EMB, CLED, BAP	– Gram-negative rods – Lactose fermenter (mucoid colonies) – Urease-positive	– Gram stain – Lactose fermentation – Urease test
Proteus mirabilis	MAC, BAP, CLED	– Gram-negative rods – Swarming motility – Urease-positive – H₂S production (TSI agar)	– Gram stain – Urease test – Swarming observation
Enterococcus spp.	CNA, BAP	– Gram-positive cocci in chains – Catalase-negative – PYR-positive	– Gram stain – Catalase test – PYR test
Pseudomonas aeruginosa	MAC, BAP	– Gram-negative rods – Non-lactose fermenter (green pigment on MAC) – Oxidase-positive – Fruity odor	– Gram stain – Oxidase test – Odor observation
Staphylococcus saprophyticus	CNA, BAP	– Gram-positive cocci in clusters – Catalase-positive – Novobiocin-resistant	– Gram stain – Catalase test – Novobiocin susceptibility
Candida albicans	SDA, CHROMagar™	– Yeast (budding cells) – Germ tube-positive – Green colonies on CHROMagar™	– Gram stain/KOH prep – Germ tube test – CHROMagar™
Streptococcus agalactiae (GBS)	CNA, BAP	– Gram-positive cocci in chains – Catalase-negative – CAMP test-positive	– Gram stain – CAMP test – Hippurate hydrolysis
Corynebacterium urealyticum	BAP, CLED	– Gram-positive rods (palisades) – Urease-positive – Alkaline urine	– Gram stain – Urease test – Extended incubation (48–72 h)
Aerococcus urinae	BAP, CNA	– Gram-positive cocci in tetrads/clusters – PYR-negative – Alpha-hemolytic	– Gram stain – PYR test – LAP (leucine aminopeptidase) test

Minimal Testing for Reporting Isolates:

Organism	Minimal Testing	Definitive ID Required?
Escherichia coli	Lactose fermentation, indole-positive	No
Enterococcus spp.	Catalase-negative, PYR-positive	No
Staphylococcus aureus	Catalase-positive, coagulase-positive	No
Pseudomonas aeruginosa	Oxidase-positive, grape-like odor	No
Candida albicans	Germ tube test, CHROMagar™ (green colonies)	Yes (species-level)
Corynebacterium urealyticum	Urease-positive, gram-positive rods	Yes

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Tables by Agar Type

1. Blood Agar (BAP)

Pathogen	Colony Appearance	Hemolysis	Key Identifier
Escherichia coli	Grayish-white, smooth, convex, shiny	Beta-hemolytic	Musty odor, Gram-negative rods, indole-positive.
Klebsiella pneumoniae	Large, mucoid, gray-white	Gamma-hemolytic	Mucoid capsule, urease-positive, lactose-fermenting.
Proteus mirabilis	Swarming motility, translucent	Gamma-hemolytic	Swarming pattern, “fishy” odor, urease-positive.
Enterococcus spp.	Small, white, non-hemolytic	Gamma-hemolytic	Gram-positive cocci in chains, catalase-negative, PYR-positive.
Pseudomonas aeruginosa	Flat, greenish (pyocyanin pigment)	Beta-hemolytic	Grape-like odor, oxidase-positive, Gram-negative rods.
Staphylococcus saprophyticus	White, opaque, round	Gamma-hemolytic	Gram-positive cocci in clusters, catalase-positive, novobiocin-resistant.
Candida albicans	Creamy, white, smooth	Non-hemolytic	Yeast cells (budding), germ tube-positive.
Streptococcus agalactiae (GBS)	Small, white, translucent	Beta-hemolytic	Gram-positive cocci in chains, CAMP test-positive.
Corynebacterium urealyticum	Small, white, dry	Gamma-hemolytic	Gram-positive rods in palisades, urease-positive, alkaline urine.
Aerococcus urinae	Small, alpha-hemolytic, greenish	Alpha-hemolytic	Gram-positive cocci in tetrads, PYR-negative.

2. MacConkey Agar (MAC)

Pathogen	Colony Appearance	Lactose Fermentation	Key Identifier
Escherichia coli	Pink, flat, dry	Yes (LF)	Indole-positive, oxidase-negative.
Klebsiella pneumoniae	Pink, mucoid, convex	Yes (LF)	Mucoid capsule, urease-positive.
Proteus mirabilis	Colorless, swarming	No (NLF)	Swarming motility, urease-positive, H₂S production.
Pseudomonas aeruginosa	Colorless, flat, green pigment	No (NLF)	Oxidase-positive, fruity odor.
Enterococcus spp.	No growth (gram-positive)	N/A	Not applicable; MacConkey selects for gram-negative bacteria.
Staphylococcus saprophyticus	No growth (gram-positive)	N/A	Not applicable.
Candida albicans	No growth (fungal)	N/A	Requires fungal-specific media (e.g., CHROMagar™).

3. Eosin Methylene Blue Agar (EMB)

Pathogen	Colony Appearance	Lactose Fermentation	Key Identifier
Escherichia coli	Metallic green sheen	Yes (LF)	Indole-positive, oxidase-negative.
Klebsiella pneumoniae	Pink, mucoid	Yes (LF)	Mucoid colonies, urease-positive.
Proteus mirabilis	Colorless	No (NLF)	Swarming motility, urease-positive.
Pseudomonas aeruginosa	Colorless, flat	No (NLF)	Oxidase-positive, green pigment (pyocyanin).

4. CLED Agar (Cystine-Lactose-Electrolyte-Deficient)

Pathogen	Colony Appearance	Lactose Fermentation	Key Identifier
Escherichia coli	Yellow	Yes (LF)	Indole-positive, non-swarming.
Klebsiella pneumoniae	Yellow, mucoid	Yes (LF)	Mucoid colonies, urease-positive.
Proteus mirabilis	Blue (non-lactose fermenter)	No (NLF)	Swarming inhibited, urease-positive.
Pseudomonas aeruginosa	Green-blue	No (NLF)	Oxidase-positive, fruity odor.
Enterococcus spp.	Small, white	No (NLF)	Gram-positive cocci, catalase-negative.

5. CNA Agar (Columbia Colistin-Nalidixic Acid)

Pathogen	Colony Appearance	Hemolysis	Key Identifier
Enterococcus spp.	Small, white, non-hemolytic	Gamma-hemolytic	Gram-positive cocci in chains, PYR-positive.
Staphylococcus saprophyticus	White, opaque	Gamma-hemolytic	Catalase-positive, novobiocin-resistant.
Streptococcus agalactiae (GBS)	Small, translucent, beta-hemolytic	Beta-hemolytic	CAMP test-positive, hippurate hydrolysis.

6. CHROMagar™

Pathogen	Colony Appearance	Key Identifier
Escherichia coli	Pink	Lactose-fermenting, indole-positive.
Klebsiella pneumoniae	Blue	Mucoid colonies, urease-positive.
Candida albicans	Green	Germ tube-positive, chlamydospore formation.
Enterococcus spp.	No growth	Not applicable; CHROMagar™ targets gram-negative bacteria and yeast.

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Tables by Pathogen

1. Escherichia coli

Agar Type	Colony Appearance	Key Feature
Blood Agar (BAP)	Grayish-white, beta-hemolytic	Musty odor, Gram-negative rods.
MacConkey Agar (MAC)	Pink, lactose-fermenting	Indole-positive.
EMB Agar	Metallic green sheen	Lactose-fermenting.
CLED Agar	Yellow	Non-swarming, lactose-fermenting.

2. Pseudomonas aeruginosa

Agar Type	Colony Appearance	Key Feature
Blood Agar (BAP)	Flat, green pigment (pyocyanin)	Grape-like odor, oxidase-positive.
MacConkey Agar (MAC)	Colorless, non-lactose fermenting	Oxidase-positive, fruity odor.
EMB Agar	Colorless	Non-lactose fermenting.

3. Proteus mirabilis

Agar Type	Colony Appearance	Key Feature
Blood Agar (BAP)	Swarming motility, translucent	Urease-positive, “fishy” odor.
MacConkey Agar (MAC)	Colorless, non-lactose fermenting	Swarming inhibited.
CLED Agar	Blue (non-lactose fermenter)	Urease-positive.

4. Enterococcus spp.

Agar Type	Colony Appearance	Key Feature
Blood Agar (BAP)	Small, white, gamma-hemolytic	Catalase-negative, PYR-positive.
CNA Agar	Small, white, gamma-hemolytic	Gram-positive cocci in chains.

Key Notes:

• Confirm identification with biochemical tests (e.g., API, MALDI-TOF) or molecular methods.
• Hemolysis patterns may vary by strain (e.g., some E. coli strains are gamma-hemolytic).

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Quality Control and Troubleshooting:

• Rejection Criteria:
  • Specimens >2 hours unrefrigerated, Foley catheter tips, or bag urine.
• Antimicrobial Inhibition: Report “Colony count unreliable due to antimicrobial inhibition” if growth occurs only in diluted areas.
• Calibration: Validate loops monthly to avoid over/underestimation of CFU/mL.

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Communication with Clinicians:

Effective collaboration prevents mismanagement:

• Clarify collection method (e.g., bagged vs. catheterized).
• Flag contaminants in pediatric or geriatric specimens.
• Highlight fastidious organisms (e.g., C. urealyticum) requiring extended incubation (48 hours).

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Biochemical Tests for Common Urinary Pathogens:

Pathogen	Key Biochemical Tests	Expected Results	Key Notes
Escherichia coli	– Indole test – Lactose fermentation (MAC/EMB) – IMViC (Indole, MR, VP, Citrate)	Indole (+) Lactose (+) IMViC: + + – –	Beta-hemolytic on BAP. Most common UTI pathogen.
Klebsiella pneumoniae	– Urease test – Lactose fermentation (MAC/EMB) – IMViC	Urease (+) Lactose (+) IMViC: – – + +	Mucoid colonies; often hospital-acquired.
Proteus mirabilis	– Urease test – Swarming motility (BAP) – H₂S production (TSI) – Phenylalanine deaminase	Urease (+) Swarming (+) H₂S (+) PAD (+)	Alkaline urine; associated with kidney stones.
Pseudomonas aeruginosa	– Oxidase test – Cetrimide test – Growth at 42°C	Oxidase (+) Cetrimide (+) Growth at 42°C (+)	Pyocyanin pigment (green); antibiotic resistance common.
Enterococcus spp.	– PYR test – Bile esculin test – Salt tolerance (6.5% NaCl)	PYR (+) Bile esculin (+) Salt tolerance (+)	Catalase-negative; vancomycin-resistant strains (VRE) require special reporting.
Staphylococcus saprophyticus	– Catalase test – Coagulase test – Novobiocin susceptibility	Catalase (+) Coagulase (–) Novobiocin-resistant	Common in young women; adheres to uroepithelial cells.
Candida albicans	– Germ tube test – CHROMagar™ (green colonies) – Urease test	Germ tube (+) Green colonies Urease (–)	Budding yeast; colonization common in catheterized patients.
Streptococcus agalactiae (GBS)	– CAMP test – Hippurate hydrolysis – Gram stain	CAMP (+) Hippurate (+) Gram-positive cocci in chains	Beta-hemolytic; critical to report in pregnancy.
Corynebacterium urealyticum	– Urease test – Catalase test – Gram stain	Urease (+) Catalase (+) Gram-positive rods (palisades)	Alkaline urine; associated with encrusted catheters.
Aerococcus urinae	– PYR test – LAP test – Gram stain	PYR (–) LAP (+) Gram-positive cocci (tetrads/clusters)	Alpha-hemolytic; resembles enterococci but PYR-negative.

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Notes:

1. Indole Test: Differentiates E. coli (positive) from Klebsiella (negative).
2. Urease Test: Identifies Proteus and Corynebacterium urealyticum (urease-positive).
3. Oxidase Test: Critical for Pseudomonas aeruginosa (positive) vs. Enterobacteriaceae (negative).
4. CAMP Test: Confirms Streptococcus agalactiae (GBS).
5. Germ Tube Test: Rapid identification of Candida albicans.

List of Abbreviations with Full Forms:

1. AST: Antimicrobial Susceptibility Testing
2. ATP: Adenosine Triphosphate
3. BAP: Blood Agar Plate
4. CAMP: Christie-Atkins-Munch-Petersen test (enhanced hemolysis near Staphylococcus aureus).
5. CFU: Colony-Forming Units
6. CHOC: Chocolate Agar
7. CNA: Columbia Colistin-Nalidixic Acid Agar
8. CLED: Cystine-Lactose-Electrolyte-Deficient Agar
9. EMB: Eosin Methylene Blue Agar
10. hpf: High-Power Field
11. IMViC: Indole (+), Methyl Red (+), Voges-Proskauer (–), Citrate (–) for E. coli.
12. LF: Lactose-fermenting.
13. MAC: MacConkey Agar
14. mL: Milliliter
15. NLF: Non-lactose-fermenting.
16. PAD: Phenylalanine Deaminase.
17. PEA: Phenylethyl Alcohol Agar
18. PYR: Pyrrolidonyl Arylamidase Test
19. rpm: Revolutions Per Minute
20. SPA: Suprapubic Aspiration
21. TB: Tuberculosis
22. TSI: Triple Sugar Iron agar.
23. UTI: Urinary Tract Infection
24. VRE: Vancomycin-Resistant Enterococci.
25. WBC: White Blood Cells
26. µL: Microliter

References:

1. American Academy of Pediatrics (2011) guidelines.
2. College of American Pathologists (CAP) Q-Probes Study (2008).
3. MLO-May 2010: Urine Cultures: Contaminants, Skin Flora, or?

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Possible References Used

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