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SOP

Bacterial Culture and Antimicrobial Susceptibility Testing

SOP for bacterial culture, isolation, identification, and antimicrobial susceptibility testing (AST).

Last verified 1 month ago 2 min read Microbiology Bacteriology Antimicrobial Susceptibility Testing Culture #microbiology #AST #Kirby-Bauer #Antimicrobial Susceptibility #culture and sensitivity #antibiotic testing
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Document Control
Version
1.0
Effective
Aug 28, 2026
Reviewed
Aug 29, 2026
Department
Microbiology
Principle

Bacterial culture isolates pathogens on appropriate media. Identification uses morphological, biochemical, and/or molecular characteristics. Antimicrobial susceptibility testing (disc diffusion) measures the ability of antibiotics to inhibit bacterial growth. Antibiotic discs diffuse into agar; bacteria grow except where antibiotic concentration exceeds the minimum inhibitory concentration (MIC). Zone diameter correlates with susceptibility.

Standard Operating Procedure: Culture and Sensitivity

1. Purpose and Scope

To isolate, identify bacterial pathogens from clinical specimens and determine their susceptibility to antimicrobial agents.

2. Specimen Requirements

  • Type: Various (blood, urine, sputum, wound, CSF, body fluids, tissue).
  • Collection: Use appropriate collection devices and transport media.
  • Storage: Process immediately or store at appropriate temperature (usually 2-8°C).
  • Rejection Criteria: Improperly labeled, contaminated, or delayed specimens.

3. Safety and Precautions

  • Wear appropriate PPE.
  • Handle all specimens as potentially infectious.
  • Work in biological safety cabinet for high-risk specimens.

4. Equipment and Reagents

  • Culture media (blood agar, MacConkey, chocolate agar, selective media).
  • Incubator (35-37°C, 5% CO₂ if needed).
  • Biochemical identification tests or automated system.
  • Mueller-Hinton agar for AST.
  • Antibiotic discs, McFarland standards.
  • Inoculating loops, sterile swabs.

5. Step-by-Step Procedure

A. Culture

  1. Inoculation: Inoculate specimen onto appropriate media using aseptic technique.
  2. Streaking: Use quadrant streak method for isolation.
  3. Incubation: Incubate at 35-37°C for 18-24 hours (or longer for slow growers).
  4. Examination: Examine for growth, colony morphology, hemolysis pattern.

B. Identification

  1. Gram stain: Perform on representative colonies.
  2. Biochemical tests: Perform appropriate tests (catalase, coagulase, oxidase, sugar fermentation, etc.) or use automated system.
  3. Confirm identification to species level when clinically relevant.

C. Antimicrobial Susceptibility Testing (Kirby-Bauer Disc Diffusion)

  1. Inoculum preparation: Pick 3-5 well-isolated colonies, suspend in saline/broth to match 0.5 McFarland standard (1-2 × 10⁸ CFU/mL).
  2. Inoculation: Within 15 minutes, dip sterile swab into suspension, remove excess, inoculate entire surface of Mueller-Hinton agar plate (3 swabs in 3 directions).
  3. Disc application: Apply antibiotic discs with sterile forceps or dispenser (center-to-center ≥24 mm, ≥15 mm from edge).
  4. Incubation: Invert plates, incubate at 35-37°C for 16-18 hours.
  5. Reading: Measure zone diameters (including disc) in mm.

D. Interpretation

  • Compare zone diameters to CLSI/EUCAST breakpoints.
  • Categorize as: S (Susceptible), I (Intermediate), R (Resistant).

6. Quality Control

  • Run QC strains (E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27853) weekly or with each new batch of media/discs.
  • Verify zone diameters fall within acceptable ranges.

7. Reporting Results

  • Report organism identification and susceptibility pattern.
  • Report critical resistance immediately (e.g., MRSA, VRE, ESBL, carbapenem-resistant).
  • Include interpretive comments when appropriate.

Frequently Asked Questions

Mueller-Hinton agar has low inhibitors, good reproducibility, and allows adequate diffusion of antibiotics.
Intermediate indicates the organism may be susceptible if antibiotic is concentrated at the infection site or if higher doses are used.