Purpose
To prepare validated serial dilutions for microbiological procedures such as enumeration, quantitative testing or assay preparation.
Scope
This procedure applies to trained microbiology laboratory personnel working within an approved quality-management and biosafety system.
Principle
Each dilution is prepared by transferring a defined portion of material into a defined volume of diluent, producing a predictable sequential reduction in concentration.
Specimen / Test Material
Appropriate specimen suspension, culture suspension or other material approved for the intended test.
Equipment and Materials
- Sterile or validated diluent
- Correctly labelled tubes or containers
- Calibrated pipettes or dispensing devices
- Vortex mixer where appropriate
- Personal protective equipment
Procedure
- Label dilution containers in sequence.
- Add the required validated volume of diluent.
- Mix the starting material as required.
- Transfer the validated aliquot to the first dilution.
- Mix appropriately.
- Continue sequential transfers using clean technique.
- Process the required dilution(s) according to the intended test.
Quality Control and Quality Assurance
- Verify pipette performance.
- Use correct dilution calculations and labelling.
- Include contamination controls where required.
Reporting
Report dilution factors and results using the approved calculation and reporting method.
Safety
Use risk-based biosafety practices, appropriate personal protective equipment and approved decontamination and waste-disposal procedures. Treat clinical specimens and cultures according to the laboratory's biological risk assessment.
References
- World Health Organization (WHO): Toolkit for Standard Operating Procedures for Bacteriology, Mycology and Antimicrobial Susceptibility Testing.
- WHO: Laboratory Biosafety Manual, 4th edition.
- WHO guidance on collection, packaging and transport of infectious substances.
- EUCAST methodology and quality-control guidance for antimicrobial susceptibility testing, where applicable.
- Current locally approved laboratory procedures and validated manufacturer instructions.