Evaluation of a Novel Cryopreservation Methodology for Quality Control and Reference Microorganisms in Microbiology Laboratories.

Introduction and Background:

Preserving bacterial and fungal cultures will maintain viability, genetic stability, and metabolic activity is crucial for research, biotechnology, clinical microbiology quality control (QC), teaching, and tackling antimicrobial resistance (AMR). Smaller laboratories often face challenges accessing and maintaining QC strains due to cost and resource limitations. This study proposes and evaluates a standardized, cost-effective cryopreservation method intended to facilitate reliable preservation and potential future distribution of QC and reference strains from central/reference laboratories to smaller facilities, enhancing quality assurance and patient safety across the diagnostic network.

Study Objectives

The primary objective is to evaluate the effectiveness of a novel cryopreservation methodology based on routine QC data. Specifically, we aim to assess:
 - Long-term viability and stability of a wide range of bacterial and fungal QC strains.
-  Preservation of genetic integrity and prevention of drift/mutation.
-  Maintenance of metabolic activity relevant to QC testing.
-  The practicality, efficiency, and cost-effectiveness of the method compared to traditional approaches.

Methodology

-  Samples: The study will utilize existing, anonymized Quality Control (QC) strains routinely used in our laboratory (e.g., ATCC strains, and internal QC strains) and de-identified, residual clinical isolates (bacterial and fungal) originally collected for diagnostic purposes, where further identification/sensitivity testing is warranted. No new samples will be collected specifically from patients for this study. All isolates are handled as part of standard laboratory procedures.
- Preservation Technique:
    . Master Stock: Microorganisms will be preserved in Brain Heart Infusion (BHI) broth supplemented with 15% glycerol and stored at -80°C.
    . Working Stock: Aliquots will be stored at -20°C.
-  QC Testing: Working stocks will be sub-cultured weekly (for QC purposes) onto Mueller Hinton Agar (MHA) or appropriate media. Fastidious organisms will be sub-cultured bi-weekly from working stocks. Viability, purity, and performance in standard QC tests (e.g., antibiotic susceptibility testing for relevant strains) will be rigorously monitored and recorded daily/weekly as per protocol.

Conclusion

The proposed method will have a potential to revolutionize the field of microbiology and broaden the applications of bacterial preservation in quality assurance, tackling antimicrobial resistance, research and training.

Recommendations

The authors recommend the methodology to be rolled out by the tertiary and reference laboratories to work with smaller upcoming laboratories for quality assurance and patient safety through time, quality and reliable microbiology results.