Automated bacterial sample preparation from agar to MALDI target for routine bacterial identification

Significance of the Topic

Matrix‐assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has revolutionized species-level microbial identification by offering rapid, accurate results. However, traditional smear‐technique sample preparation is manual, labor-intensive and susceptible to operator variability, affecting spot quality and reproducibility.

Study Objectives and Overview

The study aimed to integrate a colony-picking robot into the MALDI-TOF workflow to automate sample transfer and matrix application, comparing three preparation strategies to identify an optimized, high-throughput approach.

Methodology and Instrumentation

• Organisms: Escherichia coli for method optimization; Bacillus subtilis, Cellulomonas uda and Pantoea agglomerans for validation.
• Culture conditions: Columbia agar with 5 % horse blood, 18–24 h incubation; samples stored on porous beads.
• Matrix: α-Cyano-4-hydroxycinnamic acid (CHCA), 40 mg/mL in acetonitrile/ethanol/water with 3 % TFA.
• Target: Disposable FlexiMass-DS slides (Shimadzu).
• Automation: PIXL colony-picking robot (Singer Instruments) using pinning (‘dab’) and smear-like deposition modes.
• Mass spectrometer: iDplus Performance MALDI-TOF (Shimadzu); data interpretation via SARAMIS database.

Main Results and Discussion

• Three methods tested: A) mix colonies with matrix in microplate well; B) robot smear + manual matrix addition; C) fully automated smear and single/double matrix deposition.
• Method A failed reproducibly due to polymer contamination (m/z 2000–8000), likely from the microplate.
• Methods B and C achieved >99.9 % confidence identifications for E. coli with >200 detected peaks; double matrix dip increased peak counts (Av. 214 vs. 172).
• Method C was selected for full validation: 16 spots per slide for each bacterial species; high‐confidence IDs obtained across B. subtilis, C. uda and P. agglomerans.
• Mixed identifications for B. subtilis indicated sample heterogeneity consistent with manual controls.

Benefits and Practical Applications

• Enhanced reproducibility by minimizing user-dependent variability in sample transfer and matrix addition.
• Reduced hands-on time and labor, facilitating high-throughput workflows in microbiology laboratories.
• Improved consistency benefits quality assurance in clinical, research and industrial settings, particularly for less experienced operators.

Future Trends and Potential Applications

• Fully automated colony detection and spot selection integrated with real-time quality control.
• Expansion to multiplexed assays and direct colony analysis without prior subculture.
• Integration of AI-driven algorithms for optimized spot morphology and spectral quality.
• Linkage with laboratory information management systems (LIMS) for seamless data tracking and reporting.

Conclusion

The PIXL colony-picking robot successfully prepared MALDI-TOF samples suitable for confident microbial identification, matching the performance of skilled manual preparations. Full automation of smear deposition and matrix application enhances reproducibility and throughput in routine bacterial identification workflows.

Reference

• Nairn MD, Kirk P, Openshaw ME, Severn O, Ashley L. Automated bacterial sample preparation from agar to MALDI target for routine bacterial identification. Shimadzu & Singer Instruments.