MIDI Sherlock® Instant fatty acid methyl ester (FAME) User's Guide

Importance of Topic

Rapid and reproducible microbial identification based on fatty acid methyl ester (FAME) profiling is critical for clinical diagnostics, environmental monitoring and industrial quality control. The Instant FAME™ protocol dramatically reduces sample preparation time from 90 minutes to under 3 minutes while increasing sensitivity ten‐fold, enabling high‐throughput analysis of bacteria, mycobacteria and yeast.

Study Objectives and Overview

This user guide describes the Instant FAME extraction workflow integrated into the Sherlock® Microbial Identification System. It outlines growth conditions, extraction reagents and stepwise sample preparation. The guide compares Instant FAME with traditional methods, details library versions, and provides troubleshooting advice to ensure robust, consistent identifications.

Methodology and Instrumentation

All operations are performed in a single 2 ml GC vial using three proprietary reagents. Key steps:

• Harvest 2.5–3.0 mg wet cells from log‐phase culture (quadrant 3 of purity streak).
• Add 250 µl Reagent 1, vortex 10 s, add 250 µl Reagent 2, vortex 3 s, then add 250 µl Reagent 3 to induce phase separation.
• Transfer 70 µl of the upper organic layer into an insert using the MIDI pipette guide to avoid interface contamination.

Extracts are analyzed on an Agilent 6850/6890/7890 GC equipped with a capillary column and flame ionization detector, controlled by Sherlock MIS v6.0 or later. Periodic calibration uses the MIDI Rapid Method Calibration Mix (Part 1300-AA) and performance qualification (PQ) tables to track retention time and response drift.

Main Findings and Discussion

Instant FAME delivers comparable or improved library match scores versus standard methods, with Total Response maintained between 100 000 and 500 000. Fewer cells and reagent volumes minimize solvent waste. QC and blank controls validate reagent integrity, instrument performance and operator consistency. A detailed PQ table and troubleshooting matrix guide pressure, temperature and maintenance adjustments to sustain reliable calibrations.

Benefits and Practical Applications

• 30× faster sample prep and 10× greater sensitivity versus traditional FAME extraction.
• Elimination of water baths and tumblers reduces equipment footprint.
• Improved throughput supports large‐scale microbial surveillance and clinical laboratories.
• Lower reagent volumes and benign chemistries enhance laboratory safety and reduce waste disposal costs.

Future Trends and Possibilities

Ongoing advances may include further integration of microfluidic extraction, automated liquid handlers, expanded spectral libraries for emerging pathogens and machine‐learning algorithms for enhanced pattern recognition. Cloud-based data sharing and AI‐driven updates could streamline method validation and cross‐laboratory standardization.

Conclusion

The Instant FAME protocol in the Sherlock MIS represents a significant improvement in microbial FAME analysis, combining speed, sensitivity and ease of use. Proper adherence to growth, extraction and QC procedures ensures reliable identifications across clinical, environmental and industrial settings.

Reference

MIDI, Inc. Sherlock Software and Libraries License Agreement and User Manual. Newark, DE; 2012.