Sherlock Equipment and Consumables List Phospholipid Fatty Acid (PLFA) Extraction Method

Significance of PLFA Extraction Method

Phospholipid fatty acid profiling offers a sensitive approach to quantify living microbial biomass and assess community composition by targeting membrane lipids. This technique is widely applied in environmental monitoring, soil and wastewater analysis, and industrial bioprocess control, providing crucial insights into microbial dynamics and ecosystem health.

Objectives and Overview

The purpose of the provided protocol is to enumerate all reagents, consumables, instrumentation, gas supplies, and proprietary items required to perform the PLFA extraction procedure using the Sherlock Microbial Identification System. By detailing US-sourced part numbers and critical items without substitutes, the overview enables laboratories to reproduce the method with confidence and interchange materials when needed.

Methodology and Instrumentation

The PLFA extraction workflow involves several key stages: lipid extraction with a chloroform–methanol–water mixture, saponification and methylation of phospholipids, solid-phase extraction cleanup, and solvent concentration. High-purity solvents (chloroform, methanol, hexanes, acetone) and specific reagents (potassium hydroxide, toluene, acetic acid, phosphate buffers) ensure sample integrity. An internal standard (19:0 PLFA) is introduced to calibrate recoveries. Consumables such as glass culture tubes, GC vials with PTFE/silicone/PTFE septa, and PFA pipette tips support trace-level accuracy.

Instrumentation Used

• Adjustable-volume pipettes, bottle-top dispensers, and pipette pumps for precise volume delivery
• Digital vortex mixer and sonication bath for sample homogenization
• Lyophilizer or freeze dryer for sample drying and concentration
• Vacuum concentrator system (CentriVap) with cold trap and acid-resistant rotor
• Rotary vane or hybrid vacuum pump to support SPE procedures
• Eppendorf centrifuge with swinging-bucket rotor for phase separation
• Tube revolver and paddle attachments for gentle, uniform mixing
• Gas chromatograph fitted with certified Ultra 2 capillary columns and calibrated injection port liners
• Incubator, fume hood, pH meter, and approved solvent disposal containers for safe handling

Main Results and Discussion

Although primarily a materials compilation, the protocol underscores the impact of reagent and gas purity on analytical performance. Specifying 99.9 percent solvent grades and instrument-grade hydrogen, nitrogen, and zero-air ensures baseline stability and peak resolution in GC analyses. The absence of acceptable substitutes for key items highlights their critical role in method reproducibility.

Benefits and Practical Applications

This standardized inventory accelerates method implementation in QA/QC, environmental assessment, and microbial ecology research. By streamlining procurement and minimizing variability, laboratories can achieve consistent PLFA profiles, facilitating cross-study comparisons and reliable quality control in industrial and academic settings.

Future Trends and Opportunities

• Automation and miniaturization of PLFA workflows to boost throughput and reduce solvent use
• Integration of PLFA extraction with mass spectrometric detection for enhanced molecular specificity
• Development of on-line sampling interfaces for real-time microbial community monitoring
• Expansion of informatics platforms and databases to improve lipid fingerprint interpretation

Conclusion

Providing a detailed list of reagents, consumables, instrumentation, and gas requirements, this resource equips laboratories to perform PLFA extraction with the Sherlock MIS reliably. Adherence to specified materials and purity standards is essential for accurate, reproducible microbial lipid profiling across diverse analytical applications.