History
For more than 15 years, a substantial portion of the pharmaceutical industry has relied on the MIDI Sherlock® Microbial Identification System for identification in their microbiological testing laboratories. The Sherlock System identifies microorganisms based on gas chromatographic (GC) analysis of extracted microbial fatty acid methyl esters (FAMEs). Microbial fatty acid profiles are unique from one species to another, and this has allowed for the creation of very large microbial libraries. The current Sherlock System libraries have over 1,500 bacterial species, along with 200 species of yeast.
Procedure
Figure1. MIDI’s Fatty Acid-based Microbial Identification System Workflow.
More than 300 fatty acids and related compounds are found in bacteria. The wealth of information contained in these compounds is both in the qualitative differences (usually at genus level) and quantitative differences (commonly at species level).
The Sherlock System requires that bacteria be grown in culture. The fatty acids are extracted by a procedure which consists of saponification in dilute sodium hydroxide/methanol solution followed by derivatization with dilute hydrochloric acid/methanol solution to give the respective methyl esters (FAMEs). The FAMEs are then extracted from the aqueous phase by the use of an organic solvent and the resulting extract is analyzed by GC (Table 1).
TABLE: 1
| STEP | PURPOSE |
| Harvesting | Removal of Cells from culture media |
| Saponification | Lysis of the cells to liberate fatty acids from the cellular lipids |
| Methylation | Formation of fatty acid methyl esters (FAME’s) |
| Extraction | Transfer of the FAME’s from aqueous phase to organic phase |
| Base Wash | Aqueous wash of the organic extract prior to chromatographic analysis |
As the bacteria are killed in the saponification step of the extraction, there is little infectivity concern with handling of the sample once this step is concluded. FAMEs are more volatile than their respective fatty acids and therefore more suitable to GC analysis. The Sherlock software automates all analytical operations and uses a sophisticated pattern recognition algorithm to match the unknown FAME profile to the stored library entries for identification.
The technique used by the Sherlock System to present results is based on a Similarity Index (SI). The SI is a numerical value, which expresses how closely the fatty acid composition of an unknown compares with the mean fatty acid composition of the strains used to create the library entry listed as its match.
The Sherlock System is capable of identifying a wide range of microorganisms. Screening isolates for physiological or biochemical grouping before analysis is not required. Each library was carefully developed by collecting well-characterized strains of reference cultures from microbiologists specializing in many areas, including: clinical, environmental, industrial, drinking/waste water, and food. To provide for normal species variability, Sherlock library entries include many different strains of each organism. As an example, the library entry for B. anthracis has over 70 strains for this entry alone.
In the combined Sherlock libraries, there are nearly 2,000 microbial species, including 700 environmental aerobic species, 620 anaerobic species, and 200 species of yeasts.
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