125 Sandy Drive, Newark, Delaware 19713 USA

tel: 302-737-4297 fax: 302-737-7781 www.midi-inc.com

Sherlock v.6.3 Update – PLFA Enhancements

1. PLFAD1 method updates

a. PLFAD1 v.1.20 to v.1.30.

b. Adjustment of several peak locations.

c. Addition of three compounds: 17:1 iso ω10c, 18:4 ω3c, and 17:0 iso 3OH.

d. The default weight of the 19:0 Internal Standard is updated to 10,000 (per the Buyer standard).

2. Subtraction of background contamination

The BackgroundSubtract program eliminates a known set of background peaks from the sample. This tool is useful for

extraction procedures that leave a known set of compounds in the sample that originate from the sample

preparation.

3. PLFA Tools Category file updates

a. The Fatty Acid Type file is updated to reflect new peaks (FATYPE3).

b. The Ratio file is updated to reflect new peaks and to merge the Gram-neg stress ratios (RATIO3).

c. The Microbial type file now comes with two versions (note, customers can create own versions).

i. The 18:1 ω9c peak assigned to the fungal group (MICFUNG3).

ii. The 18:1 ω9c peak assigned to the Gram-positive group (MICSOIL3).

The Method for MICSOIL3 is automatically available for activation (instead of requiring the user to generate it).

4. Automatic post-process actions

The capability for automatically running post-process actions is shipped along with a default setup for running the

Scale and Category processes. The program EditProcessOptions is also included for editing the desired transforms. The

default setup file is shipped “off” but a single check-box in EditProcessOptions turns it on.

5. PLFA Tools Scale file updates

Sherlock 6.2B only included a PLFAMole file which adjusted for molarity but could not be used for

weights. Sherlock 6.3 includes an updated PLFAMole3 file (with the new peaks) and also a PLFAWeight3 file which

should be used when weights are desired instead of moles.

6. Data Export updates

A new crosstab is added to all Data Export databases; it uses the amounts from the run instead of the percentages.

This allows PLFA customers to determine quantitative results without having to edit queries.

SherlockTM Microbial IdentificationSystem v.6.3 with Traditional Methods & PLFA

Specification Sheet

General DescriptionThe SherlockTM Microbial IdentificationSystem (MIS) identifies fatty acid methylesters (FAMEs) by gas chromatography.The resulting FAME profile can be usedto identify the fatty acids in a sample(e.g. for soil PLFA analysis), or theresulting profile can be compared to oneof Sherlock’s microbial libraries (e.g. forplant pathogenic bacterial identification).

For a complete and automated microbialidentification and fatty acid analysissolution, the Sherlock software,methods and libraries are combined withan Agilent 6850, 7820 or 7890 modelGC and Agilent ChemStation software.

Sherlock’s pattern recognitionalgorithms, combined with its calibrationmixture, standardize each instrument.This virtually eliminates the manualcalibration adjustments associated witha GC. No chromatography knowledgeor experience is required.

Methods & LibrariesThe Sherlock MIS contains Standard, aswell as Optional Methods & Libraries.

Standard Methods & Libraries

Environmental Aerobes (RTSBA)• 916 entries.• Trypticase Soy Broth Agar (TSBA).• 28oC ± 2ºC for 24 ± 2 hours.

Clinical Aerobes (RCLIN)• 593 entries.• Trypticase Soy Agar (TSA) with 5%

defibrinated sheep blood.• 35ºC ± 2ºC for 24 ± 2 hours.

Optional Method

Phospholipid Fatty Acids (PLFAD1)• 168 fatty acids and related.• Uses any extraction protocol.• Peak naming only – no library.• Includes PLFA Tools & Background

Subtract software.

Optional Methods & Libraries

Biothreat Agents (RBTR)• 60 entries.• Trypticase Soy Agar (TSA) with

5% defibrinated sheep blood.• 35ºC ± 2ºC for 24 ± 2 hours.

Anaerobes (SMOORE)• 769 entries.• PYG Broth.• 35ºC ± 2ºC for 24 ± 2 hours.

Environmental Yeast (SYEAST)• 196 entries.• SAB Dextrose Agar.• 28oC ± 2ºC for 24 ± 2 hours.

Instrument ThroughputFollowing a short preparationprocedure (typically done inbatches), the sample vials areloaded into the instrument’sautosampler. The automatedsystem takes over and analyzeseach sample.

• The RAPID methods (methodname starts with an “R”) process6 samples per hour.

• The SENSITIVE methods(method name starts with an “S”)process 2 samples per hour.

• The PLFAD1 method processes4 samples per hour.

CulturingMicrobial Identification Only

Similar to all microbial identificationsystems, Sherlock requires purecultures. Using standard laboratorytechniques, a single subculturefrom the primary isolation plate,incubated for 24 hours, is typicallysufficient for performing theanalysis. Slow-growingorganisms typically require48-hour incubation times.

Sample PreparationMicrobial Identification Only*

Using inexpensive reagents,available from almost any chemicalsupply house, a technicianaverages only 5 minutes persample to prepare a batch of 30samples. Each sample is preparedfor analysis using a liquid-liquidextraction in a single test tube.

• Harvesting a small quantity ofcells from the culture plate is themost labor-intensive step. It willtypically take 1 hour or less toharvest cells from 30 plates into30 test tubes.

• The four-step liquid-liquidextraction process requires about1½ hours or less for a batch of30 samples. During theextraction process,approximately 35 minutes of“wait time” are available for thetechnician to do paper work andother tasks.

• The same sample preparation isused on all samples. It is notnecessary to do a Gram stain orother offline tests beforepreparing and analyzing asample.

*Note: The PLFAD1 method uses adifferent extraction protocol, whichis not discussed here.

Bio-SafetyMicrobial Identification Only

Live microbes are not introducedinto the instrument. The first step ofthe extraction procedure treats thecells with a sodium hydroxidesolution for 30 minutes in a 100oCwater bath. This kills the microbes,including both vegetative cells andspores.

125 Sandy Drive, Newark, Delaware 19713 USA

tel: 302-737-4297 fax: 302-737-7781 www.midi-inc.com

Automated Phospholipid Fatty Acid (PLFA) Analysisusing MIDI’s Sherlock PLFA Analysis Software

Application Note – Agriculture | Bioremediation

Abstract

Soil phospholipid fatty acid (PLFA) analysis can provide a real-time snapshot of the soil microbialcommunity (soil microbiota) structure. Using a high throughput PLFA extraction method1, coupledto an automated PLFA naming process reduces turnaround time and reagent use, while limitingpotential errors that can occur with “manual” PLFA analysis approaches.

Introduction

The soil microbiota is responsible for many ecosystem functions such as plant growth regulation, nutrientcycling and carbon sequestration. Additionally, the microbiota has the ability to degrade environmentalpollutants, such as PAHs and PCBs. The microbiota is highly sensitive to soil-altering processes (degradativeor beneficial) and changes can guide appropriate management procedures (conservation or restoration).

Phospholipids are an essential structural component of allmicrobial cellular membranes. Upon microbial death,phospholipids rapidly degrade. Phospholipid content in a soilsample is therefore assumed to be from the living microbiota.Phospholipid fatty acids (PLFAs) are the main structuralcomponent of the phospholipid and serve as useful biomarkersfor different microbial groups. PLFA analysis is a widely-usedtechnique for estimation of the total biomass and to observebroad changes in the soil microbiota composition. Multipledifferent Gas Chromatography (GC) and Gas Chromatography-Mass Spectroscopy (GC-MS) methods and instrument typeshave been used to determine PLFA profiles. However, most ofthis analysis is performed manually, and the analysis process islaborious and potentially error-prone.

MIDI Inc.’s Sherlock PLFA Analysis Software automatically names all the PLFAs in a sample and categorizesthem by microbial origin (e.g. Actinomycetes). This automated process yields consistent and easy-to-interpret results with less chance of errors. The sensitivity of the Sherlock method ensures that all PLFAsare measured. Further, the PLFA data can be visualized with the Sherlock 2-D Plot and Dendrogram tools orexported to Microsoft Excel® or Access® databases for further study and ease of publication.

Experimental

This note details the Sherlock PLFA analysis of a soil sample following a high throughput PLFA extractionprotocol1. A known number of moles of the internal standard (IS), 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (19:0 PC, Avanti Polar Lipids p/n 850367), were added at the beginning of the process.

After the PLFAD1 Method was selected from the available methods within the Sherlock Sample Processor,the correct method parameters were automatically loaded into the Agilent GC. The PLFA CalibrationStandard was processed first, followed by the soil sample run. The Sherlock software automaticallycalculated each peak name and weight percent.

The Sherlock PLFA Tools software was then used to calculate mole percent, scale the data to the IS,calculate the biomass (nmol/g) for each microbial type (customizable) and calculate key PLFAratios (customizable). 54 fatty acids were identified in the sample and 97.84% of the peakswere named.

Sherlock PLFA Analysis – Peak Identification Report

Method: PLFAD1 File: E132054.66CSample ID: D-PEN-13-01(24-SOIL G=2Created: 2/5/2013 12:19:21 PM

RT Response RFact ECL Peak Name %0.7232 1.656E+9 ---- 7.7257 SOLVENT PEAK ----1.0529 567 ---- 9.5153 ----2.1600 3893 1.056 13.6119 14:0 iso 0.662.3211 3753 1.035 13.9997 14:0 0.622.5421 6077 1.014 14.4399 15:1 iso w6c 0.982.5648 1236 1.011 14.4853 15:4 w3c 0.202.5878 1838 1.010 14.5310 15:1 anteiso w9c 0.302.6308 36686 1.006 14.6167 15:0 iso 5.892.6777 25287 1.002 14.7103 15:0 anteiso 4.052.7523 931 0.996 14.8588 15:1 w6c 0.152.8240 2834 0.991 15.0015 15:0 0.452.8562 2300 ---- 15.0566 ----3.0527 1199 0.978 15.3930 16:1 w7c alcohol 0.193.0832 5047 0.976 15.4453 15:0 DMA 0.793.1859 13682 0.971 15.6211 16:0 iso 2.123.2423 894 0.969 15.7176 16:0 anteiso 0.143.2719 11207 0.967 15.7684 16:1 w9c 1.733.3036 50107 0.966 15.8227 16:1 w7c 7.733.3573 26955 0.964 15.9146 16:1 w5c 4.153.4077 58996 0.962 16.0008 16:0 9.063.4396 2993 ---- 16.0501 ----3.5382 1000 ---- 16.2026 ----3.6817 57533 0.953 16.4245 16:0 10-methyl 8.763.7255 4193 0.952 16.4923 17:1 iso w9c 0.643.7523 3240 0.951 16.5338 17:1 anteiso w9c 0.493.7766 601 0.951 16.5713 17:1 anteiso w7c 0.093.8120 10652 0.950 16.6261 17:0 iso 1.623.8735 10902 0.948 16.7213 17:0 anteiso 1.653.9231 4934 0.947 16.7980 17:1 w8c 0.753.9867 19667 0.946 16.8963 17:0 cyclo w7c 2.974.0562 3581 0.944 17.0036 17:0 0.544.0825 5951 0.944 17.0414 17:1 w7c 10-methyl 0.90

4.1286 1576 ---- 17.1077 ----4.3375 3504 0.940 17.4077 17:0 10-methyl 0.534.4001 1401 ---- 17.4978 ----4.4813 4062 0.939 17.6144 18:0 iso 0.614.5593 12731 0.938 17.7264 18:2 w6c 1.914.5916 51588 0.938 17.7729 18:1 w9c 7.734.6302 79549 0.938 17.8283 18:1 w7c 11.9

14.6954 11914 0.937 17.9221 18:1 w5c 1.784.7510 17661 0.937 18.0019 18:0 2.644.8111 6320 0.937 18.0850 18:1 w7c 10-methyl 0.954.8654 2513 0.936 18.1603 17:0 iso 3OH 0.384.9590 682 0.936 18.2898 18:1 w7c DMA 0.105.0318 17852 0.936 18.3905 18:0 10-methyl 2.675.1131 759 0.936 18.5029 19:4 w6c 0.115.1541 1217 0.936 18.5597 19:3 w6c 0.185.2963 2156 ---- 18.7564 ----5.3379 1526 0.936 18.8140 19:1 w8c 0.235.4046 44722 0.936 18.9063 19:0 cyclo w7c 6.695.4748 13957 ---- 19.0033 19:0 ----5.6770 665 ---- 19.2768 ----5.7682 4816 0.938 19.4001 20:4 w6c 0.725.8238 2245 0.938 19.4753 20:5 w3c 0.345.8910 1338 0.939 19.5661 20:3 w6c 0.206.0414 6631 0.940 19.7695 20:1 w9c 1.006.2128 2615 0.941 20.0013 20:0 0.396.3548 2390 ---- 20.1937 ----6.7999 1502 0.946 20.7972 21:1 w8c 0.236.9162 1634 0.948 20.9549 21:1 w3c 0.257.1356 596 0.950 21.2533 22:5 w6c 0.097.6846 2087 0.955 22.0005 22:0 0.328.4044 837 0.960 22.9990 23:0 0.139.1112 2143 0.961 24.0007 24:0 0.339.4787 1784 ---- 24.5215 ----

Total Response: 669402 Percent Named: 97.84% Peaks Named: 54

Figure 1. Representative chromatogram for analysis of the soil sample using the Agilent 6890N GC.

Sherlock PLFA Tools – Normalizes the data for molarity, Scales to an Internal Standard,Transforms the data into Biomass (nmol/g) & user-defined Ratios

Biomass (nmol/g) by Microbial Type

Method: MICSOIL3 File: E132054.MICSample ID: D-PEN-13-01(24-SOIL G=2Created: 9/24/2015 2:19:15 PM

PLFA Origin Biomass (nmol/g)

Gram-Positive 33.21

Gram-Negative 56.30

AM Fungi 7.06

Fungi 2.96

Anaerobe 1.47

Actinomycetes 21.54

Other Eukaryote 2.73

Not Assigned 35.93

Total PLFA 161.20

Key PLFA ratios calculated automatically

Method: RATIO3A File: E132054.RATSample ID: D-PEN-13-01(24-SOIL G=2Created: 9/24/2015 2:27:22 PM

Ratio Name Ratio

Fungi/Bacteria 0.10

Predator/Prey 0.03

Gram+/Gram- 0.98

Sat/Unsat 0.84

Mono/Poly 10.54

GNeg Stress 2.14

Conclusion

PLFA analysis of soil samples via the Sherlock PLFA Analysis Software and Agilent GC provides an automatedand comprehensive method for analyzing PLFAs from the soil microbiota. Coupled to a high throughputextraction method1, the MIDI PLFA Solution results in a standardized PLFA protocol that can beimplemented by most soil science laboratories for detailed study of the soil microbiota. User-definedvariables (e.g. which fatty acids to assign to which microbial group) allow for customization of results.

min1 2 3 4 5 6 7 8 9

pA

15

20

25

30

35

40

45

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60

2.3

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2.5

42

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2.5

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2.6

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2.8

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3.1

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3.2

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3.7

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3.8

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13

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Reference

Buyer, J.S. & Sasser, M. (2012). High throughput phospholipid fatty acid analysis of soils. In Applied SoilEcology 61, 127-130.

Full Text Versionwww.sciencedirect.com/science/article/pii/S0929139312001400

GC Conditions________________________________________GC instrument Agilent 6890N SeriesAutosampler Agilent 7683 Injector and sample traySoftware MIDI Sherlock Software v.6.2B

with PLFA PackageAgilent OpenLab CDS ChemStation

Column Agilent Ultra 2, 25 m x 0.2 mm x0.33 µm film thickness (MIDI p/n Column G)

Liner Split liner, silanized (MIDI p/n 1221)Inlet temperatrure 250 °CCarrier gas Hydrogen, constant flow, 1.3 mL/minOven program 190 °C, 10 °C/min to 285 °C (9.5 min),

60 °C/min to 310 °C (0.42 min),Split ratio 30:1Injection volume 2.0 µL

FID___________________________________________________Temperature 300 °C

© MIDI, Inc., Sept 2015

Veteran-Owned Small Business

www.midi-inc.com

HardwareA Sherlock system is composed of aWindows® based computer loadedwith the MIDI Sherlock and AgilentOpenLab CDS ChemStation Editionsoftware. The computer is interfacedto one of the following Agilent GCs:

Agilent 6850 Series II GC

• 57cm x 28cm x 49cm (L x W x H).• Weight: 29kg.• Operating temp: 15ºC to 35ºC.• Operating humidity: 5% to 95%.

Agilent 7820A Series GC – Single

• 51cm x 56cm x 49cm (L x W x H).• Weight: 50kg.• Operating temp: 15ºC to 30ºC.• Operating humidity: 30% to 70%.

Agilent 7890 Series GC – Single• 51cm x 58cm x 49cm (L x W x H).• Weight: 49kg.• Operating temp: 15ºC to 35ºC.• Operating humidity: 5% to 95%.

Software PackagesThe Sherlock MIS containsStandard, as well as OptionalSoftware packages.

Note, the PLFA Software packagescome as part of the PLFA Methodpackage.

Standard Software Packages

Analysis

This software enables a user toexplore relationships betweensample data using:

• Dendrogram plots.• Neighbor-joining trees.• Principal component

analysis (PCA) with 2-D plotsand histograms.

The information can be exported toMicrosoft Office® and other packagesfor further analysis and forpublication purposes.

Data Export

This software enables a user toexport sample data, fatty acidprofiles, library match results andother information to Excel®

spreadsheets and Access®

databases. There are manyapplications for custom reports andcalculations created using Excel,Access, and other data analysistools:

• Trend analysis.• Custom reports.• Microbial population studies.

Optional Software Packages

Library Generation

This software enables a user tocreate custom libraries from anysample data. Uses for LibraryGeneration include:

• Create entries for species that arenot in the Sherlock libraries.

• Assign an identity to organismsthat do not have a publishedtaxonomy.

• Alternative growth conditions.

Tracker/Cluster

This optional software enables auser to locate the source of acontamination. Tracker andCluster operate independently ofsample identification, allowingunknown samples to be compared.

Tracker locates other samples thatare likely to be the same strain as asample of interest. Tracker searchesfor matches between the currentsample and all previous samples.

Cluster automatically finds groups(clusters) of highly related samples.

Uses for Tracker/Cluster include:

• Track a consistent, yetunnamed contaminant.

• Trend analysis.• Microbial population studies.

PLFA Analysis Tools

This software enables a user toautomatically perform complexcalculations, including the following:

• Adjusting for the molarity of differentfatty acid compounds.

• Scaling by a known amount ofinternal standard.

• Categorizing results based onBiomarker groups (e.g. G+/G-).

• Calculating key Microbial ratios (e.g.Fungi/Bacteria).

PLFA Background SubtractThis software enables a user toautomatically:

• Eliminate a known set of backgroundpeaks from the sample.

• Useful for sample preparations thatcontribute consistent contamination.

Markets Using Sherlock• Animal Science• Biodefense / Public Health• Bioremediation• Clinical Microbiology• Edible Oil Analysis• Marine Science• Microbial Culture Collections• Pharmaceutical QC• Plant Pathology / Protection• Renewable Energy• Soil Science / PLFA• Taxonomy Studies

The information in this publication issubject to change without notice.

Copyright 2016 MIDI, Inc.All Rights Reserved

Date: 01 June 2016

MIDI, Inc.125 Sandy DriveNewark, Delaware 19713 USAPhone: 302-737-4297Fax: 302-737-7781Email: sales@midi-inc.comWeb: www.midi-inc.com