Clin Infect Dis.-2013--564-72

M E D I C A L M I C R O B I O L O G Y I N V I T E D A R T I C L EMelvin P. Weinstein and L. Barth Reller, Section Editors

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in ClinicalMicrobiology

Robin Patel

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology and Division of Infectious Diseases, Department of Medicine, MayoClinic, Rochester, Minnesota

Despite widespread application of nucleic acid diagnostics, cultures remain integral in modern laboratories.Because cultures detect a large number of organism types, it is unlikely that they will disappear from clinicalpractice in the near future. Their downside is slow turn-around time, impacted by time to growth and identifi-cation of that growth. The latter is expedited using a new proteomic technology, matrix-assisted laser desorp-tion ionization–time of flight mass spectrometry (MALDI-TOF MS).

Keywords. mass spectrometry; bacteria; fungi; MALDI-TOF; matrix-assisted laser desorption ionization-timeof flight.

Despite widespread application of nucleic acid diagnos-tics, cultures remain integral in modern laboratories.Because cultures detect a large number of organismtypes, it is unlikely that they will disappear from clinicalpractice in the near future. Their downside is slowturn-around time, impacted by time to growth andidentification of that growth. The latter is expeditedusing a new proteomic technology, matrix-assistedlaser desorption ionization–time of flight mass spec-trometry (MALDI-TOF MS).

Historically, identification of cultured bacteria andfungi has been a complex, algorithmic task. Textbooks,flowcharts, and tables have been used to interpretcolony morphology as well as stains and biochemicaltests performed on colonies. Phenotypic tests may becontingent on subsequent growth, prolonging time toidentification. Manual biochemical tests (eg, coagulase,

catalase) are rapid, but identify limited organism types(eg, Staphylococcus aureus). Biochemical test panelsperformed manually (eg, API strips, bioMérieux, Dur-ham, North Carolina) or on automated instruments(eg, VITEK, bioMérieux; BD Phoenix Automated Mi-crobiology System, BD, Franklin Lakes, New Jersey; Mi-croScan, Siemens Healthcare Diagnostics, Tarrytown,New York) identify more microorganisms, but havelonger turn-around times and costly consumables, andoften require knowledge of the organism type beingtested (eg, gram-positive cocci). Finally, broad-rangePCR followed by sequencing accurately identifies bacte-ria and fungi but has a long turn-around time, and isexpensive and performed in select laboratories. WithMALDI-TOF MS, colonies growing in culture are inex-pensively and accurately identified in minutes, withouta priori knowledge of microorganism type; users donot even need to know whether a bacterium or a yeastis being tested. The product of advances in mass spec-trometry and bioinformatics, MALDI-TOF MS is revo-lutionizing bacterial and fungal identification in clinicalmicrobiology [1].

The concept of using mass spectrometry to identifybacteria (albeit not based on proteomics) was proposedin the 1970s [2];with the Nobel Prize–winning inventionof MALDI-TOF MS in the 1980s [3, 4], whole-organism

Received 29 January 2013; accepted 5 April 2013; electronically published 17April 2013.

Correspondence: Robin Patel, MD, Mayo Clinic, Division of Clinical Microbiology,200 First St SW, Rochester, MN 55905 ([email protected]).

Clinical Infectious Diseases 2013;57(4):564–72© The Author 2013. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected]: 10.1093/cid/cit247

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proteomics became possible. It took until the 1990s for develop-ment of libraries of reference spectra and software for bacterialidentification, and until the past few years for commercializationof the technology [1, 5]. MALDI-TOF MS, used for a few yearsin European clinical microbiology laboratories, and being rap-idly adopted worldwide, is likely to antiquate most biochemicalmicrobial identification in the near future.

The simplest MALDI-TOF MS applications test bacterial oryeast colonies without elaborate preparation and using minimalconsumables, providing rapid and low-cost identification.Testing begins by “picking” an unknown colony from a cultureplate to a “spot” on a MALDI-TOF MS target plate (Figure 1).The cells may be treated on the target plate (we use formicacid) and are ultimately overlain with 1–2 μL of matrix. Follow-ing a short drying period, the plate is placed in the ionizationchamber of the mass spectrometer (Figure 2).

“MALDI” refers to the matrix, which assists in the desorp-tion and ionization of microbial analytes through pulses ofenergy from an ultraviolet N2 laser. The matrix (eg, α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile and 2.5%

trifluoroacetic acid) isolates microbial molecules from eachother; microbial and matrix molecules on the target plate aredesorbed, with the majority of energy being absorbed by thematrix, converting it to an ionized state. Through random colli-sion in the gas phase, charge is transferred from matrix to mi-crobial molecules; analytes within the mass range specified aremeasured, the most abundant being ribosomal proteins. Thecloud of ionized microbial proteins is funneled through a posi-tively charged electrostatic field into a time of flight or “TOF”mass analyzer, a tube under vacuum. The ions travel toward anion detector with small analytes traveling fastest, followed byprogressively larger analytes. A mass spectrum is generated,representing the number of ions of a given mass impacting theion detector over time.

Analytes are not individually characterized; instead, the gen-erated mass spectrum provides a profile (“fingerprint”) of theunknown microorganism. Generated mass spectra are uniqueto individual microorganism types, with peaks specific togenera, species, and even strains. The test isolate’s mass spec-trum is automatically compared to a database of reference

Figure 1. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) workflow. A bacterial or fungal colony (typicallysingle) is “picked” from a culture plate to a “spot” on a MALDI-TOF MS target plate (a reusable or disposable plate with a number of test spots) using awooden or plastic stick, pipette tip, or loop. One or many isolates may be tested at one time. At our institution, cells are treated with formic acid on the targetplate. The spot is then overlain with 1–2 μL of matrix. Following a short drying period, the plate is placed in the ionization chamber of the mass spectrometerfor analysis (see Figure 2). A mass spectrum is generated and automatically compared against a database of mass spectra by the software, resulting in identifi-cation of the organism (Candida parapsilosis in position A4 in the example). More complex preparatory protein extraction, commonly used in older studies, maybe performed prior to target plate inoculation. Although this yields high-quality mass spectra [46], it is technically cumbersome; direct on-plate testing usingformic acid identifies a similar number of organisms [37]. Direct on-plate testing must be avoided with organisms hazardous to microbiology technologists (eg,Brucella species) [28]. Reproduced by permission of Mayo Foundation for Medical Education and Research. All rights reserved.

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spectra to determine the relatedness of the spectrum to spectrain the database; a list of most closely related organisms is gener-ated, each with a numeric ranking (percentage or score) indicat-ing the level of confidence in identification. Depending on howhigh the value is, the organism is identified at the family, genus,or species level. As with any identification system, well-curatedspectral databases representing comprehensive collections ofcorrectly named and clinically relevant bacteria and fungi are

necessary. In the absence of mass spectral entries in a database,entries may be manually added (assuming availability of requi-site isolates). The time from putting the target plate into the in-strument to final result is fast; for example, the Bruker systemproduces results in ≤3 minutes per isolate. No mass spectrome-try expertise is required, and the technology is “green” with re-usable target plates available and few disposables.

Commercial MALDI-TOF MS systems are available fromBruker Daltonics Inc (Billerica, Massachusetts) and bioMérieuxInc. A system used primarily in France (Andromas, Paris,France), will not be further discussed. The Bruker Biotypersystem is most well studied and includes a mass spectrometer,software, and library; over the past few years, improved softwareand progressively more comprehensive and better curated massspectral libraries have been made available. AnagnosTec(Zossen, Germany) previously marketed a microbial databasecalled SARAMIS used with Shimadzu’s AXIMA Assurancemass spectrometer (Shimadzu, Columbia, Maryland), whichwas acquired by bioMérieux in 2010; with this acquisition, thesystem’s name was changed to VITEK MS RUO. BioMérieuxdeveloped its own database/operating software/algorithms, re-ferred to as VITEK MS (including a prerelease version of theVITEK MS v1.1 database [6], the v1 system/v1.1 database [7–10],the v2.0 system/v2.0 database and algorithms, and VITEK MSPlus, which incorporates the VITEK MS v2.0 and SARAMISv4.0 databases). Rapidly implemented progressive improve-ments in both systems are enhancing the clinical laboratory’sability to identify a range of organisms but render it challengingfor those reading the literature to compare studies due to vari-able software, interpretive rules, databases, mass spectrometers,comparators, specimen preparations, organism sets (ie, en-riched for unusual organisms, or not), and so forth. Currentsystems’ databases are generally best for common aerobicgram-negative bacilli and gram-positive cocci, and may lacksome esoteric organisms, such as certain anaerobes, fungi, andmycobacteria. Given changing nomenclature and ongoing de-scription of new species and emerging microorganisms, regularand ongoing updates to databases are imperative to ensure clin-ical utility. Databases should include entries representing majorphylogenetic lineages within each species. It is possible forusers to add their own mass spectral entries to enhance existingdatabases or create their own database by including locally im-portant strains or strains not well represented in commerciallibraries.

The Bruker and bioMérieux MALDI-TOF MS systems differin databases, algorithms used to identify organisms, and instru-mentation. Numeric rankings are reported on different scales,and are not directly comparable. Bruker’s Microflex LT massspectrometer is a desktop instrument, whereas bioMérieux’s isa larger, floor model. At this time, neither system is approvedby the US Food and Drug Administration.

Figure 2. Matrix-assisted laser desorption ionization–time of flightmass spectrometer. The target plate is placed into the ionization chamberof the mass spectrometer. Spots to be analyzed are shot by an ultravioletN2 laser desorbing microbial and matrix molecules from the target plate.The majority of energy is absorbed by the matrix, converting it to anionized state. Through random collision in the gas phase, charge is trans-ferred from matrix to microbial molecules. The cloud of ionized moleculesis funneled through a positively charged electrostatic field into the time offlight mass analyzer, a tube under vacuum. The ions travel toward an iondetector with small analytes traveling fastest, followed by progressivelylarger analytes. As ions emerge from the mass analyzer, they collide withan ion detector generating a mass spectrum representing the number ofions hitting the detector over time. Although separation is by mass-to-charge ratio, because the charge is typically single for the described ap-plication, separation is effectively by molecular weight. Reproduced bypermission of Mayo Foundation for Medical Education and Research. Allrights reserved.




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Generally speaking, MALDI-TOF MS performs at least aswell as, if not better than, automated biochemical identificationfor commonly encountered bacteria and yeast (Table 1), andbetter for unusual organisms. We compared the BD PhoenixAutomated Microbiology System and Biotyper system using440 common and unusual gram-negative bacilli; the formercorrectly identified 83% and 75% of isolates to the genus andspecies levels, respectively, whereas MALDI-TOF MS correctlyidentified 93% and 82% of isolates to the genus and specieslevels, respectively [11]. We also evaluated the Biotyper systemusing 217 clinical isolates of staphylococci, streptococci, andenterococci, 98% and 79% of which were correctly identified tothe genus and species levels, respectively [12]. The performanceof the 2 commercial systems appears to be similar. Martiny et alreported 93% species-level identifications of 1003 routine bac-terial isolates with both a prerelease version of the VITEK MSv1.1 database and the Biotyper system [6].

In most cases, organisms are either correctly identified oryield a low score/percentage, indicating that identification hasnot been achieved; the latter typically implies no “match” in thedatabase but can occur due to technically poor preparation.Misidentifications are unusual but may occur with some closelyrelated organisms. Although a comprehensive review of identi-fication of specific organisms is beyond the scope of this article,some generalizations can be made. Identification of staphylo-cocci [13] and β-hemolytic streptococci is excellent [14]. Theα-hemolytic streptococci are more problematic because Strepto-coccus mitis and Streptococcus oralis may be poorly differentiat-ed from one another and from Streptococcus pneumoniae, atleast with the Bruker system [12, 15]; recent data suggest thatVITEK MS may overcome this limitation [8]. Arcanobacteriumhaemolyticum, Rhodococcus equi, and all but select closelyrelated Corynebacterium species are reliably identified [9, 16].

Some closely related organisms, such as Escherichia coli andShigella species, are not differentiated. (To avoid potential mis-identification, we attempt to avoid testing of suspect E. coli andwe perform additional testing if E. coli is suggested.) MALDI-TOF MS accurately detects Enterobacter cloacae complex,although it may not discriminate some species within thiscomplex [9, 17]. Likewise, accurate differentiation of all speciesof Acinetobacter may be challenging with current databases, al-though Acinetobacter baumannii is identifiable [18]. Similarchallenges exist with other identification systems. MALDI-TOFMS is exciting because of the breadth and depth of organismtypes that can be identified with one platform, including, butnot limited to, Aeromonas species [19], Campylobacter species,Arcobacter butzleri, and Yersinia enterocolitica [20]. MALDI-TOF MS can identify and differentiate Capnocytophaga cani-morsus from Capnocytophaga cynodegmi [21]. Although typingwithin the genus Salmonella is not generally possible, it may bepossible to distinguish Salmonella enterica subspecies entericaserovar Typhi from other S. enterica serovars [22]. And withuser-supplemented databases, HACEK organisms [23], Legion-ella species [24], and Nocardia species [25] (the last with specif-ic extraction procedures) may be identified.

Jamal et al reported species-level identification of 89% and100% of 274 routinely isolated anaerobic bacteria (enriched inBacteroides fragilis) using the Biotyper DB Update v3.3 and theVITEK MS v1 system/v1.1 database, respectively [10]. Identifi-cation of more esoteric anaerobes, including Prevotella species,has been successful in 83% of cases with user supplementationof Bruker’s Reference Library 3.2.1.0 [26]. We evaluated adiverse collection of 253 clinical anaerobic isolates using the Bi-otyper system and a user-supplemented database; 92% and71% of isolates were correctly identified to the genus andspecies levels, respectively [27]. Further commercial database

Table 1. Examples of Recent Evaluations of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for RoutineBacterial Identification

Isolates % Identification

System No. TypePeriod of

Isolate Collection Genus Level Species Level Country Comparator Reference

Bruker Biotyper 1013 Bacteria 2 mo 99% 97% France Phoenix, API, Biochemical [72]

Bruker Biotyper 468 Bacteria 3 mo 97% 92% Japan MicroScan, API, Phoenix [73]Bruker Biotyper 2781 Bacteria 1 mo 96% 85% Australia VITEK2, API, Biochemical [74]

Vitek MSa 767 Bacteria 6 wk 95% 87% France VITEK2 [8]

Bruker Biotyper/Vitek MSb

986 Bacteria 3 mo 96%/94% 93%/93% Belgium Bruker Biotypercompared toVitek MS

[6]

a v1 system/v1.1 database.b Prerelease version of v1.1 database.




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supplementation will improve anaerobic identification to alevel previously possible only with 16S ribosomal RNA gene se-quencing or gas liquid chromatography.

MALDI-TOF MS can accurately identify Francisella tularen-sis and Brucella species; however, the Biotyper library does notcontain and will not identify these organisms. Use of Bruker’s“Security Relevant” database enables Brucella species and F.tularensis identification [28].

A handful of investigators have reported using MALDI-TOFMS for mycobacteria. This requires special processing to killtested organisms (for safety), disrupt clumped cells, and breakdown cell envelopes. Current commercial libraries inadequatelyaddress the clinically relevant Mycobacterium species, but withappropriate library construction, MALDI-TOF MS should beable to identify most, with a few caveats expected. Members ofthe Mycobacterium tuberculosis complex are identified at thecomplex level [29]; some species (eg, Mycobacterium abscessus/massiliense; Mycobacterium mucogenicum/phocaicum; Myco-bacterium chimaera/intracellulare) may not be well differentiat-ed from one another [29], and identification from liquid mediamay be more challenging than from colonies on solid media [30].

Beyond bacteria, MALDI-TOF MS can identify yeast, out-performing some conventional phenotypic systems, and distin-guishing Candida dubliniensis and albicans; Candida rugosaand pararugosa; Candida norvegensis, krusei, and inconspicua;Candida parapsilosis, orthopsilosis, and metapsilosis [31]; and(with database supplementation) Cryptococcus neoformans andgattii [32–34]. Dhiman et al evaluated the Biotyper system foridentification of 138 common and 103 unusual yeast isolates,and reported 96% and 85% accurate species-level identification,respectively [35]. MALDI-TOF MS may outperform currentsystems to identify esoteric and sometimes misidentified Candidaspecies, such as Candida famata [31]. Although older studiesused preparatory extraction for yeasts, we and others use thesame direct testing strategy we use for bacteria (Figure 1) [36, 37].

Few filamentous fungi are included in current commercialdatabases. Filamentous fungi exhibit variable phenotypes;protein spectra may vary with growth conditions or with thezone of fungal mycelium analyzed. De Carolis et al developed alibrary of Aspergillus species, Fusarium species, and Mucoralesusing the Biotyper system and identified 97% of 94 isolates tothe species level [38]. Using the VITEK MS v1 system/v1.1 da-tabase and direct on-plate testing, Iriart et al identified 82% of44 Aspergillus isolates (including all isolates with species in thedatabase) [7]. Theel et al developed a dermatophyte library andused it to identify 93% and 60% of 171 dermatophyte isolatesto the genus and species levels, respectively, using the Biotypersystem [39]. Preliminary studies indicate that Pseudallescheria/Scedosporium complex species are identifiable [40]. Beyondfungi, MALDI-TOF MS has been used for Prototheca speciesidentification [41].

Compared to standard methods, MALDI-TOF MS improvesturn-around time for bacterial and fungal identification by anaverage of 1.45 days [42], and, because only a small amount oforganism is required, testing can be performed directly fromsingle colonies on primary culture plates, whereas othermethods may require subculture. Tests for screening for someenteric pathogens (eg, triple sugar iron agar for Salmonellaspecies) may be eliminated [43].

MALDI-TOF MS reagent costs are approximately $0.35/test;implementation reduces reagent and labor costs for identifica-tion, in one model, by 57% within 1 year [42]. An estimated87% of isolates are identified on the first day (compared with9% with standard techniques), with final identifications a dayearlier for most organisms, and several days earlier for bio-chemically inert, fastidious, or slow-growing organisms [42].DNA sequencing expenses are avoided for some esoteric organ-isms, waste disposal is decreased, and quality control and labo-ratory technologist training/labor for replaced/retired tests areeliminated [44].

Adoption of MALDI-TOFMS impacts laboratory workflow [9].Time at the bench assessing primary cultures is relatively unaf-fected, with subcultures needed for susceptibility testing. Gramstaining of colonies may not always be needed, as this informa-tion is not required for MALDI-TOF MS. For some organisms(eg, S. aureus), rapid tests performed at the bench will remain.

MALDI-TOF MS has limitations (Table 2). Unlike publicallyavailable sequence databases such as GenBank, MALDI-TOFMS databases are proprietary. Although low identification per-centages for some organisms may be improved by user additionof mass spectral entries of underrepresented species or strains(to cover intraspecies variability) to the database, or even read-dition of reference strain spectra [26], doing so may be beyondthe capabilities of some laboratories. Because of low scores/per-centages, repeat testing is required for approximately 10% ofisolates [42]. Growth on some media (eg, colistin-nalidixic acidagar) may be associated with low scores/percentages [45], andtiny or mucoid colonies may fail identification [9, 46]. Refinedcriteria are needed to distinguish closely related species and todifferentiate them from the next best taxon match [47]. Forcertain species of organisms, genus- or species-specific (includ-ing lowered) cutoffs may be appropriate [13, 16, 48]. Laboratoryerrors may occur, including colony inoculation in erroneoustarget plate locations, testing impure colonies, smearing be-tween spots, failure to clean target plates, and erroneous resultentry into laboratory information systems. There is a learningcurve to applying ideal colony amounts to target plates [9]. In-strument cost is comparable to that of other common laborato-ry equipment; laser, software, hardware, or mass spectrometerfailure require an appropriate service plan. Although MALDI-TOF MS is generally reproducible, sources of variability in-clude the mass spectrometer, matrix and solvent composition,




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technologists, culture conditions, and biological variability;quality control strategies are being defined. Notably, MALDI-TOF MS may yield different (generally more accurate) identifi-cation than current systems [11, 49].

Antimicrobial susceptibility is not directly determined withthe above-described strategy. By rapid organism identification,intrinsic antimicrobial resistance characteristics of particularspecies (or typical susceptibility of the identified species basedon local antibiograms) may guide therapy. Some investigatorshave incubated bacteria with antibiotics and measured antibiot-ic degradation using MALDI-TOF MS; this requires specificsystem configuration and incubation (ie, to allow antibiotic hy-drolysis) and applies only to resistance mechanisms associatedwith antibiotic degradation (eg, β-lactamases) [50]. Althoughdetection of methicillin resistance in S. aureus has been contro-versial, this does not appear to be feasible using the Biotypersoftware in detection ranges and instrument settings preset formicrobial identification [51]. Microbial strain typing is underdevelopment with no current “universal protocol”; innovationsand advances are expected. With strain typing [52, 53], strains ofS. aureus likely to be methicillin resistant (eg, USA300 [54]), en-terococci likely to be vancomycin resistant [55], or Bacteroides

fragilis likely to be carbapenem resistant (cfiA positive) [56]may be called out (as markers for possible resistance).

Although direct testing of clinical specimens is not generallyfeasible because of a high limit of detection, urine may betested directly due to the high numbers of bacteria present inclinically infected urine (although α-defensins 1, 2, and 3 mayimpair database matching [57]).

Rapid identification of microorganisms growing in bloodculture bottles is promising, but requires preparatory specimenprocessing as blood culture bottles contain macromoleculesfrom blood and growth media. Processing is accomplished withdifferential centrifugation and washings, selective lysis of bloodcells (eg, using water, ammonium chloride, saponin, sodiumdodecyl sulfate), separator gels, filtration, and so on; commer-cial blood culture bottle processing using the Sepsityper(Bruker Daltonics) is available [58–64]. Although results arevalid when obtained, the yield is generally not as good as directcolony testing with a higher percentage of gram-negative thangram-positive organisms being identified [58]; polymicrobialcultures may not be fully characterized (although strategies arebeing developed). Most Candida species are identifiable [65].An alternate approach is to subculture positive blood culturebottles to solid media and test growth after a short period (eg,2–4 hours) of incubation [66, 67]. By testing of positive bloodculture bottles using MALDI-TOF MS, the time to organismidentification may be reduced by a day or more, which mayserve as a guide for empirical treatment [68], but antimicrobialsusceptibility is not provided. Recently, MALDI-TOF MS hasbeen tied with direct antimicrobial susceptibility testing frompositive blood culture bottles [69, 70], improving time tooptimal therapy and decreasing hospital length of stay andcosts [71].

In summary, MALDI-TOF MS is a quantum leap forwardfor rapid and accurate identification of cultured bacteria andfungi in clinical microbiology. This is incontrovertibly benefi-cial to laboratories, and decreased expense is helpful to labora-tories and patients alike. MALDI-TOF MS implementationmay impact patient outcomes and/or cost of care, dependingon its application. Further, this new technology may providebetter definition of the epidemiology, pathogenesis, and antimi-crobial susceptibility of hitherto unrecognized (or misidenti-fied) microorganisms.

Notes

Acknowledgments. I thank Dr Nancy L. Wengenack, Scott A. Cun-ningham, Brenda L. Dylla, and Sherry M. Ihde for their thoughtful reviewsof this manuscript.Potential conflicts of interest. Author certifies no potential conflicts of

interest.The author has submitted the ICMJE Form for Disclosure of Potential

Conflicts of Interest. Conflicts that the editors consider relevant to thecontent of the manuscript have been disclosed.

Table 2. Advantages and Limitations of Matrix-Assisted LaserDesorption Ionization–Time of Flight Mass Spectrometry in Clini-cal Microbiology

Advantages Limitations

Rapid turn-around time Does not provide antimicrobialsusceptibility

Single colony requirement Requires isolated colonyAutomated, highthroughput

Repeat testing occasionallyrequired

Broad applicability tobacteria and fungi,including esotericorganisms

Not cleared by the US Food andDrug Administration

Databases may beexpanded by users

Identification limited by entries indatabase; databases vary, notpublicly available

Minimal consumables Laser, hardware, software, massspectrometer failure→ potentialtesting downtime

Cost-effective Instrument cost

Generalizes to bacteria andyeast

Misidentifications: failure to testpure colonies or to clean targetplates

Some closely related organismsnot differentiated (eg,Escherichia coli and Shigellaspecies)

Growth on somemediaassociated with no identification(without extraction)

Tiny or mucoid colonies may notbe identified




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