PATENT DOCUMENT

Abstract:
Rapid methods that identify sepsis-causing bacteria or yeast aid the physician in critical therapeutic decision-making, thus decreasing patient mortality rates. The methods described herein employ plating microorganisms directly on to a MALDI-MS plate, adding concentrated formic acid, and identifying the microorganism by mass spectrometry. Optionally, an organic solvent may be combined with the formic acid, or added to the sample before or after the concentrated formic acid is added thereto. The methods enable direct extraction of proteins from microorganisms without the need for liquid protein extraction methods and yields positive identification results for gram-positive bacteria, gram-negative bacteria and yeast in minutes.

Full Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a continuation of U.S. patent application Ser. No. 13/600,702, filed on Aug. 31, 2012, which claims the benefit of the filing date of U.S. Provisional Patent Application No. 61/649,483 filed May 21, 2012 and U.S. Provisional Patent Application No. 61/530,620 filed Sep. 2, 2011, the disclosures of which are hereby incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Sepsis is a serious medical condition caused by an overwhelming response of the host immune system to infection. It can trigger widespread inflammation, which can give rise to impaired blood flow. As sepsis progresses, the body&#39;s organs can be starved for oxygen and nutrients, causing permanent damage and eventual failure. Left improperly diagnosed or otherwise untreated, the heart weakens and septic shock can occur, leading to multiple organ failure and death. Blood cultures are required to detect the presence of bacteria or yeast in the blood of sepsis patients, to identify the microorganism(s) present and guide treatment. The conventional separation and identification of microorganism(s) from blood cultures takes at least 24-48 hours, which results in many of the septicemia patients being initially treated with inappropriate antibiotics. It is therefore desirable to separate and identify microorganisms from a positive culture (blood, cerebrospinal fluid etc.) rapidly. 
         [0003]    Recently, certain proteomic technologies/tools, such as Matrix-Assisted Laser Desorption Ionization Time of Flight mass spectrometry, (“MALDI-TOF MS”), have been shown to provide a rapid and accurate identification of bacteria and/or fungi from a positive blood culture (“PBC”). The microorganism in the PBC sample can be sub-cultured prior to MALDI identification. In the alternative, microorganisms can be isolated from the PBC sample using various sample preparation methods without the need for subculturing. The microorganism isolates are generally directly smeared onto a MALDI plate to yield about 70-80% identification accuracy. For isolates failing to yield any identification, a follow-up liquid extraction method is typically used to extract proteins from the microorganism for improved identification by MALDI-TOF MS. Although these liquid protein extraction methods generally yield better identification accuracy, such methods not only require several centrifugation steps, but also are time-consuming. 
         [0004]    Schmidt et al (Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry,  Eur. J. Clin. Microbiol. Infect Dis,  23 Jun. 2011) discloses a method of identifying bacteria from positive blood cultures by spotting a liquid sample of the isolated bacteria onto a MALDI plate and overlaying 25% formic acid directly to the spotted liquid sample. Therefore, the final concentration of formic acid in the bacterial sample is less than 25%. The Schmidt method results in 86.6% identification accuracy for gram-negative bacteria and 60% identification accuracy for gram-positive bacteria. Schmidt does not test this method in Yeast. 
         [0005]    Hyman et al (U.S. Patent Publication No. 2010/0120085, Published May 13, 2010), discloses a similar method as Schmidt, in which intact isolated microorganisms in solution are directly smeared onto a MALDI plate. The liquid sample is then overlaid with roughly an equal volume of 50% formic acid. Therefore, the final concentration of formic acid added to the sample is approximately 25%. This method was tested on 14 different species of bacteria and yeast. Although this method resulted in 91.1% identification, the data does not indicate how effective this method is with regard to gram-positive bacteria, gram-negative bacteria, or yeast. 
         [0006]    Haigh et al. “Improved Performance of Bacterium and Yeast Identification by a Commercial Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry System in the Clinical Microbiology Laboratory,”  J. Clin. Microbiol . (Jul. 6, 2011) describes a method in which neat formic acid is used to extract microbial proteins smeared directly onto a MALDI plate. This method, however, was unable to successfully identify all strains of yeast and gram-positive bacteria. 
         [0007]    Herendael et al. “Validation of a modified algorithm for the identification of yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)”,  Eur. J. Clin. Microbiol. Infect Dis  (23 Aug. 2011) describes two methods for the identification of yeast. The standard extraction method described in Herendael et al., is a conventional liquid extraction method (this method is the “prior art procedure” of Example 1 hereinbelow). In the short extraction method described in Herendael et al., one colony was picked from an agar plate and applied directly to the target MALDI plate. Formic acid (1 μL at 70% concentration) was added to the sample and the sample was allowed to dry. The dried sample was overlaid with MALDI matrix, allowed to dry further, and analyzed by MALDI-MS. The short extraction method provided identical results as the standard extraction method although the MALDI scores were lower with the short extraction method. Nearly all of the isolates (97.6%) could be identified with the short extraction method; however 17.1% of these identifications fell below the reliable threshold level of 1.7. 
         [0008]    Accordingly, there is a need to develop a rapid and accurate method of identifying all classes of bacteria and yeast without the need for liquid protein extraction prior to identification with MALDI-TOF MS. 
       SUMMARY OF THE INVENTION 
       [0009]    Various embodiments of the disclosed method enable direct identification of microorganisms from positive blood cultures (“PBC”) or pure isolates by mass spectrometry without the need for a liquid protein extraction. In one embodiment of this method, identification of a microorganism from a pure isolate is achieved by obtaining a sample containing at least one microorganism, depositing at least a portion of the pure isolate sample (direct smear or microbial suspension) on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry, treating the sample with a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid, drying the sample, placing a MALDI matrix solution over the treated sample, and identifying the microorganism by MALDI mass spectrometry. 
         [0010]    Optionally, a PBC sample is first processed to isolate the microorganism, followed by identification of the microorganism without performing a liquid protein extraction. This embodiment is achieved by: i) obtaining a PBC sample determined to contain at least one microorganism; ii) adding a lysis buffer to the sample to lyse the blood cells; iii) while the microorganism remains intact and viable, isolating the intact microorganism; iv) depositing the isolated microorganism on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; v) treating the sample with a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid; vi) placing a MALDI matrix solution over the treated sample; and vii) identifying the microorganism by mass spectrometry. In another embodiment, the PBC sample is sub-cultured to produce a pure culture of the microorganism prior to identifying the microorganism. In one embodiment, the sample can be dried before treating the sample with a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid; placing a MALDI matrix solution over the treated sample; and/or identifying the microorganism by mass spectrometry (i.e., before the treating step v) above). 
         [0011]    The described methods can be used to isolate and/or identify a spectrum of microorganisms including but not limited to, gram-positive bacteria, gram-negative bacteria, fungi, mycobacterium, or yeast. 
         [0012]    In another embodiment, a kit is provided for the detection of microorganisms in a sample. The kit includes, for example, one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]      FIG. 1  is a flowchart that illustrates the prior art method of extracting proteins from pure microbial isolates for identification by mass spectrometry. 
           [0014]      FIG. 2  illustrates one embodiment of the disclosed method in which proteins are extracted from isolated microorganisms directly on a MALDI plate using concentrated formic acid. 
           [0015]      FIG. 3  illustrates one embodiment of the disclosed method in which proteins are extracted from isolated microorganisms directly on a MALDI plate using concentrated formic acid in an organic solvent. 
       
    
    
     DETAILED DESCRIPTION 
       [0016]      FIG. 1  illustrates the prior art methods of identifying microorganisms from PBC. These methods are known to one having ordinary skill in the art. For example, these methods are described in Nassiff et al WO 2010/100612 A1, the disclosure of which is incorporated herein by reference. A positive blood culture  100  is obtained. The microorganism is isolated from the remaining blood cells and debris either by liquid separation  110  or by sub-culturing the microorganism  120  to produce a pure culture of microorganism  130 . To identify the microorganism, microorganism separated by liquid separation  110  or the pure culture  130  can be directly smeared onto a MALDI plate  140  for MALDI-MS identification  170  or undergo liquid protein extraction  150  prior to transferring to the MALDI plate  160  and identification by MALDI-MS  170 . 
         [0017]    In one embodiment of the invention, direct identification of microorganisms is achieved from pure isolates or a PBC sample by mass spectrometry without requiring a liquid protein extraction. In one embodiment, the microorganism from pure isolates is identified by: i) obtaining a sample suspected to contain at least one microorganism; ii) depositing at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry in a manner that controls any dilution of a volatile acid and/or an organic solvent to be combined with the deposited sample; iii) treating the sample with at least one reagent; such reagents including a volatile acid, an organic solvent, and/or a combination of organic solvent and a volatile acid; iv) placing a MALDI matrix solution over the treated sample; and v) identifying the microorganism by mass spectrometry. In one embodiment the volatile acid is at least 70% formic acid. In another embodiment the volatile acid is at least 80% formic acid. In another embodiment the volatile acid is at least 90% formic acid. Unless otherwise specified herein, the formic acid solutions are aqueous solutions. In another embodiment the volatile acid is at least 100% formic acid (e.g. neat). In another embodiment, the sample is treated with at least 70% formic acid in an organic solvent such as acetonitrile, methanol, ethanol, acetone, or ethyl acetate prior to placing a MALDI matrix solution over the sample. In another embodiment, the sample is treated with at least 80% formic acid in an organic solvent such as acetonitrile, methanol, ethanol, acetone, or ethyl acetate prior to placing a MALDI matrix solution over the sample. In another embodiment, the sample is treated with at least 90% formic acid in an organic solvent such as acetonitrile, methanol, ethanol, acetone, or ethyl acetate prior to placing a MALDI matrix solution over the sample. In one embodiment, the sample deposited on the solid surface is allowed to dry prior to adding the volatile acid, to prevent the sample from diluting the volatile acid. In another embodiment, the volatile acid is dried prior to placing the MALDI matrix solution over the sample. Examples of the volatile acids that may be used in the various embodiments of the invention include, but are not limited to, formic acid, acetic acid, trifluoracetic acid and hydrochloric acid. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0018]    In another embodiment, the disclosed methods are used to isolate and/or identify gram-positive bacteria, gram-negative bacteria, or yeast. In another embodiment, the disclosed methods are used to isolate and/or identify  Streptococcus pneumoniae . In another embodiment, the disclosed methods are used to isolate and/or identify yeast, including  Cryptococcus neoformans.    
         [0019]    In one embodiment, identification of microorganisms having thickened or hydrophobic cell wall complexes, for example, yeast and mycobacteria, is performed by using any of the methods described herein, which deploy an organic solvent, among other reagents, for on-plate extraction. In a preferred embodiment,  Cryptococcus neoformans  is the microorganism targeted for identification. Without being bound by a particular theory, it is believed that the organic solvent dissolves or disperses interfering substances in the cell wall and/or bound extracellular material, such as polysaccharides or lipids, and makes the cell wall and intracellular proteins more susceptible and more accessible, respectively, to the extraction methods. 
         [0020]    After the sample is combined with the reagent(s), the combination of sample and reagents is dried. Drying is defined as allowing the liquid to evaporate sufficiently so as not to dilute any liquid subsequently added. While the sample can be dried in ambient air, a heating source, such as a heating block, hot plate, heating oven or infrared heating lamp can be used to accelerate the evaporation of the liquid portion of the combined sample and reagents. These drying methods do not change the spectrum of the sample upon identification by MALDI. 
         [0021]    After the extracted sample is dried, it is combined with additional reagents for downstream sample assay using MALDI. Any MALDI matrix solution known to those skilled in the art can be used in the disclosed methods. These matrix solutions include, but are not limited to, α-cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (SPA), 3-hydroxypicolinic acid (HPA), 3.4-dihydroxycinnamic acid, 2-(4-hydroxyphenylazo)-benzoic acid, 2-amino-4-methyl-5-nitropyridine, and 2,4,6-trihydroxy acetophonone (THAP). 
         [0022]    In one embodiment, the methods described herein can also include using a non-ionic detergent capable of solubilizing proteins as an extraction reagent in addition to those described above. The detergent does not interfere with the MALDI ionization process. These detergents can be incorporated into the initial microorganism suspension or subsequent volatile acid and/or organic solvent treatment. Typical detergents suitable for MALDI include, for example, n-octyl-fl-D-glucopyranoside (OG), saponin, triton, and those described in International Publication No. WO 2010/100612, incorporated herein by reference. In one embodiment, the concentration of detergent is in the range of 1% to 5%. In another embodiment, the concentration of detergent is 2%. 
         [0023]    Referring to  FIG. 2 , which illustrates the protein extraction method of one embodiment of the disclosed methods, an isolated microorganism  200  is obtained (i.e. pure culture from sub-culturing or liquid separation as described in  FIG. 1 ). The isolated microorganism  200  is added directly to the MALDI plate  210 . The isolated microorganism  200  on MALDI plate  210  is allowed to air dry  220  to produce dried, isolated microorganism  230 . Ninety percent formic acid  240  is applied to the dried, isolated microorganism sample  230  to obtain extracted microorganism sample  250  which contains solubilized proteins of the microorganism. Extracted microorganism sample  250  is allowed to air dry  260  to produce dried, extracted microorganism sample  270 . MALDI matrix  280  is added to dried, extracted microorganism  270 , allowed to air dry  285 , and identified by MALDI-MS  290 . 
         [0024]    In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing by direct smear at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) drying the sample; iv) treating the sample with at least 70% formic acid in an aqueous solution or at least 70% formic acid in an organic solvent; v) drying the sample; vi) placing a MALDI matrix solution over the treated sample; vii) drying the sample; and viii) identifying the microorganism by mass spectrometry. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0025]    In yet another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing by direct smear at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) treating the sample with an organic solvent; iv) drying the sample; vii) treating the sample with formic acid and an organic solvent; v) drying the sample; vi) placing a MALDI matrix solution over the treated sample; vii) drying the sample; and viii) identifying the microorganism by mass spectrometry. In one embodiment, the organic solvent is ethanol, methanol, isopropanol, or acetone. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0026]    In yet another embodiment, the sample suspected of containing at least one microorganism is resuspended in a solution prior to deposition onto the solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry. One skilled in the art will appreciate that the concentration of the microbial suspension will be optimized for the various disclosed embodiments. For example, in one embodiment, a higher concentration of microbial suspension may be required, such as a concentration greater than 0.5 McFarland (for example, about 0.75 McFarland and higher), to better ensure proper identification of the microorganism (or a reliable indication of the absence of the microorganism) by MALDI-MS. In another embodiment, the concentration of the microbial suspension can be adjusted for use in the various disclosed embodiments as well as in additional assays, such as those described in U.S. patent application Ser. No. 13/177,031 (which is incorporated by reference herein), which include, for example, antimicrobial susceptibility testing (AST). In one embodiment, the microbial suspension is at least about 2.0 McFarland or higher. 
         [0027]    One skilled in the art is aware that MALDI identification results are affected by the amount of cells deposited onto the MALDI plate. A standardized microbial suspension provides a uniform dispersion of cells, leading to more precise and reproducible results. In one embodiment, the microbial suspension is standardized prior to deposition onto the solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry. The microbial suspension can be optionally adjusted to a certain McFarland standard. Creation of the standardized microbial suspension can be accomplished by various methods well known to those skilled in the art, for example, using an inoculation loop, microdropper, or other physical methods. In a preferred embodiment, a microbial suspension is adjusted to a McFarland standard of at least 0.5 prior to deposition onto a MALDI plate. In another embodiment, a microbial suspension is adjusted to a McFarland standard in the range of 0.5-10 McFarland prior to deposition onto a MALDI plate. 
         [0028]    Referring to  FIG. 3 , which illustrates the protein extraction method of one embodiment of the disclosed methods, an isolated microorganism  300  is obtained (i.e. pure culture from sub-culturing or liquid separation as described in  FIG. 1 ). The isolated microorganism  300  is resuspended in organic solvent  305  to produce resuspended microorganism  310 . The isolated microorganism  300  can be resuspended in any suitable organic solvent  305  such as, for example, ethanol, methanol, acetone, or ethyl acetate. Any concentration of organic solvent  305  may be used, and the appropriate concentration for a particular application is readily determined by one skilled in the art. In alternative embodiments, the concentration of organic solvent  305  is in the range of about 30% to about 70%. In another alternative embodiment, the organic solvent is ethanol. The resuspended microorganism  310  is deposited onto a MALDI plate  315 . The resuspended microorganism  310  on MALDI plate  315  is allowed to air dry  320  to produce dried, isolated microorganism  325 . Seventy percent formic acid in 30% organic solvent  330  is used to extract the dried, isolated microorganism  325 . The dried, isolated microorganism  325  on MALDI plate  315  with 70% formic acid in 30% organic solvent  330  is allowed to air dry  335  to produce dried, extracted microorganism  340 . MALDI matrix  345  is added to dried, extracted microorganism  340  and allowed to air dry  350  before identification by MALDI-MS  355 . 
         [0029]    The isolated microorganism  300  is obtained according to any of the exemplary methods described herein or by other methods known to one having ordinary skill in the art. 
         [0030]    In one embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in an organic solvent; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with formic acid; vi) drying the sample; vii) placing a MALDI matrix solution over the treated sample; viii) drying the sample; and ix) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in an organic solution at a concentration greater than 0.5 McFarland. On one embodiment, the concentration of formic acid is at least 70%. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0031]    In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in water; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with formic acid and an organic solvent; vi) drying the sample; vii) placing a MALDI matrix solution over the treated sample; viii) drying the sample; and ix) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in an aqueous solution at a concentration greater than 0.5 McFarland. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0032]    In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in an organic solvent solution; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with formic acid in the organic solvent; vi) drying the sample; vii) placing a MALDI matrix solution over the treated sample; viii) drying the sample; and ix) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in an organic solvent solution at a concentration greater than 0.5 McFarland. In one embodiment, the sample is resuspended in organic solvent solution with an organic solvent concentration of about 30% to about 70%. In another embodiment, the organic solvent is ethanol. In yet another embodiment, the microorganism to be detected is yeast. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0033]    In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in water; iii) combining the microorganism resuspended in water with an organic solvent so that the final concentration of organic solvent in the combined solution is in the range of 30%-70%; iv) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; v) drying the sample; vi) treating the sample with an aqueous formic acid solution or formic acid in the organic solvent; vii) drying the sample; viii) placing a MALDI matrix solution over the treated sample; ix) drying the sample; and x) identifying the microorganism by mass spectrometry. In this embodiment, the sample suspected of containing at least one microorganism is resuspended in water at a concentration greater than about 0.5 McFarland. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0034]    In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) resuspending the sample in water; iii) depositing at least a portion of the suspension on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iv) drying the sample; v) treating the sample with an organic solvent; vi) drying the sample; vii) treating the sample with formic acid in the organic solvent; viii) drying the sample; ix) placing a MALDI matrix solution over the treated sample; x) drying the sample; and xi) identifying the microorganism by mass spectrometry. In this embodiment, the organic solvent is ethanol and the sample suspected of containing at least one microorganism is resuspended in water at a concentration greater than 0.5 McFarland. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0035]    In yet another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing by direct smear or suspension at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) treating the sample with a mixture of formic acid, an organic solvent such as ethanol or acetonitrile, and a MALDI matrix solution; iv) drying the sample; and v) identifying the microorganism by mass spectrometry. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0036]    In another embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) drying the sample; iv) treating the sample with an organic solvent solution that is at least about 70% organic solvent; v) drying the sample; vi) treating the sample with a formic acid solution that is at least about 70% formic acid; vii) drying the sample; viii) treating the sample with concentrated (100%) organic solvent; ix) drying the sample, x) placing a MALDI matrix solution over the treated sample; xi) drying the sample; and xii) identifying the microorganism by mass spectrometry. In one embodiment, the organic solvent is ethanol. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
         [0037]    In an alternative embodiment, the microorganism is identified by: i) obtaining a sample suspected of containing at least one microorganism; ii) depositing, via direct smear or from a microbial suspension, at least a portion of the sample on a solid surface adapted to be placed in an apparatus configured to determine the identity of microorganisms by MALDI mass spectrometry; iii) fixing the microorganism with an organic solvent, e.g. ethanol, a fixative, e.g. formaldehyde, or by applying heat, generally up to about 37° C. (i.e. approximately body temperature); iv) treating the fixed sample with at least 70% formic acid; v) drying the sample; vi) placing a MALDI matrix solution over the treated sample; vii) drying the sample; and viii) identifying the microorganism by mass spectrometry. Fixatives such as formaldehyde are well known to one skilled in the art and are not described in detail herein. In another embodiment, a kit is provided comprising one or more of the reagents and/or buffers described herein for processing a sample known to contain at least one microorganism for downstream identification. 
       EXAMPLES 
       [0038]    In all examples below, the MALDI matrix solution was prepared by dissolving 2.5 mg of HCCA in 250 μL of a solution that is 2.5% trifluoroacetic acid, 47.5% deionized water, and 50% acetonitrile. All the mass spectrometry data was recorded on Bruker Microflex LT with Biotyper software with the following MALDI score key definition. A MALDI ID score of greater than 2.0 indicates a satisfactory identification to the Genus and Species level. A MALDI ID score of 1.7-1.999 indicates a satisfactory identification to the Genus level. A MALDI ID score of less than 1.699 indicates an unacceptable or not reliable identification. 
       Example 1: Comparison of Extraction Methods 
     Prior Art Procedure: 
       [0039]    The method and results from this prior art procedure are reported in and describe the findings of Haigh et al. “Improved Performance of Bacterium and Yeast Identification by a Commercial Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry System in the Clinical Microbiology Laboratory”,  J. Clin. Microbiol . (Jul. 6, 2011) and Bizzini et al. “Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Bacterial Strains Routinely Isolated in a Clinical Microbiology Laboratory”, J. Clin. Microbiol. (Mar. 10, 2010). A portion of isolated microorganism(s), including gram-positive and gram-negative bacteria, is directly smeared onto a MALDI-TOF MS plate and air dried. The dried sample is overlaid with 1 μL of matrix solution (HCCA) and air dried before identification by MALDI-TOF MS. 
         [0040]    The MALDI analysis results in an overall correct identification of 70.3-75.6%. Therefore, at least 25% of the samples are not identified using this method (referred to as the “direct smear method”). 
         [0041]    For those samples not identified by the direct smear method above, a liquid extraction method is performed on the microorganism sample prior to identification. Water (300 μL) is added to an Eppendorf microcentrifuge tube. A large single colony from a sub-culture of the microorganism is transferred to the centrifuge tube containing water and the sample is vortexed thoroughly. Ethanol (900 μL) is added to the tube and vortexed thoroughly. The mixture is centrifuged at maximum speed for 2 minutes at 10,000×g in a microcentrifuge. The ethanol is decanted and the sample is centrifuged again for 2 minutes at 10,000×g speed in a microcentrifuge. Excess ethanol is removed with a pipette. Formic acid (70% solution; 50 μL) is added to the pellet and vortexed thoroughly. Acetonitrile (100% solution; 50 μL) is added to sample. The sample is centrifuged at 10,000×g for 2 minutes in a microcentrifuge. The supernatant (1 μL) is pipetted onto a MALDI plate and allowed to air dry. The dried sample is overlaid with 10 μL of MALDI matrix solution (HCCA) and air dried before identification by MALDI-MS. The liquid extraction procedure results in up to 100% identification of those microorganisms not identified by the direct smear method. 
       Exemplary Embodiment: 
       [0042]    From a PBC sample, the pellet of isolated microorganisms was resuspended in 600 μL water or 2 mM OG solution in a microcentrifuge tube. The turbidity was visually adjusted to greater than 0.5 McFarland. Each sample (1-1.5 μL) was spotted onto the MALDI plate. The sample was allowed to air dry. Formic acid (1-2 μL of 70% formic acid solution) was overlaid onto the dried, spotted sample and allowed to air dry. The dried sample was overlaid with 1 μl of matrix solution (HCCA) and allowed to air dry before identification by MALDI-TOF MS. 
         [0043]    From a sub-culture, a single colony was touched with a toothpick and a very thin layer of microorganism was smeared directly onto MALDI target plate. The sample was allowed to air dry. Formic acid (1-2 μL of a 70% aqueous solution) was overlaid onto the sample and allowed to air dry. The dried sample was overlaid with 1 μl of MALDI matrix solution (HCCA) and allowed to air dry before identification by MALDI-MS. 
         [0044]    The list of organisms tested can be found in Table 1 below. The MALDI-TOF MS analysis from both the microorganism isolated from a PBC sample and from a sub-culture resulted in 95% correct identification, including 95% identification of gram-positive bacteria (organism and type), 100% identification of gram-negative (organism and type), and 100% identification of yeast samples. These results illustrate that the methods described in this example can accurately identify microorganism(s) from a PBC sample or from a sub-culture without the need for the complex and time-consuming prior art liquid extraction methods previously described. Specifically, no separate extraction step was required to identify microorganisms not identified by the methods in this example. In addition, the results demonstrate that these methods result in significantly higher percent identification of a variety of organisms including gram-positive bacteria and gram-negative bacteria, compared to the direct smear method. 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Organism 
                 Organism Type 
                 Strain Number 
               
               
                   
               
             
             
               
                 
                   Acinetobacter baumanii 
                 
                 gram-negative 
                 ENF 11091 
               
               
                 
                   Enterobacter aerogenes 
                 
                 gram-negative 
                 13048 
               
               
                 
                   Enterobacter cloacae 
                 
                 gram-negative 
                 35030 
               
               
                 
                   Escherichia coli 
                 
                 gram-negative 
                 25922 
               
               
                 
                   Escherichia coli 
                 
                 gram-negative 
                 35218 
               
               
                 
                   Klebsiella pneumoniae 
                 
                 gram-negative 
                 33495 
               
               
                 
                   Klebsiella pneumoniae 
                 
                 gram-negative 
                 700603 
               
               
                 (ESBL-producer) 
                   
                   
               
               
                 
                   Proteus mirabilis 
                 
                 gram-negative 
                 29906 
               
               
                 
                   Pseudomonas aeruginosa 
                 
                 gram-negative 
                 27853 
               
               
                   Pseudomonas aeruginosa  (Carbap-R) 
                 gram-negative 
                 ENF 14620 
               
               
                 
                   Serratia marcescens 
                 
                 gram-negative 
                 SCENF FR197 
               
               
                 
                   Stenotrophomonas maltophilia 
                 
                 gram-negative 
                 13637 
               
               
                 
                   Enterococcus faecalis 
                 
                 gram-positive 
                 29212 
               
               
                   Enterococcus faecalis  VRE 
                 gram-positive 
                 51299 
               
               
                 
                   Enterococcus faecium 
                 
                 gram-positive 
                 19434 
               
               
                   Enterococcus faecium  VRE 
                 gram-positive 
                 700221 
               
               
                 
                   Staphylococcus aureus 
                 
                 gram-positive 
                 25923 
               
               
                 
                   Staphylococcus aureus 
                 
                 gram-positive 
                 29213 
               
               
                   Staphylococcus aureus  MRSA 
                 gram-positive 
                 POS 3421 
               
               
                   Staphylococcus aureus  MRSA 
                 gram-positive 
                 43300 
               
               
                 
                   Staphylococcus epidermidis 
                 
                 gram-positive 
                 SCPOS 3568 
               
               
                 
                   Staphylococcus epidermidis 
                 
                 gram-positive 
                 14990 
               
               
                 
                   Staphylococcus haemolyticus 
                 
                 gram-positive 
                 POS 3569 
               
               
                 
                   Staphylococcus sciuri 
                 
                 gram-positive 
                 29062 
               
               
                 
                   Streptococcus agalactiae 
                 
                 gram-positive 
                 12386 
               
               
                 
                   Streptococcus agalactiae 
                 
                 gram-positive 
                 13813 
               
               
                 
                   Streptococcus pneumoniae 
                 
                 gram-positive 
                 49619 
               
               
                 
                   Streptococcus pneumoniae 
                 
                 gram-positive 
                 6303 
               
               
                   Streptococcus pneumoniae  (P- 
                 gram-positive 
                 700670 
               
               
                 resistant) 
                   
                   
               
               
                 
                   Streptococcus pyogenes 
                 
                 gram-positive 
                 19615 
               
               
                   Viridans Streptococci  (Strep 
                 gram-positive 
                 POS 3177 
               
               
                 salivarius) 
                   
                   
               
               
                   Viridans Streptococci  (Strep mitts) 
                 gram-positive 
                 49456 
               
               
                 
                   Candida albicans 
                 
                 yeast 
                 18804 
               
               
                 
                   Candida albicans 
                 
                 yeast 
                 24433 
               
               
                 
                   Candida glabrata 
                 
                 yeast 
                 2001 
               
               
                 
                   Candida parapsilosis 
                 
                 yeast 
                 22019 
               
               
                   
               
             
          
         
       
     
       Example 2: Comparison of Extraction Protocols 
     Prior Art Procedure: 
       [0045]    A liquid sample (0.5 μL) was applied to a target plate and covered with 0.5 μL of formic acid (FA: 25%; AnagnosTec) and allowed to air dry. Therefore, the total formic acid concentration was 12.5%. The dried sample was overlaid with 0.5 μL of 20 mg 2, 5-dihydroxybenzoic acid (DHB; AnagnosTec). The matrix sample was crystallized by air drying at room temperature for 5 minutes. Measurements were performed with a Shimadzu Biotech AXIMA Assurance™ mass spectrometer equipped with a 337-nm nitrogen laser. This method correctly identified 60% of gram-positive bacteria and 86.6% gram-negative bacteria. The method and results of this example summarize the findings of Schmidt et al., “Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry,”  Eur. J. Clin. Microbiol Infect Dis,  23 Jun. 2011. 
       Exemplary Embodiment: 
       [0046]    The pellet of isolated microorganism from a PBC sample was resuspended in 600 μL water or 2 mM OG solution in a microcentrifuge tube. The turbidity was visually adjusted to greater than 0.5 McFarland. Sample (1-1.5 μL) was spotted onto the MALDI plate. The sample was allowed to air dry. Formic acid (1-2 μL of a 70% solution) was overlaid onto the sample and allowed to air dry. The dried sample was overlaid with 1 μL of matrix solution (HCCA) and allowed to air dry before identification by MALDI-TOF MS. This method correctly identified 95% of gram-positive bacteria and 100% gram-negative bacteria. These results illustrate that the use of formic acid at a concentration well above 12.5% significantly increases the rate of identification of both gram-positive and gram-negative bacteria. 
       Example 3: Comparison of Extraction Methods with Various Yeast Strains 
     Prior Art Method: 
       [0047]    Yeast colonies were resuspended in 600 μl of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Sample (0.5 μL) was pipetted onto a MALDI plate. Formic acid (0.5 μL of a 25% solution in water) was pipetted onto the sample and allowed to air dry. Therefore, the final concentration of formic acid was 12.5%. Matrix solution (1 μL HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS. This prior art method is the same method as disclosed in Schmidt et al., Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry,  Eur. J. Clin. Microbiol. Infect Dis,  23 Jun. 2011. 
       Exemplary Control Method 1: 
       [0048]    Yeast colonies were resuspended in 600 μl of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Sample (1 μL) was pipetted onto a MALDI plate and allowed to air dry. Formic acid (1 μL of a 50% solution in water) was pipetted onto the dried sample and allowed to air dry. MALDI matrix solution (1 μL of HCCA) was pipetted onto the dried sample and allowed to air dry before identification by MALDI-MS. 
       Exemplary Control Method 2: 
       [0049]    Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Sample (1-1.5 μL) was pipetted onto a MALDI plate and allowed to air dry. No formic acid extraction was performed; rather the dried sample was directly overlaid with matrix solution (1 μL of HCCA) and allowed to air dry before identification by MALDI-TOF MS. 
       Exemplary Embodiment: 
       [0050]    Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Sample (1-1.5 μL) was pipetted onto a MALDI plate and allowed to air dry. Formic acid (1-1.5 μL of a 60%, 70%, or 90% solution of formic acid in water) was pipetted onto the dried sample and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS. 
         [0051]    The results are summarized in Table 2 below and indicate a positive identification, “Yes”, or a failed identification, “No”, by MALDI-TOF MS. The results illustrate that the on-plate extraction methods described herein, when used with at least 70% formic acid, are sufficient to identify various yeast strains. In contrast, the prior art and control methods, which utilize 50% or less formic acid, failed to identify any of the yeast strains. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                   
                 12.5% 
                 50% 
                 60% 
                   
                   
               
               
                   
                   
                 No  
                 Formic  
                 Formic 
                 Formic 
                   
                   
               
               
                   
                   
                 Formic 
                 Acid 
                 Acid 
                 Acid  
                 70% 
                 90% 
               
               
                   
                 Strain 
                 Acid 
                 (Prior Art 
                 (Control 
                 (Control 
                 Formic 
                 Formic 
               
               
                 Organism  
                 Number 
                 Extraction 
                 Method) 
                 Method 1) 
                 Method 2) 
                 Acid 
                 Acid 
               
               
                   
               
             
             
               
                 
                   Candida 
                 
                 ATCC 
                 No 
                 No 
                 No 
                 No 
                 Yes 
                 Yes 
               
               
                 
                   albicans 
                 
                 145 
                   
                   
                   
                   
                   
                   
               
               
                 
                   Candida 
                 
                 ATCC 
                 No 
                 No 
                 No 
                 No 
                 Yes 
                 Yes 
               
               
                 
                   parapsilosis 
                 
                 147 
                   
                   
                   
                   
                   
                   
               
               
                 
                   Candida 
                 
                 ATCC 
                 No 
                 No 
                 No 
                 No 
                 Yes 
                 Yes 
               
               
                 
                   glabrata 
                 
                 394 
               
               
                   
               
             
          
         
       
     
       Example 4: Comparison of Exemplary Extraction Protocols with 90% Formic Acid+/−Ethyl Acetate for the Identification of Various Yeast Strains 
     Exemplary Embodiment: 
       [0052]    Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Duplicate samples (1-1.5 μL each) were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1-1.5 μL) of either a 90% solution in water or a 90% solution in ethyl acetate was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. In all, four samples (two samples for each formic acid solution) for each organism were prepared and subjected to MALDI-TOF MS analysis 
         [0053]    The MALDI score results are summarized in Table 3 below. These results illustrate an improved identification performance when formic acid extraction buffer is prepared in an organic solvent, for example, ethyl acetate, compared to formic acid in an aqueous solution. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                   
                 90%  
                 90%  
               
               
                   
                   
                   
                 Formic  
                 Formic  
               
               
                   
                   
                   
                 Acid in 
                 Acid in 
               
               
                   
                 Organism 
                 Strain Number 
                 Water 
                 Ethyl Acetate 
               
               
                   
               
             
             
               
                   
                 
                   Candida glabrata 
                 
                 YST 
                 2.098 
                 2.123 
               
               
                   
                   
                 26 
                 2.211 
                 2.038 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 
                 1.579 
                 1.494 
               
               
                   
                 
                   neoformans 
                 
                 54 
                 1.425 
                 1.573 
               
               
                   
                 
                   Candida parapsilosis 
                 
                 YST 
                 1.626 
                 2.006 
               
               
                   
                   
                 194 
                 1.826 
                 2.014 
               
               
                   
                 
                   Candida parapsilosis 
                 
                 YST 
                 1.766 
                 1.856 
               
               
                   
                   
                 792 
                 1.532 
                 1.805 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 1162 
                 1.437 
                 1.561 
               
               
                   
                 
                   neoformans 
                 
                   
                 1.481 
                 1.741 
               
               
                   
                 
                   Candida albicans 
                 
                 YST 1235 
                 1.754 
                 1.837 
               
               
                   
                   
                   
                 1.656 
                 1.813 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 1479 
                 1.344 
                 1.386 
               
               
                   
                 
                   neoformans 
                 
                   
                 1.436 
                 1.442 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 1481 
                 1.552 
                 1.617 
               
               
                   
                 
                   neoformans 
                 
                   
                 1.693 
                 1.772 
               
               
                   
               
             
          
         
       
     
       Example 5: Comparison of Exemplary Extraction Protocols with Formic Acid+/− Acetonitrile or Formic Acid+/−OG Buffer for the Identification of Various Yeast Strains 
     Exemplary Embodiment: 
       [0054]    Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Duplicate samples (1-1.5 μL) were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Various formic acid extraction buffers were prepared including: 70% formic acid in water; 90% formic acid in water; 70% formic acid in 20 mM OG buffer in water; and, 90% formic acid in 20 mM OG buffer in water. A volume of the extraction buffer (1-1.5 μL) was pipetted onto the dried samples and allowed to air dry. In one set of samples treated with 90% formic acid in water, a subsequent extraction step was performed by overlaying the dried sample with 50% acetonitrile in water and allowing the sample to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. A total of eight samples were prepared for each organism (two for each extraction buffer) and evaluated by MALDI-TOF MS. 
         [0055]    The MALDI-TOF MS score results are summarized in Table 4 below. The results illustrate that even microorganisms that are often difficult to identify, such as yeast, can be reliably identified by using an extraction buffer containing 90% formic acid, by preparing the formic acid extraction buffer in OG buffer or by performing a subsequent solvent only extraction step using an organic solvent such as acetonitrile. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                   
                   
                 90% formic 
                   
                   
               
               
                   
                   
                   
                   
                 acid in water 
                 70% formic 
                 90% formic 
               
               
                   
                   
                   
                   
                 followed by 
                 acid in 
                 acid in 
               
               
                   
                   
                 70% 
                 90% 
                 50% 
                 20 mM OG 
                 20 mM OG 
               
               
                   
                 Strain 
                 formic acid  
                 formic acid  
                 acetonitrile 
                 buffer in 
                 buffer in 
               
               
                 Organism  
                 Number 
                 in water 
                 in water 
                 in water 
                 water 
                 water 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   Candida 
                 
                 ATCC 
                 1.89 
                 1.845 
                 1.956 
                 1.856 
                 1.884 
               
               
                 
                   glabrata 
                 
                 2001 
                 1.932 
                 1.96 
                 1.968 
                 1.931 
                 1.979 
               
               
                 
                   Candida 
                 
                 ATCC 
                 1.673 
                 2.093 
                 1.932 
                 2.016 
                 2.019 
               
               
                 
                   albicans 
                 
                 18804 
                 1.643 
                 1.927 
                 2.009 
                 1.998 
                 2.093 
               
               
                 
                   Candida 
                 
                 ATCC 
                 &lt;0 
                 1.718 
                 1.642 
                 1.787 
                 2.036 
               
               
                 
                   parapsilosis 
                 
                 22019 
                 &lt;0 
                 2.131 
                 1.861 
                 1.871 
                 1.976 
               
               
                 
                   Candida 
                 
                 ATCC 
                 1.58 
                 1.687 
                 1.874 
                 1.684 
                 2.06 
               
               
                 
                   albicans 
                 
                 24433 
                 1.356 
                 1.835 
                 1.9 
                 1.788 
                 1.893 
               
               
                 
                   Candida 
                 
                 BQ141 
                 2.076 
                 1.997 
                 2.088 
                 2.205 
                 2.229 
               
               
                 
                   glabrata 
                 
                   
                 1.912 
                 2.064 
                 2.051 
                 2.172 
                 2.171 
               
               
                 
                   Candida 
                 
                 BP154 
                 1.941 
                 1.845 
                 1.996 
                 2.154 
                 2.165 
               
               
                 
                   glabrata 
                 
                   
                 1.867 
                 2.034 
                 2.181 
                 1.977 
                 2.199 
               
               
                   
               
             
          
         
       
     
       Example 6: Identification of Various Bacteria According to One Embodiment 
     Exemplary Embodiment: 
       [0056]    Bacterial colonies from a sub-culture were resuspended in 600 μL water. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three aliquots of the bacterial suspension (1.5 μL) for each microorganism were spotted onto a MALDI-TOF MS plate, and then immediately followed by sequential additions of 1.5 μL of 70% formic acid in ethanol and 1.5 μL of matrix solution (HCCA). The samples were allowed to air dry before identification by MALDI-TOF MS. The same experiment was repeated on a subsequent day by preparing a second bacterial suspension for each microorganism and performing the extraction procedure again preparing three samples for each microorganism. 
         [0057]    The MALDI-TOF MS score results are summarized in Table 5 below. These results illustrate that drying the sample after applying the extraction buffer and before applying the matrix solution is not required to obtain positive identification of the microorganism. In addition, these results demonstrate the reproducibility of the extraction procedure. Specifically, overall, two different sample suspensions produce substantially similar identification results. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Organism 
                 Strain Number 
                 Suspension 1 
                 Suspension 2 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 
                   Escherichia coli 
                 
                 25922 
                 2.292 
                 2.140 
               
               
                   
                   
                   
                 2.260 
                 2.254 
               
               
                   
                   
                   
                 2.207 
                 2.246 
               
               
                   
                 
                   Enterococcus faecalis 
                 
                 29212 
                 2.445 
                 2.567 
               
               
                   
                   
                   
                 2.385 
                 2.565 
               
               
                   
                   
                   
                 2.263 
                 2.524 
               
               
                   
                 
                   Staphylococcus 
                 
                 3569 
                 2.275 
                 2.194 
               
               
                   
                 
                   haemolyticus 
                 
                   
                 2.098 
                 2.181 
               
               
                   
                   
                   
                 2.005 
                 2.227 
               
               
                   
                 
                   Enterococcus faecium 
                 
                 700221 
                 2.316 
                 2.482 
               
               
                   
                   
                   
                 2.377 
                 2.444 
               
               
                   
                   
                   
                 0.000* 
                 2.320 
               
               
                   
                 
                   Staphylococcus 
                 
                 2885 
                 1.253 
                 1.931 
               
               
                   
                 
                   haemolyticus 
                 
                   
                 1.638 
                 2.010 
               
               
                   
                   
                   
                 1.532 
                 1.939 
               
               
                   
                 
                   Streptococcus 
                 
                 13813 
                 2.276 
                 2.527 
               
               
                   
                 
                   agalactiae 
                 
                   
                 2.320 
                 2.419 
               
               
                   
                   
                   
                 2.196 
                 2.442 
               
               
                   
                 
                   Streptococcus 
                 
                 49456 
                 2.154 
                 2.196 
               
               
                   
                 
                   pneumoniae 
                 
                   
                 2.286 
                 2.191 
               
               
                   
                   
                   
                 2.157 
                 2.138 
               
               
                   
                 
                   Klebsiella 
                 
                 700603 
                 2.189 
                 0.000* 
               
               
                   
                 
                   pneumoniae 
                 
                   
                 2.054 
                 2.006 
               
               
                   
                   
                   
                 2.126 
                 2.057 
               
               
                   
               
               
                 *These results are considered anomalies in the overall context of the data, and not viewed as a failure in the extraction protocol. 
               
             
          
         
       
     
       Example 7: Exemplary Embodiments of the Disclosed Methods Compared to Control Methods for the Identification of Yeast Strains 
     Exemplary Control Method (Process 22): 
       [0058]    Yeast colonies were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 2.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Neat formic acid (1 μL) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. 
         [0000]    Exemplary Embodiment—on Plate Extraction from Ethanol Suspension (Process 18): 
         [0059]    Yeast colonies from a sub-culture were resuspended in 600 μL of 50% ethanol in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% solution in ethanol) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. 
         [0000]    Exemplary Embodiment—on Plate Extraction from Water Suspension Followed by Ethanol (Process 19): 
         [0060]    Yeast colonies from a sub-culture were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI plate and allowed to air dry. Ethanol (1 μL of 70% concentration) was pipetted onto the dried samples and allowed to air dry. A solution of 70% formic acid/30% ethanol was pipetted (1 μL) onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. 
         [0000]    Exemplary Embodiment—on Plate Extraction from Acetone Suspension (Process 23): 
         [0061]    Yeast colonies from a sub-culture were resuspended in 600 μL of 50% acetone in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% concentration in acetone) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. 
         [0000]    Exemplary Embodiment—on Plate Extraction from Water Suspension Followed by Acetone (Process 24): 
         [0062]    Yeast colonies from a sub-culture were resuspended in 600 μL of water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. Three samples (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Acetone (1 μL of 70% concentration) was pipetted onto the dried samples and allowed to air dry. Formic acid (1 ul of 70% formic acid in acetone) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-MS. 
         [0063]    The MALDI-TOF MS score results for each of the methods in this example are summarized in Table 6 below. The results illustrate that 70% formic acid with an organic solvent produces superior results than with neat formic acid alone for the identification of yeast strains independent of the suspension used. The type of organic solvent used with formic acid did not affect the overall results. In addition, the organic solvent can be added during suspension of the sample, prior to treatment with formic acid, and/or during formic acid treatment without decreasing the rate of identification of various yeast strains. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                   
                 Strain 
                 Process 
                 Process 
                 Process 
                 Process 
                 Process 
               
               
                 Organism 
                 Number 
                 22 
                 18 
                 19 
                 23 
                 24 
               
               
                   
               
             
             
               
                 
                   Candida 
                 
                 YST 
                 1.693 
                 2.060 
                 2.067 
                 2.145 
                 2.022 
               
               
                 
                   glabrata 
                 
                 26 
                 1.752 
                 2.095 
                 2.137 
                 2.167 
                 1.913 
               
               
                   
                   
                 1.766 
                 2.097 
                 2.129 
                 2.172 
                 1.898 
               
               
                 
                   Cryptococcus 
                 
                 YST 
                 1.823 
                 1.870 
                 1.826 
                 1.862 
                 1.525 
               
               
                 
                   neoformans 
                 
                 54 
                 1.794 
                 0.000 
                 1.990 
                 1.917 
                 1.282 
               
               
                   
                   
                 1.918 
                 1.816 
                 1.872 
                 1.902 
                 0.000 
               
               
                 
                   Candida 
                 
                 YST 
                 1.927 
                 1.842 
                 1.949 
                 1.942 
                 1.814 
               
               
                 
                   parapsilosis 
                 
                 194 
                 1.968 
                 1.881 
                 2.093 
                 2.007 
                 1.864 
               
               
                   
                   
                 2.053 
                 1.974 
                 2.105 
                 1.926 
                 1.860 
               
               
                 
                   Candida 
                 
                 YST 
                 1.399 
                 1.784 
                 1.743 
                 1.763 
                 1.650 
               
               
                 
                   parapsilosis 
                 
                 792 
                 1.255 
                 1.871 
                 1.771 
                 1.932 
                 1.706 
               
               
                   
                   
                 1.645 
                 1.733 
                 1.835 
                 1.836 
                 1.769 
               
               
                 
                   Cryptococcus 
                 
                 YST 
                 1.325 
                 1.550 
                 1.681 
                 1.815 
                 1.591 
               
               
                 
                   neoformans 
                 
                 1162 
                 1.489 
                 1.776 
                 1.657 
                 1.671 
                 1.431 
               
               
                   
                   
                 1.504 
                 1.623 
                 1.741 
                 1.566 
                 1.562 
               
               
                 
                   Candida 
                 
                 YST 
                 1.148 
                 2.072 
                 2.024 
                 1.744 
                 1.968 
               
               
                 
                   albicans 
                 
                 1235 
                 1.460 
                 2.065 
                 2.004 
                 1.700 
                 1.960 
               
               
                   
                   
                 1.365 
                 2.037 
                 1.987 
                 1.837 
                 1.981 
               
               
                 
                   Cryptococcus 
                 
                 YST 
                 1.390 
                 0.000 
                 1.899 
                 1.913 
                 1.705 
               
               
                 
                   neoformans 
                 
                 1479 
                 1.445 
                 1.931 
                 1.821 
                 1.948 
                 1.689 
               
               
                   
                   
                 1.413 
                 1.941 
                 1.918 
                 2.069 
                 1.712 
               
               
                 
                   Cryptococcus 
                 
                 YST 
                 1.299 
                 0.000 
                 2.071 
                 2.020 
                 1.860 
               
               
                 
                   neoformans 
                 
                 1481 
                 1.315 
                 2.139 
                 2.076 
                 1.905 
                 1.824 
               
               
                   
                   
                 1.485 
                 2.022 
                 2.093 
                 1.981 
                 1.799 
               
               
                 
                   Candida 
                 
                 YST 
                 1.478 
                 2.176 
                 2.213 
                 2.140 
                 2.147 
               
               
                 
                   albicans 
                 
                 145 
                 1.783 
                 2.226 
                 2.045 
                 1.926 
                 2.005 
               
               
                   
                   
                 1.598 
                 2.103 
                 2.228 
                 1.860 
                 2.068 
               
               
                 
                   Candida 
                 
                 YST 
                 2.073 
                 2.113 
                 2.074 
                 2.202 
                 2.084 
               
               
                 
                   parapsilosis 
                 
                 147 
                 1.882 
                 2.021 
                 2.052 
                 2.169 
                 2.107 
               
               
                   
                   
                 2.021 
                 2.010 
                 2.064 
                 2.137 
                 2.070 
               
               
                 
                   Candida 
                 
                 YST 
                 2.022 
                 2.010 
                 2.076 
                 2.171 
                 2.188 
               
               
                 
                   parapsilosis 
                 
                 200 
                 1.961 
                 2.003 
                 1.946 
                 2.155 
                 1.992 
               
               
                   
                   
                 1.914 
                 2.013 
                 2.105 
                 2.202 
                 1.938 
               
               
                 
                   Candida 
                 
                 YST 
                 2.039 
                 2.284 
                 2.036 
                 2.073 
                 1.997 
               
               
                 
                   parapsilosis 
                 
                 214 
                 2.012 
                 2.082 
                 2.082 
                 2.163 
                 2.050 
               
               
                   
                   
                 1.991 
                 2.034 
                 2.182 
                 2.156 
                 2.030 
               
               
                 
                   Candida 
                 
                 YST 
                 1.408 
                 1.741 
                 1.520 
                 1.708 
                 1.341 
               
               
                 
                   dubliniensis 
                 
                 305 
                 1.492 
                 1.791 
                 1.467 
                 1.676 
                 1.220 
               
               
                   
                   
                 1.565 
                 1.639 
                 1.575 
                 1.262 
                 1.249 
               
               
                 
                   Candida 
                 
                 YST 
                 1.928 
                 2.213 
                 1.970 
                 2.024 
                 2.141 
               
               
                 
                   glabrata 
                 
                 394 
                 1.745 
                 2.255 
                 2.089 
                 2.022 
                 2.108 
               
               
                   
                   
                 2.011 
                 2.279 
                 2.128 
                 2.094 
                 2.136 
               
               
                 
                   Candida 
                 
                 YST 
                 1.499 
                 2.096 
                 2.130 
                 1.535 
                 1.911 
               
               
                 
                   albicans 
                 
                 1033 
                 1.366 
                 2.107 
                 2.015 
                 1.747 
                 1.863 
               
               
                   
                   
                 1.503 
                 2.070 
                 2.135 
                 1.496 
                 2.012 
               
               
                 
                   Candida 
                 
                 YST 
                 1.538 
                 2.154 
                 2.148 
                 2.047 
                 1.973 
               
               
                 
                   albicans 
                 
                 1035 
                 1.796 
                 2.120 
                 2.187 
                 1.887 
                 2.033 
               
               
                   
                   
                 1.760 
                 2.130 
                 2.236 
                 1.933 
                 1.921 
               
               
                 
                   Candida 
                 
                 YST 
                 2.029 
                 2.276 
                 2.278 
                 2.251 
                 2.219 
               
               
                 
                   glabrata 
                 
                 1045 
                 1.904 
                 2.286 
                 2.179 
                 2.232 
                 2.175 
               
               
                   
                   
                 1.986 
                 2.247 
                 2.297 
                 2.253 
                 2.145 
               
               
                 
                   Cyptococcus 
                 
                 YST 
                 1.405 
                 2.084 
                 1.786 
                 1.699 
                 1.918 
               
               
                 
                   neoformans 
                 
                 1074 
                 1.298 
                 2.057 
                 1.954 
                 1.850 
                 1.832 
               
               
                   
                   
                 1.427 
                 2.068 
                 1.940 
                 1.812 
                 1.890 
               
               
                 
                   Candida 
                 
                 YST 
                 1.454 
                 2.109 
                 2.122 
                 2.020 
                 2.040 
               
               
                 
                   albicans 
                 
                 1383 
                 1.560 
                 2.198 
                 2.096 
                 1.987 
                 2.054 
               
               
                   
                   
                 1.752 
                 2.159 
                 2.096 
                 1.758 
                 2.027 
               
               
                 
                   Candida 
                 
                 YST 
                 1.808 
                 2.283 
                 2.265 
                 2.147 
                 2.008 
               
               
                 
                   glab*rata 
                 
                 1454 
                 1.969 
                 2.257 
                 2.274 
                 2.143 
                 2.097 
               
               
                   
                   
                 1.862 
                 2.220 
                 2.301 
                 2.128 
                 1.949 
               
               
                   
               
             
          
         
       
     
       Example 8: Comparison of Exemplary Embodiments of the Disclosed Methods with Control Methods for the Identification of Yeast Strains 
     Exemplary Control: 
       [0064]    Yeast colonies from a pure culture plate were directly smeared onto a MALDI-TOF MS plate. Without drying, the matrix solution (1 μl of HCCA) was overlaid onto the sample and allowed to air dry before identification by MALDI-TOF MS. 
       Exemplary Embodiment—70% Formic Acid in Water: 
       [0065]    Yeast colonies from a pure culture plate were directly smeared onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% formic acid solution in water) was pipetted onto the smear and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS. 
       Exemplary Embodiment—70% Organic Solvent Followed by 70% Formic Acid in Water: 
       [0066]    Yeast colonies from a pure culture plate were directly smeared onto a MALDI-TOF MS plate and allowed to air dry. Organic solvent (1 μL of 70% solution in water) was pipetted onto the smear and allowed to air dry. The organic solvent was methanol, ethanol, isopropanol, or acetone. Formic acid (1 μL of 70% solution in water) was pipetted onto the dried sample and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried sample and allowed to air dry before identification by MALDI-TOF MS. 
         [0067]    The MALDI-TOF MS score results for each of the methods in this example are summarized in Table 7 below. The results indicate that for various yeast strains, using formic acid as an on-plate extraction solution provides a consistently higher rate of identification than the direct smear method without the use of formic acid. In addition, an additional extraction step with an organic solvent, prior to the formic acid on-plate extraction, results in a higher MALDI-TOF MS score than using the formic acid extraction alone. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 Direct 
                   
                   
                   
                   
                   
               
               
                   
                   
                 Smear 
                   
                 Methanol 
                 Ethanol 
                 Isopropanol 
                 Acetonel 
               
               
                   
                   
                 with no 
                 Formic 
                 Followed 
                 Followed 
                 Followed 
                 Followed 
               
               
                   
                 Strain 
                 Formic 
                 Acid 
                 by Formic 
                 by Formic 
                 by Formic 
                 by Formic 
               
               
                 Organism 
                 Number 
                 Acid 
                 Only 
                 Acid 
                 Acid 
                 Acid 
                 Acid 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   Candida 
                 
                 ATCC 
                 0 
                 1.709 
                 2.044 
                 2.083 
                 2.098 
                 2.01 
               
               
                 
                   albicans 
                 
                 18804 
                   
                   
                   
                   
                   
                   
               
               
                 
                   Candida 
                 
                 ATCC 
                 1.308 
                 1.619 
                 2.277 
                 2.289 
                 2.108 
                 2.13 
               
               
                 
                   albicans 
                 
                 24433 
                   
                   
                   
                   
                   
                   
               
               
                 
                   Candida 
                 
                 ATCC 
                 1.329 
                 1.889 
                 2.128 
                 2.209 
                 2.18 
                 2.172 
               
               
                 
                   glabrata 
                 
                 2001 
                   
                   
                   
                   
                   
                   
               
               
                 
                   Candida 
                 
                 ATCC 
                 0 
                 1.736 
                 1.887 
                 1.775 
                 1.883 
                 1.951 
               
               
                 
                   parapsilosis 
                 
                 22019 
                   
                   
                   
                   
                   
                   
               
               
                 
                   Candida 
                 
                 ATCC 
                 0 
                 1.421 
                 1.779 
                 1.886 
                 1.83 
                 1.949 
               
               
                 
                   neoformans 
                 
                 60234 
               
               
                   
               
             
          
         
       
     
       Example 9: Identification of Various Yeast and Bacteria Strains Using Exemplary Extraction Methods with Formic Acid/Ethanol Solution 
     Exemplary Embodiment: 
       [0068]    Yeast or bacterial colonies were resuspended in water in a microcentrifuge tube. The turbidity of the sample was visually adjusted to greater than 4.0 McFarland. An aliquot of the suspension was diluted 1:1 with 95.5% ethanol. Three aliquots of the diluted suspension (1 μL) for each microorganism were pipetted onto a MALDI-TOF MS plate and allowed to air dry. Formic acid (1 μL of 70% solution in ethanol) was pipetted onto the dried samples and allowed to air dry. Matrix solution (1 μL of HCCA) was overlaid onto the dried samples and allowed to air dry before identification by MALDI-TOF MS. 
         [0069]    The MALDI-TOF MS score results are summarized in Table 8 (yeast strains) and Table 9 (bacterial strains) below. Table 8 and 9 illustrate that on-plate extraction methods in which a microbial suspension is prepared by resuspending a microbial pellet in an organic solvent, for example, ethanol, followed by extraction with formic acid in ethanol provide 100% identification by MALDI-TOF MS for various yeast and bacteria strains. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                   
                   
                   
                 MALDI-TOF 
               
               
                   
                 Organism 
                 Strain Number 
                 MS score 
               
               
                   
               
             
             
               
                   
                 
                   Candida glabrata 
                 
                 YST 
                 2.013 
               
               
                   
                   
                 26 
                 2.184 
               
               
                   
                   
                   
                 2.189 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 
                 1.856 
               
               
                   
                 
                   neoformans 
                 
                 54 
                 2.068 
               
               
                   
                   
                   
                 1.912 
               
               
                   
                 
                   Candida parapsilosis 
                 
                 YST 
                 1.929 
               
               
                   
                   
                 194 
                 1.957 
               
               
                   
                   
                   
                 2.113 
               
               
                   
                 
                   Candida parapsilosis 
                 
                 YST 
                 1.962 
               
               
                   
                   
                 792 
                 1.831 
               
               
                   
                   
                   
                 1.844 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 
                 1.733 
               
               
                   
                 
                   neoformans 
                 
                 1162 
                 1.688 
               
               
                   
                   
                   
                 1.728 
               
               
                   
                 
                   Candida albicans 
                 
                 YST 
                 2.031 
               
               
                   
                   
                 1235 
                 1.860 
               
               
                   
                   
                   
                 1.949 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 
                 1.966 
               
               
                   
                 
                   neoformans 
                 
                 1479 
                 1.974 
               
               
                   
                   
                   
                 1.966 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 
                 2.115 
               
               
                   
                 
                   neoformans 
                 
                 1481 
                 2.186 
               
               
                   
                   
                   
                 2.136 
               
               
                   
                 
                   Candida albicans 
                 
                 YST 
                 2.176 
               
               
                   
                   
                 145 
                 2.226 
               
               
                   
                   
                   
                 2.103 
               
               
                   
                 
                   Candida parapsilosis 
                 
                 YST 
                 2.113 
               
               
                   
                   
                 147 
                 2.021 
               
               
                   
                   
                   
                 2.010 
               
               
                   
                 
                   Candida parapsilosis 
                 
                 YST 
                 2.010 
               
               
                   
                   
                 200 
                 2.003 
               
               
                   
                   
                   
                 2.013 
               
               
                   
                 
                   Candida parapsilosis 
                 
                 YST 
                 2.284 
               
               
                   
                   
                 214 
                 2.082 
               
               
                   
                   
                   
                 2.034 
               
               
                   
                 
                   Candida dubliniensis 
                 
                 YST 
                 1.741 
               
               
                   
                   
                 305 
                 1.791 
               
               
                   
                   
                   
                 1.639 
               
               
                   
                 
                   Candida glabrata 
                 
                 YST 
                 2.213 
               
               
                   
                   
                 394 
                 2.255 
               
               
                   
                   
                   
                 2.279 
               
               
                   
                 
                   Candida albicans 
                 
                 YST 
                 2.096 
               
               
                   
                   
                 1033 
                 2.107 
               
               
                   
                   
                   
                 2.070 
               
               
                   
                 
                   Candida albicans 
                 
                 YST 
                 2.154 
               
               
                   
                   
                 1035 
                 2.120 
               
               
                   
                   
                   
                 2.130 
               
               
                   
                 
                   Candida glabrata 
                 
                 YST 
                 2.276 
               
               
                   
                   
                 1045 
                 2.286 
               
               
                   
                   
                   
                 2.247 
               
               
                   
                 
                   Cryptococcus 
                 
                 YST 
                 2.084 
               
               
                   
                 
                   neoformans 
                 
                 1074 
                 2.057 
               
               
                   
                   
                   
                 2.068 
               
               
                   
                 
                   Candida albicans 
                 
                 YST 
                 2.109 
               
               
                   
                   
                 1383 
                 2.198 
               
               
                   
                   
                   
                 2.159 
               
               
                   
                 
                   Candida glabrata 
                 
                 YST 
                 2.283 
               
               
                   
                   
                 1454 
                 2.257 
               
               
                   
                   
                   
                 2.220 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                   
                   
                 Strain 
                 MALDI-TOF  
               
               
                   
                 Organism 
                 Number 
                 MS score 
               
               
                   
               
             
             
               
                   
                 
                   Acinetobacter 
                 
                 ENF 
                 2.359 
               
               
                   
                 
                   baumanii 
                 
                 11091 
                 2.403 
               
               
                   
                   
                   
                 2.356 
               
               
                   
                 
                   Enterobacter 
                 
                 13048 
                 2.410 
               
               
                   
                 
                   aerogenes 
                 
                   
                 2.409 
               
               
                   
                   
                   
                 2.427 
               
               
                   
                 
                   Enterobacter cloacae 
                 
                 35030 
                 2.178 
               
               
                   
                   
                   
                 2.097 
               
               
                   
                   
                   
                 2.042 
               
               
                   
                 
                   Escherichia coli 
                 
                 25922 
                 2.374 
               
               
                   
                   
                   
                 2.231 
               
               
                   
                   
                   
                 2.334 
               
               
                   
                 
                   Escherichia coli 
                 
                 35213 
                 2.387 
               
               
                   
                   
                   
                 2.164 
               
               
                   
                   
                   
                 2.219 
               
               
                   
                 
                   Klebsiella 
                 
                 33495 
                 2.282 
               
               
                   
                 
                   pneumoniae 
                 
                   
                 2.335 
               
               
                   
                   
                   
                 2.351 
               
               
                   
                 
                   Klebsiella 
                 
                 700603 
                 2.200 
               
               
                   
                 
                   pneumoniae 
                 
                   
                 2.046 
               
               
                   
                   
                   
                 2.190 
               
               
                   
                 
                   Proteus mirabilis 
                 
                 29906 
                 2.261 
               
               
                   
                   
                   
                 2.298 
               
               
                   
                   
                   
                 2.324 
               
               
                   
                 
                   Pseudomonas 
                 
                 27853 
                 2.276 
               
               
                   
                 
                   aeruginosa 
                 
                   
                 2.367 
               
               
                   
                   
                   
                 2.325 
               
               
                   
                 
                   Pseudomonas 
                 
                 ENF 
                 2.258 
               
               
                   
                 
                   aeruginosa 
                 
                 14620 
                 2.275 
               
               
                   
                   
                   
                 1.999 
               
               
                   
                 
                   Serratia marcescens 
                 
                 FR197 
                 2.139 
               
               
                   
                   
                   
                 2.081 
               
               
                   
                   
                   
                 2.054 
               
               
                   
                 
                   Stenotrophomonas 
                 
                 13637 
                 2.128 
               
               
                   
                 
                   maltophilia 
                 
                   
                 1.784 
               
               
                   
                   
                   
                 2.139 
               
               
                   
                 
                   Enterococcus faecalis 
                 
                 29212 
                 2.318 
               
               
                   
                   
                   
                 2.216 
               
               
                   
                   
                   
                 2.269 
               
               
                   
                 
                   Enterococcus faecalis 
                 
                 51299 
                 2.272 
               
               
                   
                 (VRE) 
                   
                 2.291 
               
               
                   
                   
                   
                 2.353 
               
               
                   
                 
                   Enterococcus 
                 
                 19434 
                 2.373 
               
               
                   
                 
                   faecium 
                 
                   
                 2.389 
               
               
                   
                   
                   
                 2.271 
               
               
                   
                 
                   Enterococcus 
                 
                 700221 
                 1.862 
               
               
                   
                   faecium  VRE 
                   
                 1.977 
               
               
                   
                   
                   
                 1.894 
               
               
                   
                 
                   Staphylococcus 
                 
                 25923 
                 2.379 
               
               
                   
                 
                   aureus 
                 
                   
                 2.285 
               
               
                   
                   
                   
                 2.391 
               
               
                   
                 
                   Staphylococcus 
                 
                 29213 
                 2.331 
               
               
                   
                   aureus  (MSSA) 
                   
                 2.305 
               
               
                   
                   
                   
                 2.387 
               
               
                   
                 
                   Staphylococcus 
                 
                 3421 
                 2.324 
               
               
                   
                   aureus  (MRSA) 
                   
                 2.323 
               
               
                   
                   
                   
                 2.370 
               
               
                   
                 
                   Staphylococcus 
                 
                 4330 
                 2.245 
               
               
                   
                   aureus  (MRSA) 
                   
                 2.187 
               
               
                   
                   
                   
                 2.200 
               
               
                   
                 
                   Staphylococcus 
                 
                 3568 
                 1.953 
               
               
                   
                 
                   epidermidis 
                 
                   
                 1.777 
               
               
                   
                   
                   
                 1.861 
               
               
                   
                 
                   Staphylococcus 
                 
                 77 
                 2.051 
               
               
                   
                 
                   epidermidis 
                 
                   
                 2.039 
               
               
                   
                   
                   
                 2.001 
               
               
                   
                 
                   Staphylococcus 
                 
                 356 
                 0.000 
               
               
                   
                 
                   haemolyticus 
                 
                   
                 2.071 
               
               
                   
                   
                   
                 1.883 
               
               
                   
                 
                   Staphylococcus sciuri 
                 
                 29062 
                 1.910 
               
               
                   
                   
                   
                 1.751 
               
               
                   
                   
                   
                 1.865 
               
               
                   
                 
                   Streptococcus 
                 
                 12386 
                 2.227 
               
               
                   
                 
                   agalactiae 
                 
                   
                 2.134 
               
               
                   
                   
                   
                 2.269 
               
               
                   
                 
                   Streptococcus 
                 
                 13813 
                 2.229 
               
               
                   
                 
                   agalactiae 
                 
                   
                 2.189 
               
               
                   
                   
                   
                 2.304 
               
               
                   
                 
                   Streptococcus 
                 
                 49619 
                 2.143 
               
               
                   
                 
                   pneumoniae 
                 
                   
                 1.923 
               
               
                   
                   
                   
                 2.067 
               
               
                   
                 
                   Streptococcus 
                 
                 6303 
                 2.124 
               
               
                   
                 
                   pneumoniae 
                 
                   
                 2.001 
               
               
                   
                   
                   
                 2.019 
               
               
                   
                 
                   Streptococcus 
                 
                 700670 
                 2.058 
               
               
                   
                 
                   pneumoniae 
                 
                   
                 2.321 
               
               
                   
                   
                   
                 2.185 
               
               
                   
                 
                   Streptococcus 
                 
                 19615 
                 2.380 
               
               
                   
                 
                   pyogenes 
                 
                   
                 1.721 
               
               
                   
                   
                   
                 2.474 
               
               
                   
                 
                   Streptococcus 
                 
                 3177 
                 1.864 
               
               
                   
                 
                   salivarius 
                 
                   
                 1.712 
               
               
                   
                   
                   
                 1.943 
               
               
                   
                 
                   Streptococcus mitis 
                 
                 49456 
                 2.119 
               
               
                   
                   
                   
                 2.021 
               
               
                   
                   
                   
                 1.995 
               
               
                   
               
             
          
         
       
     
         [0070]    The various embodiments described and illustrated herein, demonstrate various methods for identifying microorganism(s) in a sample resulting in a consistently superior rate of identification with fewer steps compared to the prior art methods. The on-plate extraction methods are easily amenable to automated procedures as there is no need for centrifugation. In addition, the extraction plate can be heated in order to increase the speed of the various drying steps resulting in a fast and accurate process for identifying microorganism(s) in a sample. 
         [0071]    Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.