1887

Simulation of MICROBACT Strip Assay Using Colored Liquids to Demonstrate Identification of Unknown Gram-Negative Organisms in Undergraduate Laboratory

    Authors: Latifa B. Khan1,*, Simon Swift1, Tania Kamal1,3, Hannah M. Read1,2
    VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Auckland 1023, New Zealand; 2: Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland 1023, New Zealand; 3: Auckland Cancer Society Research Centre, The University of Auckland, Auckland 1023, New Zealand
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Received 19 December 2017 Accepted 20 April 2018 Published 29 June 2018
    • ©2018 Author(s). Published by the American Society for Microbiology.
    • [open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.

    • Supplemental materials available at http://asmscience.org/jmbe
    • *Corresponding author. Mailing address: School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 1023, New Zealand. Phone: 64-09-3737599, ext 84492. E-mail: [email protected].
    Source: J. Microbiol. Biol. Educ. June 2018 vol. 19 no. 2 doi:10.1128/jmbe.v19i2.1565
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    Abstract:

    Identification of unknown microorganisms to the species level is an important component of a microbiology course. Modern technologies such as matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing offer a rapid species level identification when compared to conventional phenotype-based methods, however they rely on a well-established taxonomy database and phenotypic assays can still play an important role in species determination. Another major limitation is the up-front cost of purchasing these modern specialized instruments and the requirement of skilled personnel to operate specialized equipment and software, which makes them unsuitable to use in the undergraduate teaching laboratory. Commercial biochemical identification systems such as the Oxoid Microbact™ GNB 12A/12E/24E kit is used for the identification of Enterobacteriaceae and other Gram negative bacteria in clinical and veterinary diagnostic laboratories, and food industries for disease control and treatment. In this article, we describe a method of reliably simulating this Microbact™ strip assay using artificial color liquids (which are affordable and easy to source) for the demonstration of phenotypic characterization of unknown Gram negative organisms while providing a safe teaching environment as no bacteria are used, familiarizing students with the concept of using Microbact™ 12A kit to identify a range of unknown Gram negative organisms.

References & Citations

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2. World Health Organization (WHO) 31 October 2017 posting date Food safety [Fact sheet] www.who.int/mediacentre/factsheets/fs399/en/ Accessed October 2017
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5. Singhal N, Kumar M, Kanaujia PK, Virdi JS 2015 MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis Front Microbiol 6 791 10.3389/fmicb.2015.00791 26300860 4525378 http://dx.doi.org/10.3389/fmicb.2015.00791
6. Segawa S, Sawai S, Murata S, Nishimura M, Beppu M, Sogawa K, Watanabe M, Satoh M, Matsutani T, Kobayashi M, Iwadate Y, Kuwabara S, Saeki N, Nomura F 2014 Direct application of MALDI-TOF mass spectrometry to cerebrospinal fluid for rapid pathogen identification in a patient with bacterial meningitis Clin Chim Acta 435 59 61 10.1016/j.cca.2014.04.024 24797349 http://dx.doi.org/10.1016/j.cca.2014.04.024
7. Ng W 2013 Teaching microbial identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and bioinformatics tools J Microbiol Biol Educ 14 103 106 10.1128/jmbe.v14i1.494 23858360 3706139 http://dx.doi.org/10.1128/jmbe.v14i1.494
8. Mazzeo MF, Sorrentino A, Gaita M, Cacace G, Di Stasio M, Facchiano A, Comi G, Malorni A, Siciliano RA 2006 Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms Appl Environ Microbiol 72 1180 1189 10.1128/AEM.72.2.1180-1189.2006 16461665 1392959 http://dx.doi.org/10.1128/AEM.72.2.1180-1189.2006
9. Giebel RA, Fredenberg W, Sandrin TR 2008 Characterization of environmental isolates of Enterococcus spp. by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Water Res 42 931 940 10.1016/j.watres.2007.09.005 http://dx.doi.org/10.1016/j.watres.2007.09.005
10. Siegrist TJ, Anderson PD, Huen WH, Kleinheinz GT, McDermott CM, Sandrin TR 2007 Discrimination and characterization of environmental strains of Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) J Microbiol Methods 68 554 562 10.1016/j.mimet.2006.10.012 http://dx.doi.org/10.1016/j.mimet.2006.10.012
11. Mugg P, Hill A 1981 Comparison of the Microbact-12E and 24E systems and the API-20E system for the identification of Enterobacteriaceae J Hygiene 87 287 297 10.1017/S0022172400069503 http://dx.doi.org/10.1017/S0022172400069503
12. Vithanage NR, Yeager TR, Jadhav SR, Palombo EA, Datta N 2014 Comparison of identification systems for psychrotrophic bacteria isolated from raw bovine milk Int J Food Microbiol 189 26 38 10.1016/j.ijfoodmicro.2014.07.023 25113043 http://dx.doi.org/10.1016/j.ijfoodmicro.2014.07.023
13. Fall B, Lo CI, Samb-Ba B, Perrot N, Diawara S, Gueye MW, Sow K, Aubadie-Ladrix M, Mediannikov O, Sokhna C, Diemé Y, Chatellier S, Wade B, Raoult D, Fenollar F 2015 The ongoing revolution of MALDI-TOF mass spectrometry for microbiology reaches tropical Africa Am J Trop Med Hygiene 92 641 647 10.4269/ajtmh.14-0406 http://dx.doi.org/10.4269/ajtmh.14-0406
14. Mailafia S, Olabode O, Okoh G, Jacobs C, Adamu S, Amali Onyilokwu S 2017 Microbact™ 24E system identification and antimicrobial sensitivity pattern of bacterial flora from raw milk of apparently healthy lactating cows in Gwagwalada, Nigeria J Coastal Life Med 5 8 356 359 10.12980/jclm.5.2017J7-46 http://dx.doi.org/10.12980/jclm.5.2017J7-46
15. Brightwell G, Clemens R, Urlich S, Boerema J 2007 Possible involvement of psychrotolerant Enterobacteriaceae in blown pack spoilage of vacuum-packaged raw meats Int J Food Microbiol 119 334 339 10.1016/j.ijfoodmicro.2007.08.024 17870199 http://dx.doi.org/10.1016/j.ijfoodmicro.2007.08.024
16. Becker B, Weiss C, Holzapfel WH 2009 An evaluation of the use of three phenotypic test-systems for biochemical identification of Enterobacteriaceae and Pseudomonadaceae Food Control 20 815 821 10.1016/j.foodcont.2008.10.014 http://dx.doi.org/10.1016/j.foodcont.2008.10.014
17. Alsheikh ADI, Mohammed GE, Abdalla MA 2012 First isolation and identification of listeria monocytogenes from fresh raw dressed broiler chicken in Sudan Res J Microbiol 7 319 326 10.3923/jm.2012.319.326 http://dx.doi.org/10.3923/jm.2012.319.326
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2018-06-29
2019-08-25

Abstract:

Identification of unknown microorganisms to the species level is an important component of a microbiology course. Modern technologies such as matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing offer a rapid species level identification when compared to conventional phenotype-based methods, however they rely on a well-established taxonomy database and phenotypic assays can still play an important role in species determination. Another major limitation is the up-front cost of purchasing these modern specialized instruments and the requirement of skilled personnel to operate specialized equipment and software, which makes them unsuitable to use in the undergraduate teaching laboratory. Commercial biochemical identification systems such as the Oxoid Microbact™ GNB 12A/12E/24E kit is used for the identification of Enterobacteriaceae and other Gram negative bacteria in clinical and veterinary diagnostic laboratories, and food industries for disease control and treatment. In this article, we describe a method of reliably simulating this Microbact™ strip assay using artificial color liquids (which are affordable and easy to source) for the demonstration of phenotypic characterization of unknown Gram negative organisms while providing a safe teaching environment as no bacteria are used, familiarizing students with the concept of using Microbact™ 12A kit to identify a range of unknown Gram negative organisms.

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Figures

Image of FIGURE 1

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FIGURE 1

Results of simulated MICROBACT strip assay for four sample unknowns: gram-negative bacteria (B, C, D, and E) and an un-inoculated control strip.

Source: J. Microbiol. Biol. Educ. June 2018 vol. 19 no. 2 doi:10.1128/jmbe.v19i2.1565
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Image of FIGURE 2

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FIGURE 2

Schematic illustration of simulating MICROBACT strip assay for demonstration to identify unknown gram-negative isolates. Notes: See Table 1, Appendix 1 . See Table 1, Appendix 2 (along with MICROBACT color reference chart that comes with the kit). See Appendix 1 . See Table 2, Appendix 2 , and Table 1, Appendix 3 .

Source: J. Microbiol. Biol. Educ. June 2018 vol. 19 no. 2 doi:10.1128/jmbe.v19i2.1565
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