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Cited by 7,264Bacterial Identification and Susceptibility TestingAntimicrobial agents and applicationsEssential Oils and Antimicrobial Activity

Methods for in vitro evaluating antimicrobial activity: A review

Open access
Balouiri, Mounyr, Sadiki, Moulay, Ibnsouda, Saad Koraichi·2016·Journal of Pharmaceutical Analysis
DOI 10.1016/j.jpha.2015.11.005PMC PMC5762448
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Abstract

In recent years, there has been a growing interest in researching and developing new antimicrobial agents from various sources to combat microbial resistance. Therefore, a greater attention has been paid to antimicrobial activity screening and evaluating methods. Several bioassays such as disk-diffusion, well diffusion and broth or agar dilution are well known and commonly used, but others such as flow cytofluorometric and bioluminescent methods are not widely used because they require specified equipment and further evaluation for reproducibility and standardization, even if they can provide rapid results of the antimicrobial agent's effects and a better understanding of their impact on the viability and cell damage inflicted to the tested microorganism. In this review article, an exhaustive list of in vitro antimicrobial susceptibility testing methods and detailed information on their advantages and limitations are reported.

AI snapshot

Written from the paper’s full text.

TL;DR

This review surveys in vitro antimicrobial susceptibility testing methods—diffusion, dilution, TLC-bioautography, time-kill, ATP bioluminescence, and flow cytofluorometry—and details their advantages and limitations. It stresses that CLSI/EUCAST standardization improves reproducibility, while non-standardized approaches for natural products make result comparison difficult.

Key findings

  • Disk-diffusion is official in many clinical labs and uses ~6 mm discs; CLSI conditions include bacteria at (0.5 McFarland) (1–2)×10^8 CFU/mL, 35±2°C for 16–18 h, yeast (1–5)×10^6 CFU/mL for 20–24 h, and molds (0.4–5)×10^6 CFU/mL. It cannot determine MIC or distinguish bactericidal from bacteriostatic effects; Etest strips cost about $2–3 each.
  • Dilution methods are most appropriate for MIC determination; broth microdilution uses bacteria at 5×10^5 CFU/mL for 20 h, yeast (0.5–2.5)×10^3 CFU/mL for 24–48 h, molds (0.4–5)×10^4 CFU/mL for 48 h, and agar dilution uses 10^4 CFU/spot for 16–20 h. MBC is defined as 99.9% kill; MFC as 98%–99.9% killing.
  • TLC-bioautography localizes active constituents on the TLC plate; time-kill test reveals time- or concentration-dependent killing, with bactericidal effect at 90% lethality for 6 h equivalent to 99.9% for 24 h. ATP bioluminescence can give antimycobacterial results in 3–5 days versus 3–4 weeks for conventional dilution.
  • Flow cytofluorometry can discriminate dead, viable, and injured cells and gives results in 2–6 h versus 24–72 h for microdilution, but its widespread use is limited by equipment access. CLSI/EUCAST standards exist for bacteria, yeasts, and molds, yet natural product testing often requires protocol modifications.

Methods

  • Narrative review; no primary experimental sample or systematic search strategy is described. It synthesizes published methods and CLSI/EUCAST guidelines for in vitro antimicrobial susceptibility testing.
  • Describes procedures, recommended culture media, inoculum sizes, incubation conditions (Table 1), and advantages/limitations of diffusion, dilution, TLC-bioautography, time-kill, ATP bioluminescence, and flow cytofluorometric methods.
  • Main analysis is qualitative description and comparison; no quantitative meta-analysis or primary data are reported.

Limitations

  • Paper states: flow cytofluorometric and bioluminescent methods are not widely used because they require specified equipment and further evaluation for reproducibility and standardization; flow cytometry equipment is inaccessible in various laboratories.
  • Paper states: disk-diffusion cannot distinguish bactericidal and bacteriostatic effects and is not appropriate for MIC; macrodilution is tedious, error-prone, and requires large reagent/space; microdilution results are significantly influenced by approach.
  • Paper states: comparison of natural product results is often difficult due to non-standardized inoculum preparation, inoculum size, growth medium, incubation conditions, and endpoints. Evident from text: as a narrative review, it lacks a systematic search strategy, inclusion criteria, quality assessment, and primary data.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.jpha.2015.11.005

Cites

94 of 94 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 103 cited sources checked · 7 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Previous studies have shown a good correlation between MIC values determined by Etest and those obtained by broth dilution or agar dilution.

    “Several previous studies have shown a good correlation between the MIC values determined by Etest and those obtained by broth dilution or agar dilution method [25], [26], [27].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [25] Baker, 1991, Comparison of the E Test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria, J. Clin. Microbiol. source
    • cites [26] Berghaus, 2015, Comparison of Etest, disk diffusion, and broth macrodilution for in vitro susceptibility testing of Rhodococcus equi, J. Clin. Microbiol. source
    • cites [27] Gupta, 2015, Comparative evaluation of disc diffusion and E-test with broth micro-dilution in susceptibility testing of amphotericin B, voriconazole and caspofungin against clinical Aspergillus isolates, J. Clin. Diagn. Res. source

      The Agreement section (page not specified) reports 75–100% agreement in susceptibility results between E-test and broth micro-dilution for clinical Aspergillus isolates, supporting the broth-comparison part broadly. It does not establish a correlation between the numerical MIC values or compare E-test with agar dilution. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    The advantages of disk-diffusion, mainly simplicity and low cost, have contributed to its common use for antimicrobial screening of plant extracts, essential oils and other drugs.

    “The above-mentioned advantages of this method, mainly simplicity and low cost, have contributed to its common use for the antimicrobial screening of plant extracts, essential oils and other drugs [20], [21], [22], [23].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [20] Fourati-Ben Fguira, 2005, Purification and structure elucidation of antifungal and antibacterial activities of newly isolated Streptomyces sp. strain US80, Res. Microbiol. source
    • cites [21] Konaté, 2012, Antibacterial activity against β-lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: Fractional Inhibitory Concentration Index (FICI) determination, Ann. Clin. Microbiol. Antimicrob. source

      The Methods section (In vitro antibacterial activity; page not provided) describes a disc-diffusion assay using plant-derived alkaloid compounds and antibiotic controls, supporting the method’s use in antimicrobial testing. It does not say the method is simple or inexpensive, establish that those advantages explain its common use, or describe disc-diffusion screening of essential oils. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [22] De Billerbeck, 2007, Huiles Essentielles et Bactéries Résistantes aux Antibiotiques, Phytotherapie source
  • Premisecitation

    The time-kill test reveals a time-dependent or a concentration-dependent antimicrobial effect.

    “The time-kill test reveals a time-dependent or a concentration-dependent antimicrobial effect [55].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] Pfaller, 2004, Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization, Clin. Microbiol. Rev. source
  • Premisecitation

    The time-kill method can be used to determine synergism or antagonism between drugs (two or more) in combinations.

    “In addition, this method can be used to determine synergism or antagonism between drugs (two or more) in combinations [28], [55].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [28] White, 1996, Comparison of three different in vitro methods of detecting synergy : time-kill, checkerboard, and E test, Antimicrob. Agents Chemother. source
    • cites [55] Pfaller, 2004, Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization, Clin. Microbiol. Rev. source
  • Premisecitation

    The ATP bioluminescence technique can provide results in 3-5 days for antimycobacterial tests in comparison with the conventional dilution technique, which requires 3-4 weeks of incubation.

    “Indeed it has been demonstrated that this technique can provide results in 3–5 days for antimycobacterial tests [90], [91] in comparison with the conventional dilution technique, which requires 3–4 weeks of incubation [90,91].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [90] Andreu, 2012, Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence, J. Antimicrob. Chemother. source

      The Abstract Results report that bioluminescence yielded MIC results in 3 days, supporting the claim of faster antimycobacterial testing. But the Introduction assigns the 3–5-day range to an optical-density microdilution assay and the 3–4-week period to colony formation on solid medium, not to the bioluminescence and conventional dilution techniques, respectively. The paper does not identify its method as ATP bioluminescence. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [91] Beckers, 1985, Evaluation of a bioluminescence assay for rapid antimicrobial susceptibility testing of mycobacteria, Eur. J. Clin. Microbiol. source
  • Classified as probably not a substantial claim. 30
  • May combine two claims. 23

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Antimicrobial resistance has considerable impact, with treatment failures associated with multidrug-resistant bacteria, and is a global public health concern.

    “Currently, its impact is considerable with treatment failures associated with multidrug-resistant bacteria and it has become a global concern to public health [2], [3].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [2] Guschin, 2015, Treatment efficacy, treatment failures and selection of macrolide resistance in patients with high load of Mycoplasma genitalium during treatment of male urethritis with Josamycin, BMC Infect. Dis. source

      The Results (page unavailable) report three josamycin treatment failures in patients whose Mycoplasma genitalium strains had macrolide-resistance mutations during treatment. The paper does not establish that those failures involved multidrug-resistant bacteria or that antimicrobial resistance generally is a global public-health concern. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [3] Martin, 2015, Antimicrobial resistance to Neisseria gonorrhoeae in Canada: 2009–2013, Can. Commun. Dis. Rep. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Agar disk-diffusion testing was developed in 1940 and is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing.

    “Agar disk-diffusion testing developed in 1940 [8], is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [8] Heatley, 1944, A method for the assay of penicillin, Biochem. J. source

      In “Possible Modifications and Applications of the Method,” Heatley says that a filter-paper disk method was proposed in 1940. The paper describes a penicillin assay using cylinders, however, and does not say that disk diffusion was developed in 1940 or became an official method for routine clinical antimicrobial susceptibility testing. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Direct bioautography is the easiest technique for detection of antifungal substances and gives consistent results for spore-producing fungi such as Aspergillus, Penicillium and Cladosporium.

    “It is the easiest technique for the detection of antifungal substances, and also gives consistent results for spore-producing fungi such as Aspergillus, Penicillium and Cladosporium [51], [52].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [51] Suleiman, 2010, Detection of antimicrobial compounds by bioautography of different extracts of leaves of selected South African tree species, Afr. J. Tradit. Complement. Altern. Med. source

      The Introduction (page not specified) describes bioautography as fast, cheap and effective for detecting antimicrobial compounds, which supports its general use for detecting antifungal substances. It does not call direct bioautography the easiest technique or report consistent results for spore-producing fungi. The Microorganisms and medium section lists Aspergillus fumigatus, but not Penicillium or Cladosporium. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [52] Homans, 1970, Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic substances, J. Chromatogr. A source

  • May combine two claims.

    Premisecitation

    TLC-bioautography offers a rapid technique for screening a large number of samples for bioactivity and in bioactivity-guided fractionation.

    “the TLC–bioautography offers a rapid technique for the screening of a large number of samples for bioactivity and in the bioactivity-guided fractionation [45].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [45] Dewanjee, 2015, Bioautography and its scope in the field of natural product chemistry, J. Pharm. Anal. source

  • May combine two claims.

    Premisecitation

    Generally, the bactericidal effect is obtained with a lethality percentage of 90% for 6 h, which is equivalent to 99.9% of lethality for 24 h.

    “Generally, the bactericidal effect is obtained with a lethality percentage of 90% for 6 h, which is equivalent to 99.9% of lethality for 24 h [21].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [21] Konaté, 2012, Antibacterial activity against β-lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: Fractional Inhibitory Concentration Index (FICI) determination, Ann. Clin. Microbiol. Antimicrob. source

  • May combine two claims.

    Interpretationcitation

    Development of methodologic standards does not guarantee clinical relevance of testing, but it allows the bioassay to be performed in a standardized approach to evaluate clinical relevance of results.

    “The development of such methodologic standards does not guarantee the clinical relevance of such testing. Nevertheless, it does allow the bioassay to be performed in a standardized approach in order to evaluate the clinical relevance of results [55].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] Pfaller, 2004, Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization, Clin. Microbiol. Rev. source

  • May combine two claims.

    Interpretationreasoning

    Disk-diffusion cannot distinguish bactericidal and bacteriostatic effects because bacterial growth inhibition does not mean bacterial death.

    “However, since the bacterial growth inhibition does not mean the bacterial death, this method cannot distinguish bactericidal and bacteriostatic effects.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Synergy in the Etest combination method is detected by a decrease of the MIC of the combination by at least two dilutions compared to the most active antibiotic tested alone.

    “The synergy is detected by a decrease of the MIC of the combination by at least two dilutions compared to that of the most active antibiotic tested alone [29].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Denes, 2009, Synergie et Antagonisme en Antibiothérapie, Antibiotiques source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    TLC-bioautography combines TLC with both biological and chemical detection methods.

    “This technique combines TLC with both biological and chemical detection methods.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts.

    “Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts [32], [33].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] Magaldi, 2004, Well diffusion for antifungal susceptibility testing, Int. J. Infect. Dis. source
    • cites [33] Valgas, 2007, Screening methods to determine antibacterial activity of natural products, Braz. J. Microbiol. source

  • May combine two claims.

    Premisecitation

    TLC-bioautography is a simple, effective and inexpensive technique for separation of a complex mixture and localizes active constituents on the TLC plate.

    “Overall, TLC–bioautography is a simple, effective and inexpensive technique for the separation of a complex mixture, and at the same time, it localizes the active constituents on the TLC plate.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Marston, 2011, Thin-layer chromatography with biological detection in phytochemistry, J. Chromatogr. A source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Time-kill test and flow cytofluorometric methods are recommended to study the antimicrobial effect of an agent in depth, providing information on the nature of the inhibitory effect and the cell damage inflicted to the test microorganism.

    “To further study the antimicrobial effect of an agent in depth, time-kill test and flow cytofluorometric methods are recommended, which provide information on the nature of the inhibitory effect (bactericidal or bacteriostatic) (time-dependent or concentration-dependent) and the cell damage inflicted to the test microorganism.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Not all fastidious bacteria can be tested accurately by disk-diffusion, but standardization has been made to test certain fastidious bacterial pathogens using specific culture media, incubation conditions and interpretive criteria for inhibition zones.

    “Although not all fastidious bacteria can be tested accurately by this method, the standardization has been made to test certain fastidious bacterial pathogens like streptococci, Haemophilus influenzae, Haemophilus parainfluenzae, Neisseria gonorrhoeae and Neisseria meningitidis, using specific culture media, various incubation conditions and interpretive criteria for inhibition zones [9].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard, 7th ed., CLSI document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cross streak method is used to rapidly screen microorganisms for antagonism.

    “Cross streak method is used to rapidly screen microorganisms for antagonism [36].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Lertcanawanichakul, 2008, A comparison of two methods used for measuring the antagonistic activity of Bacillus species, Walailak J. Sci. Tech.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Agar diffusion, also known as agar contact method, is the least-employed of the three TLC-bioautographic techniques.

    “Also known as agar contact method, it is the least-employed one of the techniques.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In 1946, Goodall and Levi combined paper chromatography with contact bioautography to detect different penicillins.

    “In 1946, Goodall and Levi [40] combined paper chromatography method (PC) with contact bioautography to detect different penicillins for their determination.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Goodall, 1946, A microchromatographic method for the detection and approximate determination of the different penicillins in a mixture, Nature source

  • May combine two claims.

    Premisecitation

    Etest strips cost about $2-3 each, so the approach becomes costly if numerous drugs are tested.

    “However, Etest® strips cost about $2–3 each. Therefore, this approach becomes costly if numerous drugs are tested [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Jorgensen, 2009, Antimicrobial susceptibility testing: a review of general principles and contemporary practices, Clin. Infect. Dis. source

  • May combine two claims.

    Premisecitation

    Agar overlay bioassay provides well-defined growth inhibition zones and is not sensitive to contamination.

    “It provides well-defined growth inhibition zones and is not sensitive to contamination [44].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Marston, 2011, Thin-layer chromatography with biological detection in phytochemistry, J. Chromatogr. A source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    The most known and basic methods for evaluating in vitro antimicrobial activity are disk-diffusion and broth or agar dilution.

    “The most known and basic methods are the disk-diffusion and broth or agar dilution methods.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A standardized antifungal disk-diffusion approach is used to test non-dermatophyte filamentous fungi.

    “Currently, a standardized antifungal disk-diffusion approach is used to test non-dermatophyte filamentous fungi [18].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] CLSI, Method for Antifungal Disk Diffusion Susceptibility Testing of Nondermatophyte Filamentous Fungi, Approved guideline, CLSI document M51-A. Clincal and Laboratory Standards Institute, 950 West Valley Roead, Suite 2500, Wayne, Pennsylvania 19087, USA, 2010.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisenone given

    Disk-diffusion assay offers advantages over other methods: simplicity, low cost, ability to test enormous numbers of microorganisms and antimicrobial agents, and ease of interpreting results.

    “Nevertheless, disk-diffusion assay offers many advantages over other methods: simplicity, low cost, the ability to test enormous numbers of microorganisms and antimicrobial agents, and the ease to interpret results provided.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Accepted and approved standards for disk-diffusion testing of bacteria and yeasts have been published by the Clinical and Laboratory Standards Institute (CLSI).

    “Nowadays, many accepted and approved standards are published by the Clinical and Laboratory Standards Institute (CLSI) for bacteria and yeasts testing [9], [10].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9] CLSI, Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard, 7th ed., CLSI document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [10] CLSI, Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts, Approved Guideline. CLSI document M44-A. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2004.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Fischer and Lautner introduced TLC in the same field.

    “Thereafter, Fischer and Lautner [41] introduced TLC in the same field.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Fischer, 1961, On the paper chromatographic detection of penicillin preparations, Arch. Pharm. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several works have screened organic extracts, mainly plant extracts, for antibacterial and antifungal activity by TLC-bioautography.

    “Several works have been done on the screening of organic extracts, mainly plant extracts, for antibacterial and antifungal activity by TLC–bioautography [42], [43].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [42] Horváth, 2010, Antimicrobial activity of essential oils: the possibilities of TLC-bioautography, Flavour Fragr. J. source
    • cites [43] Mehrabani, 2013, Evaluation of antifungal activities of Myrtus communis L. by bioautography method, Jundishapur J. Microbiol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Microbial and plant products occupy the major part of the antimicrobial compounds discovered until now.

    “Microbial and plant products occupy the major part of the antimicrobial compounds discovered until now [4].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Berdy, 2005, Bioactive microbial metabolites, J. Antibiot. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tetrazolium salts undergo conversion to corresponding intensely colored formazan by dehydrogenases of living cells.

    “These salts undergo a conversion to corresponding intensely colored formazan by the dehydrogenases of living cells [46], [47].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [46] Choma, 2011, Bioautography detection in thin-layer chromatography, J. Chromatogr. A source
    • cites [47] Grzelak, 2011, Development of a novel direct bioautography-thin-layer chromatography test: optimization of growth conditions for gram-negative bacteria, Escherichia coli, J. AOAC Int. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    For bacteria, the time-kill test has been well standardized and described in M26-A document of CLSI.

    “For bacteria, this test has been well standardized and described in M26-A document of CLSI [75].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [75] CLSI, Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved Guideline, CLSI document M26-A. Clinical and Laboratory Standards Institute, 950 West Valley Roadn Suite 2500,Wayne, Pennsylvania 19087, USA, 1998.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationnone given

    If multiple isolates are tested against a single compound, or if the compound or extract tested masks detection of microbial growth in liquid medium with its coloring, agar dilution is often preferred to broth dilution for MIC determination.

    “If multiple isolates are being tested against a single compound, or if the compound (or extract) tested masks the detection of microbial growth in the liquid medium with its coloring, agar dilution method is often preferred to broth dilution for the MIC determination.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Inoculum size, type of growth medium, incubation time and inoculum preparation method can influence MIC values.

    “It is well known that the inoculum size [65], the type of growth medium [66], the incubation time and the inoculum preparation method can influence MIC values [67], [68].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [65] Gehrt, 1995, Effect of increasing inoculum sizes of pathogenic filamentous fungi on MICs of antifungal agents by Broth microdilution method, J. Clin. Microbiol. source
    • cites [66] Meletiadis, 2001, Analysis of growth characteristics of filamentous fungi in different nutrient media, J. Clin. Microbiol. source
    • cites [67] Gomez-Lopez, 2005, Analysis of the influence of tween concentration, inoculum size, assay medium, and reading time on susceptibility testing of Aspergillus spp, J. Clin. Microbiol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several studies have been carried out to evaluate different test parameters for determination of MFC of various drugs against Candida isolates, Aspergillus and other molds.

    “Several studies have been carried out for evaluation of different test parameters for determination of MFC of various drugs against Candida isolates [76], Aspergillus [77] and other molds [78].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [76] Cantón, 2003, Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species, Diagn. Microbiol. Infect. Dis. source
    • cites [77] Espinel-Ingroff, 2002, Testing conditions for determination of minimum fungicidal concentrations of new and established antifungal agents for Aspergillus spp.: NCCLS collaborative study, J. Clin. Microbiol. source
    • cites [78] Espinel-Ingroff, 2002, Optimal testing conditions for determining MICs and minimum fungicidal concentrations of new and established antifungal agents for uncommon molds: NCCLS collaborative study, J. Clin. Microbiol. source

  • May combine two claims.

    Premisecitation

    Bioluminescence assay has a large range of applications, such as cytotoxicity test, in situ evaluation of the impact of biofilms in situ, and drug screening on Leishmania.

    “Bioluminescence assay has a large range of applications, such as cytotoxicity test [86], in situ evaluation of the impact of biofilms in situ [87], and drug screening on Leishmania [88].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [86] Crouch, 1993, The use of ATP Bioluminescence as a measure of cell proliferation and cytotoxicity, J. Immunol. Methods source
    • cites [87] Bozorg, 2015, Bacterial bioluminescence to evaluate the impact of biofilm on porous media hydraulic properties, J. Microbiol. Methods source
    • cites [88] Paloque, 2013, A new, rapid and sensitive bioluminescence assay for drug screening on Leishmania, J. Microbiol. Methods source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Premisenone given

    Broth micro- or macro-dilution is one of the most basic antimicrobial susceptibility testing methods.

    “Broth micro- or macro-dilution is one of the most basic antimicrobial susceptibility testing methods.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    The most recognized standards for dilution antimicrobial susceptibility testing are provided by CLSI and EUCAST.

    “The most recognized standards are provided by the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The main disadvantages of macrodilution are tedious manual undertaking, risk of errors in preparation of antimicrobial solutions for each test, and comparatively large amount of reagents and space required.

    “Unlike microdilution method, the main disadvantages of the macrodilution method are the tedious, manual undertaking, risk of errors in the preparation of antimicrobial solutions for each test, and the comparatively large amount of reagents and space required [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Jorgensen, 2009, Antimicrobial susceptibility testing: a review of general principles and contemporary practices, Clin. Infect. Dis. source

  • May combine two claims.

    Premisecitation

    For conidium and spore forming fungi, CLSI microdilution uses an inoculum adjusted spectrophotometrically to 0.4×10^4-5×10^4 CFU/mL, while the EUCAST assay adjusts inoculum to (2-5)×10^5 CFU/mL by haemocytometer counting.

    “As regards to the conidium and spores forming fungi, the microdilution standardized by CLSI involves an inoculum of spores adjusted spectrophotometrically to 0.4×104–5×104 CFU/mL. However, in the EUCAST assay, the inoculum can be adjusted to (2–5)×105 CFU/mL by haemocytometer counting [72].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [72] Lass-Flörl, 2006, Antifungal susceptibility testing in Aspergillus spp. according to EUCAST methodology, Med. Mycol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Agar dilution is often recommended as a standardized method for fastidious organisms such as anaerobes and Helicobacter species.

    “Agar dilution is often recommended as a standardized method for fastidious organisms [79] such as anaerobes and Helicobacter species.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [79] CLSI, Methods for Antimicrobial Dilution and Disk Susceptibility of Infrequently Isolated or Fastidious Bacteria, Approved Guideline, 2nd. ed., CLSI document M45-A2. Clinical and Laboratory Standards Institute, 950 West Valley Roadn Suite 2500,Wayne, Pennsylvania 19087, USA, 2010.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Commercially produced inoculum replicators are available and can transfer between 32 and 60 different bacterial inocula to each agar plate.

    “Nowadays, commercially produced inoculum replicators are available and can transfer between 32 and 60 different bacterial inocula to each agar plate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    Either broth or agar dilution method may be used to quantitatively measure in vitro antimicrobial activity against bacteria and fungi.

    “Either broth or agar dilution method may be used to quantitatively measure the in vitro antimicrobial activity against bacteria and fungi.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several antifungal substances were studied by the time-kill method.

    “Similarly, several antifungal substances were studied by this method [84], [85].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [84] Clancy, 2006, Characterizing the effects of caspofungin on Candida albicans, Candida parapsilosis, and Candida glabrata isolates by simultaneous time-kill and postantifungal-effect experiments, Antimicrob. Agents Chemother. source
    • cites [85] Klepser, 1998, Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods, Antimicrob. Agents Chemother. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    These guidelines provide a uniform procedure for testing that is practical to perform in most clinical microbiology laboratories.

    “As advised, these guidelines provide a uniform procedure for testing that is practical to perform in most clinical microbiology laboratories.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The EUCAST broth dilution method is principally similar to that of CLSI, with modifications usually concerning inoculum preparation, inoculum size, and MIC reading method, which is visual in CLSI and spectrophotometric in EUCAST guidelines.

    “The EUCAST broth dilution method is principally similar to that of CLSI with modifications usually concerning some of the test parameters such as inoculum preparation, inoculum size, and the MIC reading method which is visual in CLSI assay and spectrophotometric in EUCAST guidelines [71].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [71] Arikan, 2007, Current status of antifungal susceptibility testing methods, Med. Mycol. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Injured cells are described as stressed cells exhibiting cellular components damages and subsequent impairment of reproductive growth.

    “The injured cells are described as stressed cells exhibiting cellular components damages and subsequent impairment of reproductive growth [100].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [100] Yousef, 2003, Basics of stress adaptation and implications in new-generation foods source

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    Methods for antimicrobial susceptibility testing and discovering novel antimicrobial agents have been extensively used and continue to be developed.

    “Therefore, methods for antimicrobial susceptibility testing and discovering novel antimicrobial agents have been extensively used and continue to be developed.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Microbial infections are an important clinical threat with significant associated morbidity and mortality, mainly due to the development of microbial resistance to existing antimicrobial agents.

  • May combine two claims.

    Premisecitation

    Flow cytofluorometric method gives reproducible results rapidly (2-6 h compared to 24-72 h for the microdilution method).

    “Moreover, it gives reproducible results rapidly (2–6 h compared to 24–72 h for the microdilution method) [96].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [96] Ramani, 2000, Flow cytometry antifungal susceptibility testing of pathogenic yeasts other than Candida albicans and comparison with the NCCLS broth microdilution test, Antimicrob. Agents Chemother. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Propidium iodide (PI), a fluorescent and intercalating agent, is widely used as DNA stain in flow cytometry.

    “Therefore, propidium iodide (PI), a fluorescent and intercalating agent, is widely used as DNA stain [96].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [96] Ramani, 2000, Flow cytometry antifungal susceptibility testing of pathogenic yeasts other than Candida albicans and comparison with the NCCLS broth microdilution test, Antimicrob. Agents Chemother. source

  • May combine two claims.

    Premisecitation

    Flow cytofluorometric method allows the detection of antimicrobial resistance and estimates the impact of the molecule tested on the viability and cell damage of the tested microorganism.

    “Indeed, flow cytofluorometric method allows the detection of antimicrobial resistance and estimates the impact of the molecule tested on the viability and cell damage of the tested microorganism [101].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [101] Tang, 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Interpretationnone given

    When testing natural products, some modifications of the standardized protocols are often requested.

    “However, when testing natural products, some modifications of the standardized protocols are often requested.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Some techniques have been subjected to standardization by the CLSI and EUCAST.
47 of 47

  • The paper relies on this without support.

    ATP bioluminescence assay measures adenosine triphosphate (ATP) produced by bacteria or fungi.

    “ATP bioluminescence assay is based on the capacity to measure adenosine triphosphate (ATP) produced by bacteria or fungi.”

  • The paper relies on this without support.

    ATP is present in more or less a constant amount in a cell because it is the chemical form of energy of all living cells.

    “As ATP is the chemical form of energy of all living cells, it is present in more or less a constant amount in a cell.”

  • The paper relies on this without support.

    Microbial infections are an important clinical threat with significant associated morbidity and mortality, mainly due to the development of microbial resistance to existing antimicrobial agents.

    “Currently, microbial infections have become an important clinical threat, with significant associated morbidity and mortality which is mainly due to the development of microbial resistance to the existing antimicrobial agents.”

  • The paper relies on this without support.

    Some techniques have been subjected to standardization by the CLSI and EUCAST.

    “Some techniques were subjected to standardization by the CLSI and EUCAST, marking the major remarkable steps on this field.”
4 of 4

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. Mayers, 2009, vol. 2
  2. Caron, 2012, Antimicrobial susceptibility testing : a four facets tool for the clinician, J. Des. Anti-Infect., 14
  3. Brantner, 1997, Influence of various parameters on the evaluation of antibacterial compounds by the bioautographic TLC assay, Pharm. Pharmacol. Lett., 7, 152
  4. Ouedrhiri, 2015, Chemical composition of Citrus aurantium L. Leaves and zest essential oils, their antioxidant, antibacterial single and combined effects, J. Chem. Pharm. Res., 7, 78

Cited by

Sorted by citations

Related

Authors
Balouiri, M., Sadiki, M., & Ibnsouda, S. K.
Affiliations
Sidi Mohamed Ben Abdellah University (MA)
Corresponding author
Balouiri, Mounyr
ORCID
Venue
Journal of Pharmaceutical Analysis
Publisher
Elsevier BV
Published
2016-04
Volume
6
Issue
2
Pages
71-79
ISSN
2095-1779 (ISSN-L)
DOI
10.1016/j.jpha.2015.11.005
PMC
PMC5762448
OpenAlex
W2185564569
Semantic Scholar
4981ba0c192f65f457e8aebd7c10e8211ba3e3d3
Cited by
7,264
References
94
Citations by year
2026: 808 · 2025: 917 · 2024: 1085 · 2023: 961 · 2022: 879 · 2021: 803 · 2020: 665 · 2019: 519 · 2018: 349 · 2017: 221 · 2016: 44 · 2015: 1 · 2014: 2
FWCI
135.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Pharmaceutical Analysis (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Bacterial Identification and Susceptibility Testing
  • Physical Sciences › Chemistry › Organic Chemistry › Antimicrobial agents and applications
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Essential Oils and Antimicrobial Activity
Keywords
antimicrobial activity, antimicrobial susceptibility testing, disk diffusion, broth dilution, agar dilution, flow cytometry, bioluminescence, microbial resistance, method standardization

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Cited sources are checked by gpt-6-sol.

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