“Modified String Test to Improve and Confirm by Molecular Characterization for Bacterial Identification”

  1. Muhammad Dawood Mian
  2. Saadullah Jan Khan
  3. Rehana Rani
  4. Sara Sadiq
  5. Laila Jafri
  6. Bushra Jamil

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Abstract

Rapid and precise identification of bacteria is crucial in both clinical and environmental microbiology. Traditional Gram staining methods, while foundational in bacterial classification, often suffer from delays and require additional confirmatory tests, raising concerns about the potential for misidentification. To address these limitations, we present a modified KOH string test enhanced with Methylene Blue, aimed at improving the visualization and differentiation of Gram-positive and Gram-negative bacteria. In our study, we analyzed approximately 185 samples collected from hospitals, soil, salt mines, and honeycombs. Initially, we employed conventional Gram staining for preliminary bacterial identification, followed by our modified KOH string test. This innovative procedure involved mixing a drop of 3% KOH with a bacterial colony on a slide. After one minute, the addition of a dye significantly enhanced the visibility of string formation, facilitating the distinction between bacterial types. The results were compelling: 100% of Gram-negative bacteria displayed a visible string, while all Gram-positive bacteria exhibited no string formation. To further validate our findings, selected environmental samples were analyzed using 16S rRNA PCR, reinforcing the effectiveness of our modified KOH string test. This enhanced method is not only cost-effective and straightforward but also reliable, positioning it as an excellent supplementary or alternative approach for bacterial identification. By improving diagnostic accuracy and efficiency, our innovative technique represents a valuable advancement in microbiological applications.

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  1. Access Microbiology

    Comments to Author

    Thank you for submitting your manuscript entitled "Modified String Test to Improve and Confirm by Molecular Characterization for Bacterial Identification" to Access Microbiology. Your study proposes a practical and low-cost enhancement to the traditional KOH string test by incorporating methylene blue dye for improved visualization, and validates the results using 16S rRNA gene sequencing. The manuscript presents a relevant approach for bacterial differentiation, particularly in resource-limited settings. Below are my comments for your consideration: Strengths： 1.Practical value: The study addresses a real-world challenge in basic microbiological diagnostics by improving an existing method with minimal resources. 2.Simple and low-cost methodology: The improved KOH test offers a feasible solution for preliminary bacterial identification, especially in laboratories lacking molecular facilities. 3.Adequate experimental validation: The use of 185 diverse samples and the confirmation by 16S rRNA sequencing increase the credibility of your results. 4.Clear structure: The manuscript is generally well-organized and easy to follow, with appropriate supplementary figures supporting the findings. Areas for Improvement 1.Lack of statistical performance metrics: While you state a 100% accuracy rate in distinguishing Gram-negative bacteria using the modified KOH test, the manuscript does not provide statistical indicators such as sensitivity, specificity, and overall accuracy. Please consider including these metrics to strengthen your claims. 2.Limited comparison with other diagnostic methods: The discussion primarily focuses on comparison with Gram staining. It would be beneficial to include brief comparisons with other rapid identification methods, such as biochemical kits (e.g., API), to provide a broader context. 3.Discussion on dye selection: Although methylene blue improved string visualization, other dyes were mentioned only in the supplementary materials. A brief discussion in the main text comparing their effectiveness would enhance the scientific rigor. 4.Language and phrasing: Some expressions (e.g., "string visualization was improved by many folds") are too informal or vague. Please revise such sentences for scientific clarity and precision. A professional language edit is recommended. 5.Ethical considerations: Although the samples were anonymized, a formal statement from an institutional review board or ethics committee confirming that ethical approval was not required would increase transparency. In brief, this study provides a useful and implementable improvement to a well-known diagnostic method, with potential applications in clinical and environmental microbiology. If the above issues—particularly statistical validation and minor clarifications—are addressed, the manuscript would be suitable for publication in Access Microbiology.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  2. Access Microbiology

    This short communication describes a modified KOH string test incorporating methylene blue to enhance visualisation of DNA release and improve discrimination between Gram-negative and Gram-positive bacteria. The approach is technically straightforward and clearly described, and it is evaluated on an appropriate sample size and diverse set of clinical and environmental isolates. Comparison with conventional Gram staining, together with 16S rRNA sequencing of selected isolates, provides a reasonable framework for assessing the assay’s performance. The experimental procedure is robust and clearly described. The conclusion that the modified KOH string test reliably distinguishes Gram-negative from Gram-positive bacteria is generally supported by the data. Nonetheless, assertions of “100% accuracy” and broad applicability would benefit from more cautious framing, given the lack of blinded assessment, inter-operator variability analysis, and testing against Gram-variable or difficult-to-identify taxa, as well as the absence of quantitative performance metrics. Overall, the conclusions are directionally supported but somewhat overextended relative to the validation presented. With modest tempering of claims and clearer articulation of limitations, the study would represent a useful and technically sound contribution, particularly for low-resource or high-throughput laboratory settings. The manuscript would benefit from a clear operational definition of a “positive” string test. For example is there a minimum string length? Is there a time window for interpretation? is viscosity alone or visible filament formation required for a positive result? This is particularly important if the method is promoted for low-training or field use. A subset of isolates underwent 16S rRNA sequencing, but the criteria for selection are not stated. Please clarify how many isolates were sequenced, if they were randomly selected or chosen based on discordant/representative phenotypes, and if sequencing confirmed Gram status only, or species-level identity. While the method is clearly useful as a supplementary and/or screening tool, the authors’ claims suggesting it could replace Gram staining are far-fetched. The discussion should more clearly acknowledge that the assay does not provide morphological information and cannot resolve Gram-variable or atypical organisms (at least there are no data in the manuscript to contradict this). Figure legend for figure 1, with specific details for each plate Figure 2. what percentage of methylene blue was used? Figure 4 legend states visualisation was improved many fold. I don’t believe there was quantifications, therefore it cannot be stated that a fold change exists. Please modify language to reflect the observational nature of the finding. Line 247. Please include the time for Gram staining here The manuscript refers to methylene blue at 1% in some sections and 2% in others. Please correct. Use either “Gram-positive/Gram-negative” consistently throughout. Similarly, “Modified String Test,” “modified KOH string test,” and “string test” are used interchangeably. Please standardise terminology. In the discussion and conclusion sections claims comparing Gram staining and KOH testing are reiterated multiple times and could be condensed for clarity and impact. Some figure captions describe conclusions (excellent binding, improved by many folds) rather than observations. Please revise so figure legends and results are neutral, descriptive captions and reserve interpretation for the Results or Discussion.

  3. Version published to 10.1099/acmi.0.000965.v1 on Access Microbiology