Alternate inks for arbuscular mycorrhizal root staining

  1. Thomas I Wilkes

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Abstract

Alternate methods for root staining of arbuscular mycorrhizal (AM) fungi have recently gained more attention for the reduction of hazard exposure to the user. Sheaffer® blue ink has been employed for such an identification and quantification, having shown and increased degree of image clarity. However, sourcing Sheaffer® blue ink is becoming problematic, leading to the need to find alternative inks that are readily available. Parker® ink is a well-known brand, providing comparable colour option to Sheaffer®. Two Parker® inks, blue and washable blue, were employed alongside Sheaffer® blue for comparative AM fungal root staining. From quantified AM fungal vesicles and arbuscles, along with the degree of stained image clarity under microscopy, none of the inks utilised for this comparison produce a significantly (P=0.97) different AM fungal quantification or change in image clarity. Therefore, results of the present communication suggest that Parker® blue and washable blue inks are alternative ink stains for the viewing and quantification of AM fungi in host cortical root tissues.

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  3. Version published to 10.1099/acmi.0.000618.v3 on Access Microbiology
  4. Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data Satisfactory 2. Presentation of results Satisfactory 3. How the style and organization of the paper communicates and represents key findings Satisfactory 4. Literature analysis or discussion Satisfactory 5. Any other relevant comments

    Please rate the manuscript for methodological rigour

    Satisfactory

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

    Satisfactory

    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

  5. Access Microbiology

    Comments to Author

    I thank the reviewer for their responses, but my main concern regarding the citation of previous work was denied. In my previous review, pointed out the importance of citing similar work which had used the same dye for the similar purpose of staining the fungi in the plant roots. In fact, Yon et al (2015) used Parker QuinK blue ,washable in water, and this work used Parker washable blue and Parker standard blue inks. Proper citations will make readers aware of the similar work and the increase the credibility of the current work.

    Please rate the manuscript for methodological rigour

    Satisfactory

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

    Satisfactory

    To what extent are the conclusions supported by the data?

    Partially 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

  6. Version published to 10.1099/acmi.0.000618.v4 on Access Microbiology
  7. Version published to 10.1099/acmi.0.000618.v2 on Access Microbiology
  9. Access Microbiology

    Comments to Author

    In Title Suggestion: In the title, please do not use "root staining for arbuscular mycorrhizal". The short communication is interesting and can be a differential access material for teaching activities at higher levels, young scientists at the beginning of their careers, in addition to other functions in science, however the way it is understood suggests that fungi have roots, which does not occurs. It is convention among mycologists to try their best not to associate botanical structures with fungi. Please always use "roots colonized by MA fungi". Do this for all text. In Abstract Review previous comment and apply to lines 13;19-20. The p for significance, on line 22, is lower case. In Introduction Revise previous comment and apply to line 43. 1. Methodological rigour, reproducibility and availability of underlying data While using the wheat variety for the text (line 47), please provide the name of the wheat species to increase the replicability of the work. Please cite the authorship of the R software package used for this analysis. In Results Please, put the p of significance, on line 58, in lower case. Same thing for the F value and F critical. In References Please add https://doi.org/ on line 102. Some newspaper names are abbreviated and others are not. Please check this in all references. 2. Any other relevant comments The coloring really worked, but the photos I had access to in the file for review didn't turn out well. If possible, please replace with better quality images. The circles used to present the structures of the AM fungi are too thick, added to the colors, they are points of distraction. It would be interesting to make them less evident by drawing attention to the structures, which are the targets. These structures were barely visible, possibly due to the quality of the image. If you could check that too, that would be great. It would be interesting to standardize the position of the scales in the images.

    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

  10. Access Microbiology

    Comments to Author

    In this short communication, the author tests suitability of Parker washable blue and Parker Blue as alternatives to stain AM in winter wheat roots due to the scarcity of Sheaffer blue in the market. The research objectives are clearly stated in the introduction and has justified the underlying reasons for the research. However, the citations are not representing the broad literature available and very similar previous work is not cited. For example, use of Parker Blue dye to stain MA by Yon et al in 2015. Therefore highly recommend to include similar work done previously in this topic. Yon etal, DOI: 10.13140/RG.2.2.10232.65287 Research methodology is not concisely presented to reproduce the experiment. For example, important factors like ink pre-preparation conditions, the staining time is not mentioned. Without this information, this method cannot be reproduced. It is also important to state if the inks are used as received or any dilution or filtration is carried out prior to staining procedure. Though the statistics of counts are presented, microscopic image acquisition parameters, nor the number of images analysed are not revealed in the methods section. For the analysis of dye performance, and comparison between different dyes, intensity of the signal is a valid parameter rather reporting the number of features identified, which is adapted in this experiment. Instead, recommend to include signal intensity which will provide a qualitative measurement, therefore, dye performance can be correctly quantified across samples. It would also be interesting to discuss reports on toxicity of the inks being used (if available) in this study.

    Please rate the manuscript for methodological rigour

    Poor

    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

  11. Version published to 10.1099/acmi.0.000618.v1 on Access Microbiology