Browsing by Author "Wilkes, Thomas I."

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    Alternate inks for arbuscular mycorrhizal root staining
    (The Microbiology Society, 2023-08-31) Wilkes, Thomas I.
    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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    Ergosterol extraction: a comparison of methodologies
    (The Microbiology Society, 2023-04-28) Wilkes, Thomas I.
    Ergosterol is a component of the cell membrane of mycorrhizal fungi and is frequently used to quantify their biomass. Arbuscular mycorrhizal (AM) fungi and ectomycorrhizal (ECM) fungi establish a symbiotic relationship with a respective host plant. Several methods are currently employed for quantification of ergosterol; however, these utilise a series of potentially hazardous chemicals with varying exposure times to the user. The present comparative study aims to ascertain the most reliable method to extract ergosterol whilst limiting hazard exposure to the user. Chloroform, cyclohexane, methanol and methanol hydroxide extraction protocols were applied to a total of 300 samples of root samples and a further 300 growth substrate samples across all protocols. Extracts were analysed via HPLC methodologies. Chromagraphic analysis showed chloroform-based extraction procedures produced a consistently higher concentration of ergosterol in both root and growth substrate samples. Methanol hydroxide, without the addition of cyclohexane, produced a very low concentration of ergosterol, with a reduction of quantified ergosterol of between 80 and 92 % compared to chloroform extractions. Hazard exposure was greatly reduced following the chloroform extraction protocol when compared with other extraction procedures.