Amazonian açai and food dyes for staining arbuscular- micorrhizal fungi

Abstract

<span>A microscopia de Micorrizas Arbusculares requer coloração diferencial de estruturas típicas. Alguns corantes empregados, como Trypan Blue, oferecem riscos à saúde e ambiente. Corantes alternativos como tintas de caneta e anilina foram propostos, porém, com eficácia de coloração variável. Neste trabalho o descolorante de raízes KOH foi substituído por Soda Cáustica e foram testados os corantes naturais Açaí, Urucum e Açafrão, comparados a Trypan Blue e tintas de caneta. Houve diferença significativa entre os corantes quanto às estruturas micorrízicas coradas e qualidade de imagens fotografadas. Açaí foi considerado o melhor corante alternativo, com resultados similares ao Trypan Blue.</span>

Alternative and safe dyes for staining Arbuscular Mycorrhizal fungi

“…Although it is possible to reduce the risks of the traditional technique (VIERHEILIG et al, 1998;ISHIL et al, 2004;SILVA et al, 2015), it is still not possible to fully replace the Trypan Blue dye, being one of the causes to avoid activities involving AM fungi by users with little experience. Grace et al (1991) and Vierheilig et al (1998) proposed less harmful dyes formulated from pen inks of different colours such as purple (Waterman), green (Reynolds), red (Parker; Lamy; Pelikan), blue (Pelikan; Kreuzer; Shaeffer) and black (Pelikan; Carrefour; Shaeffer; Cross) for AM fungal staining.…”

“…To clarify the roots, it was used the methodology suggested by Silva et al (2015) in a modification of the Phillips et al (1970) technique, where fragments of fresh root were submerged in a commercial caustic soda solution (10%) in a bain marie (± 90°C). After each period of discolouration, different for each plant species tested (Table 1), the roots were washed under running water, acidified in vinegar for one minute and then prepared for staining.…”

“…The roots, together with 20 ml of P a g e | 403 Revista Ibero-Americana de Ciências Ambientais v.11 -n.7  Dez 2020 each dye solution, were placed in 50 ml test tubes and heated in a bain marie (± 90°C) for five minutes. As a control, the natural dye of açaí pulp, obtained in the local market, was diluted in commercial vinegar at 5% (v/v), according to Silva et al (2015), and the Trypan Blue dye (0.05%), formulated using the traditional technique. After staining, the roots were transferred to new tubes of equal capacity, containing 20 ml of glycerin solution (10%) and kept at room temperature until analysis.…”

“…Based on this concern, some scientists have modified the traditional mycorrhiza staining technique for less harmful and more accessible options. Following the example of Vierheilig et al (1998) who suggested the use of vinegar to replace HCl, Ishil et al (2004) who modified the technique by Koske et al (1989), eliminating acidification with HCl, but maintaining the clarification step with KOH and staining with Trypan Blue and Silva et al (2015) who replaced KOH with caustic soda and HCl with vinegar, both in their commercial form, thereby obtaining good quality in the decolourisation of the roots.…”

“…However, these synthetic substitutes can be expensive, difficult to access and present health risks just like Trypan Blue (COMBES et al, 1982), in addition to the low efficiency in the colouration of specific structures of AM fungi (VIERHEILIG et al, 2005). P a g e | 402 Revista Ibero-Americana de Ciências Ambientais v.11 -n.7  Dez 2020 Silva et al (2015) proposed the use of natural food dyes from Amazonian products, such as Euterpe oleracea Mart pulp. (açaí), Bixa orellana L. (annatto) and Crocus sativus L. (saffron), in contrasts for visualisation of AM fungi under microscopy.…”