Staining and microscopy of mycorrhizal fungal colonization in preserved ericoid plant roots

BACKGROUND: Visualization of ericoid mycorrhizal colonization using traditional methods relies on either fresh or KOH stored samples. Increasing interest in studying ericoid mycorrhization has highlighted a need for methods which can be used for preserved samples and are simple to implement with commonly available equipment. OBJECTIVE: The aim of this study was to improve on traditional techniques for staining ericoid mycorrhizal fungi and microscopically visualizing ericoid mycorrhizal roots which have been preserved. METHODS: Ericoid mycorrhizal roots were placed in KOH or frozen at -20 degrees C for long-term storage. Traditional Trypan Blue staining methods were modified to reduce damage to fine mycorrhizal hyphae and cortical cells. A high light-intensity dark-field microscopy technique was applied to clearly visualize stained mycorrhizae. The novel application was compared to other commonly used practices. RESULTS: Trypan Blue staining without KOH storage or clearing allowed for successful staining of ericoid mycorrhizal roots stored at -20 degrees C. The application of high light-intensity dark-field microscopy provided high contrast visualization of mycorrhizal structures. CONCLUSIONS: The modified Trypan Blue staining method was effective on frozen root samples, with dark-field microscopy being particularly effective at visualizing dark colored roots. Advantages to this method are lowcost and relatively fast application time. Therefore, this method is a realistic option for large scale analyses with many samples which require long-term preservation.