Ultrastructural analysis of the early development of nematodes is hampered by the impermeability of the eggshell to most commonly used fixatives. High-pressure freezing (HPF), a physical cryo-fixation method, facilitates a fast rate of fixation, and by using this method the issue of the uneven delivery of fixative is circumvented. Although HPF results in a superior preservation of the fine structure, the equipment costs impede a wider application of this method. Self-pressurised rapid freezing (SPRF) is an alternative low-cost cryo-fixation method, and its usefulness was evaluated in an ultrastructural study of the eggshell and the cuticle of unhatched second-stage juveniles (J2) of Globodera rostochiensis and Heterodera schachtii. A comparison with conventional (chemical) fixation demonstrates that SPRF fixation results in a remarkably well-preserved ultrastructure of the entire egg including both the eggshell and the cellular details of the unhatched J2. Therefore, SPRF fixation is proposed as an affordable, relatively easy-to-use and time-efficient technique to study the ultrastructure of unhatched J2 and eggs of nematodes.
Claeys, M., Handayani, N. D., Yushin, V. V., Lestari, P., Dikin, A., Helder, J., & Bert, W. (2019). Self-pressurised rapid freezing, a time-efficient and affordable cryo-fixation method for ultrastructural observations on unhatched cyst nematodes. Nematology, 22(1), 103-110. https://doi.org/10.1163/15685411-00003287