Self-pressurised rapid freezing, a time-efficient and affordable cryo-fixation method for ultrastructural observations on unhatched cyst nematodes

Abstract

SummaryUltrastructural 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.

Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects

“…The success of specimen fixation and contrasting is largely determined by the rate of penetration of the reagents into the sample 11 , 22 , which is considerably slowed down in heterogeneous samples with the presence of a poorly permeable integument or of tissues with a high lipid content. Such problems with sample preparation are noted for insects 23 , 24 and other arthropods 25 , other invertebrates 25 – 28 and vertebrates 29 , as well as plants 30 .…”