Use of Hot Formaldehyde Fixative in Processing Plant-Parasitic Nematodes for Electron Microscopy

Abstract

A preparative technique is formulated for processing plant-parasitic nematodes of the order Tylenchida for electron microscopy. A population of Dolichodorus heterocephalus is used as test objects. One and a half grams of paraformaldehyde are dissolved in 25 ml of water at 60 C. Five drops of 1 N sodium hydroxide are added to clear the solution, which is then cooled to room temperature. Two and a half milliliters of 25% glutaraldehyde are added with 23 ml 0.1 M phosphate buffer, pH 7.3, and 0.2 M with respect to sucrose. The final solution contains 3% formaldehyde and 1% glutaraldehyde and is pH 7.2. It is heated to 70 C, poured over specimens, and allowed to cool to 4 C in 2 hr. The nematodes are then incised in a fixative containing 2% glutaraldehyde and 5% dimethyl sulfoxide at 4 C for 16-24 br. Five milliliters of 25% glutaraldehyde and 2.5 ml of dimethyl sulfoxide are combined in 17.5 ml of water. Twenty-five milliliters of phosphate buffer (supplemented as above) are added. The final pH is 7.2. The glutaraldehyde, aided by dimethyl sulfoxide, uniformly and permanently fixes the nematode tissues. The specimens are embedded in agar. Following a 30-min buffer wash (4 C) they are postfixed in buffered 2% osmium tetroside for 2 hr at room temperature, washed, and dehydrated through an ethanol series and two acetone baths. Dehydration includes a 2-hr stop in 75% ethanol containing 2% uranyl acetate. After embedding in Spurr's epoxy resin, specimens are sectioned and poststained in 0.5% aqueous uranyl acetate for 6 min and saturated aqueous lead citrate 3-4 min.

This technique reduces killing time to less than 2 sec, straightens specimens for easier orientation, and eliminates the typically high internal pressure of nematodes which causes displacement of internal structures observed with other fixation techniques.