Technique for en-masse cryo-fixation and processing of second-stage juveniles of Meloidogyne incognita for scanning electron microscopy

Large number of second-stage juveniles of root-knot nematode, Meloidogyne incognita was prepared for scanning electron microscopy over a millipore filter (pore size 0.45 mu m). The J2 juveniles were quickly immobilized and killed before fixation using the cryo-fixation technique followed by the primary fixation in 2.5% glutaraldehyde pre-cooled at 4-8[degrees]C and then secondary vapor fixation in 1% osmium tetraoxide and drying with the series of ethanol without subsequent critical point drying. The specimens were vacuum dried in a desiccator for a period of 24-48 h. Cryo-fixation emphasizes the need to quickly stop cellular activity and thus prevent any surface artifacts that occur due to conventional fixation techniques.