African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12227

Short Communication

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

Shikha Sharma*
  • Shikha Sharma*
  • Punjab Agricultural University, Ludhiana-141001, India.
  • Google Scholar
Rajdeep K. Sidhu
  • Rajdeep K. Sidhu
  • Punjab Agricultural University, Ludhiana-141001, India.
  • Google Scholar
V. R. Parshad
  • V. R. Parshad
  • Punjab Agricultural University, Ludhiana-141001, India.
  • Google Scholar


  •  Received: 06 September 2013
  •  Accepted: 14 July 2014
  •  Published: 03 September 2014

Abstract

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 µm). 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°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.

 

Key words: Scanning electron microscopy, cryo-fixation, root-knot nematode, Meloidogyne incognita, surface morphology.