Nematode E. dianae - Critical Point Drying Protocol for SEM

Application Note for Leica EM CPD300 - Life Science Research

November 16, 2016

Species: Eubostrichus dianae
Critical point drying of nematode Eubostrichus dianae to detect the ectosymbiotic bacteria layer with subse­quent gold coating and SEM analysis.

Sample Holder:
Samples were placed into the filter discs and porous pots holder.

Fixation and Dehydration

2.5% Glutaraldehyde in 0.1M Cacodylate Buffer2 h
0.1M Cacodylate Buffer3x10 min.
1% OsO4 in 0.1M Cacodylate Buffer4-12 h
0.1M Cacodylate Buffer3x10 min.
Ethanol series: 30%, 50%, 70%, 80%, 80%, 90%, 90%, 100%, 100%10 min.
1:1 Mix Ethanol / Acetone10 min.
100% Acetone10 min.

Gold: 10-20 nm

Fig.1: Eubostrichus with ectosymbiotic bacteria layer