Bacteria Protocol - Critical Point Drying of E. coli for SEM

Application Note for Leica EM CPD300 - Life Science Research

October 13, 2016

Introduction
Species: Escherichia coli
Critical point drying of E. coli with subsequent platinum / palladium coating and SEM analysis.

Procedure
Sample Holder:
Sample was inserted into a filter disc (Pore size: 16 - 40 μm) and placed into the filter discs and porous pots holder.

Cultivation
Cultivate fungi and bacteria on agar containing growth medium for 3 days. Selected parts of the colonies of bacteria.

Fixation and Dehydration

3% Glutaraldehyde in PBS, pH 7.3 at 4°C16 h
Distilled water3x10 min.
1% aqueous OsO4, at 4°C16 h
Distilled water3x10 min.
Ethanol series: 30%, 50%, 70%, 80%, 90%, 96%, 100% at 25°C1x10 min.
Acetone series: 30%, 50%, 100%1x10 min.

Leica EM CPD300 auto program

Coating
Mount the dried samples on stubs containing carbon adhesives.
Platinum / Palladium coating: 6 nm

Fig.1: E. coli
Fig.2: E. coli

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