Cholera SEM on Nucleopore filters

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CHOLERA SEM Preparation on Nucleopore Filters
method new in 2008

1. Cholera suspension in 4% GTA in 0.1M NaCacodylate pH 7.4 for 1 hour. Pre-wet 13mm 0.1ľ Nucleopore filter with in 0.1M NaCacodylate pH 7.4. Add ~0.8 ml to one ml syringe attached to wetted filter assembly and add aliquot of chorea suspension to syringe. Slowly push fluid through syringe/filter assembly. Make sure filter doesn't dry-out; i.e. Leave some fluid in syringe before disassembly and removal of filter.

2. Rinse in 0.1M NaCacodylate pH 7.4

3. Post fix in 1% OsO4 in 0.1M NaCacodylate pH 7.4 for 1 hour.

4. Rinse in 0.1M NaCacodylate pH 7.4

5. Dehydrate 5 mins. each in 50%, 70% ETOH

6. Three 10 mins. washes with 100% ETOH.

7. Wash 3x10 min 1:1 ETOH/HMDS or 1:1ETOH/TMS

8. Wash 3x10 min in 1:2 ETOH/HMDS or 1:2ETOH/TMS

9. Wash 1x 20 min. in 100% HMDS. Immediately move coverslips to new dish containing HMDS.

10. Wash 2x HMDS 20 min each.

11. Remove HMDS until filter is almost uncovered and vacuum dessicate overnight.

12. Coat: @3nm with Osmium plasma coater

13. Store samples in a desiccator