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TEM PREP - Clostridia thermicellum

DAVID HOGSETT

1. Concentrate/dilute whole cell sample to OD660 = 0.8

2. Centrifuge chilled for 5 minutes at 15,900 g.

3. Remove supernatant and re-suspend pellet with dH2O

4. Repeat steps 2 and 3 twice more for three washings total.

5. Concentrate/dilute sample to approximately OD660 = 80

Negative Stain:

1. Sample concentration should be 108. Dilute this 1:10 in dH2O and bring chilled sample to EM lab ASAP.

2. Place Formvar grid or 100? indirect C grid on drop of bacteria. Four drops:
no dilution
1:10 with dH2O
1:10 with dH2O
1:10 with dH2O

3. Leave 4 minutes.

4. Transfer after wicking off excess moisture (DO NOT LET GRID DRY) grid to drop of 2% Uranyl Acetate (aqueous).

5. Stain 2 minutes

6. Remove excess UA; observe on TEM within 24 hours.

Last Updated: 10/2/08