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.
