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Revision as of 06:49, 26 September 2012

Preparation of Escherichia Coli for the energy dispersive X-ray spectroscopy (EDX)

NOTE: The pellet must be about 0.25 g and the preparation takes about 4 days

Primary Fixation-step

• give about 1 ml pre-fixationmixure to your pellet and incubate at room temperature for at least 1 hour

NOTE: you have to centrifuged your pellet after each step (about 30-45 sec at max. rpm)

Washing-step

• wash your pellet 4 times with about 750µL 0.1 M Cacodylate-buffer (CaCo) pH 7.4 for at least 15 min at RT

Secondary fixation-step

• give about 750µL 0.5 % Osmium in 0.1 M CaCo Puffer pH 7.4 to your pelett and incubate at RT at least 1 hour

washing-step

• wash your pellet 2 times with about 750µL 0.1 M CaCo buffer pH 7.4 for at least 10 min and 2 times with bidest H20 for 10 min

dehydration-step

• give to your pellett about 750µL 30%,50%,90%,100% acetone and incubate each pellet at RT for at least 10 min
• give to your pelett about 750µL 100% acetone (dried) and incubate 2 times at RT for at least15 min

Infiltration-step

• give to your pelett about 500µL 25 % SPURR (in acetone) and incubate at RT for at least 45 min
• give to your pelett about 500µL 50 % SPURR (in acetone) and incubate at RT for at least 45 min
• give to your pelett about 500µL 75 % SPURR (in acetone) and incubate at RT for at least 45 min
• give to your pelett 2 times about 500µL 100 % SPURR (in acetone) and incubate at RT for at least
• give to your pelett about 500µL 100 % SPURR (in acetone) and incubate at 4 °C overnight.

Polymerisation-step

NOTE: your pellet have to be on the bottom of the capsule

• give to your pelett fresh mixed SPURR and fill a part of your pellet into a capsule and incubate at 74°C over night.

cutting-step

• cut your polymererised capsule with a.... in a thicker than 100 nm rectangles
• your sample ist ready for EDX

Mixtures

Pre-fixationmedia

1. 0.1 M Cocadynaltebuffer pH 7.4 (with acentic acid) (C2H6AsO2Na x 3 H2O Dimethyle arsine acid sodium salt, 214. 13 g/mol) for 50 mL 2.14 g Dimethyle arsine acid sodium salt 2. 25% Glutaralaldehyde solution 3. 10 % Formalaldehyde solution freshly mixed: annul 0.5 g Paraformaldehyde in 5 mL ddH2O and heat up to 60 °C. Add dropwise NaOH until a clear solution occurs. 4. ddH2O

Amount for 10 mL pre-fixationmedia

1. 5 mL (endconcentration 0.1 M) 2. 1 mL (endconcentration 2.5 %) 3. 2 mL (endconcentration 2 %) 4. 2 mL

E.R.L-embedding media

1. 3,4 epoxycyclohexyl methyl carboxylate 2.5 g 2. D.E.R. 736 epoxy resin 1.0 g 3. Nonenyl-succinic acid 6.5 g 4. Dimetyl-aminomethanol 0.1 g