Team:TU Darmstadt/Protocols/SamplePreparationEDX

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

For the EDX measument the cells have to be fixed, embedded in a SPURR-embedding medium (resin) and cut in to small rectangles smaller than 100 nm.

Fixation

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 750 µL 0.1 M Cacodylate-buffer (CaCo) pH 7.4 for at least 15 min at RT

Secondary fixation-step

• give about 750 µL 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 750 µL 0.1 M CaCo buffer pH 7.4 for at least 10 min and 2 times with bidest. H₂O for 10 min

Dehydration

• 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

• 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

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

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

Mixtures

Pre-fixation medium

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
4. ddH₂O

´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.

Amount for 10 mL pre-fixation medium

1. 5 mL (endconcentration 0.1 M)
2. 1 mL (endconcentration 2.5 %)
3. 2 mL (endconcentration 2 %)
4. 2 mL ddH₂O

E.R.L-embedding media (SPURR)

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