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#Three disadvantages of electron microscope manual

The usage of NaOH pellets to minimize the precipitation is a commonly used technique.

#Three disadvantages of electron microscope manual

The major problem of the manual staining procedure is the precipitation of the metallic salts during the staining process, especially the precipitation of lead. After wicking and drying, grids can be analysed.

After staining, the grids are rinsed again in several water baths and by using a jet of water (Figure 9c). In addition, the rinse water should be CO 2-free (double distilled). In order to prevent precipitation of the lead citrate by exposure to CO 2, pellets of NaOH are added to the staining dish (here a Petri dish) to exclude atmospheric CO 2. Then the grids are rinsed in water several times before being placed on drops of lead citrate for incubation (Figures 9a and 9b). Here, the grids are placed on drops of uranyl acetate (UA) solution and removed after a certain incubation time with forceps (Figures 8a and 8b). In the preparation of specimens for electron microscopy, the manual staining of sections is the step in which most failures are experienced. If the pH varies by more than 0.1 unit from pH 12.0, poor staining or precipitation will occur. Note: An exact pH is extremely important with this stain. Before use, centrifuge (5,000 × g/10 min) or passage through a microfilter.

The solution has a shelf life up to 6 months if sealed tightly.

After pH has been verified, add CO 2-free water to the volumetric flask to bring the solution to a final volume of 50 ml.

If the pH is too low add more NaOH to the clear solution in the flask of step 2.

Withdraw a small amount of clear stain and check the pH using a pH meter.

Solid waste that is contaminated with UA should be disposed of in a solid waste container set aside for radioactive wastes (contact lab safety officer!). If the solution still does not turn clear, something is wrong and the stain should be discarded! Rinse contaminated glassware into a waste bottle for UA solution and store separate from laboratory glassware. If not, add a few more drops of NaOH up to total 8 ml.

Add 5 to 7 ml of 1 N NaOH to the suspension while stirring.

After dissolving let the solution cool down to room temperature.

Stir on stir plate until uranyl acetate dihydrate crystals (Figure 3) are dissolved. If one sees large chunks, continue shaking until the particles are dissociated, or start over. This will generate a milky white suspension of lead stain with no large particles. Shake the solution vigorously for several minutes then 5–6 times over a 30 min period.

Combine 1.33 g lead nitrate, 1.76 g sodium citrate and 30 ml CO 2-free double-distilled water in the 50 ml volumetric flask.

Automated contrasting with ready-made pre-packaged UA and lead citrate stains ensures minimum user contact with reagents and eliminates nearly all traditional problems of manual staining such as uranyl or lead precipitates, health risks and high reagent consumption. The scientific demand of reproducibility, high sample quality with low reagent consumption on one side and the request of safe working conditions by avoiding contact with hazardous reagents on the other side can be satisfied with a standardised, closed and automated process.Īccording to these demands, Leica Microsystems developed the fully automated Leica EM AC20. Avoiding CO 2 in a manual process is difficult for which a special procedure is needed.

The lead citrate, which enhances the contrast by interacting with proteins and glycogens, will form a waterinsoluble toxic white precipitate (lead carbonate), if not used strictly under CO 2-free conditions. The uranyl acetate (UA), which enhances the contrast by interaction with lipids and proteins, forms a yellow, needle-like crystal precipitate if not used in the right concentration and if redundant UA is not removed from the section. Besides manual handling, especially the properties of used chemicals, which can cause precipitation artefact, are the error factors. Although this technique is well known contrasting is one of the most critical steps of specimen preparation for electron microscopy. Therefore, the double contrast method of ultrathin sections on grids with uranyl acetate and lead citrate is the standard routine contrasting technique for electron microscopy.