Some Improvements in Zoological Microtechnique for Electron Microscopy
Cleaning of Staining Solutions, etc
Cleaning of Staining Solutions, etc.
Particles of extraneous "inert" material must surely be regarded as one of the main sources of annoyance to an electron microscopist working at the magnifications usually employed. These have the perverse habit of depositing themselves in the page 2 most undesired of places. It is cause for wonder that methods seen in reference works do not mention the necessity for filtering staining solutions, embedding materials and wash liquids to rid them of this type of contamination. We reckoned there were enough problems in the technique without having to endure this added burden which could be so easily eliminated. Maybe it is unwritten law in electron microscope laboratories to filter solutions before use. If this is the case, the textbooks are of little help to the beginner if they omit the necessity for filtration. Some idea of the amount of suspended matter can be got by putting a strong pin-point light source under the container of stain or other liquid being used. This is the test we employed to evaluate the quality of our filtration.
For this reason, we filter all solutions except the staining fluid through a sintered glass filter of porosity grade 4, making sure that the container collecting the filtrate is the only one used for storing its contents. The staining fluid is filtered through a sintered bacterial filter. The filtering apparatus is shown in figure 1. It is simple to assemble and dismantle since all joints are standard taper glass joints. Under no circumstances should filter paper be used.
The embedding material we use is Ciba Araldite and we have adopted the following method for removing suspended foreign matter from it. The Araldite is diluted with an equal volume of benzene, and this mixture is passed several times through the filter. The diluted Araldite is collected in a flask which can then be attached to a rotary evaporator. The benzene is recovered under vacuum, leaving the Araldite at about the same consistency that it had initially—but very much cleaner. The hardener is treated in the same way. Benzene was chosen because of its capacity to form an azeotropic mixture with water. We thought that traces of water left in the Araldite after manufacture might be responsible for much of the early difficulty we had in getting Araldite-embedded sections to remain stable under the electron beam. The washed Araldite has proved to be so stable that no supporting film of Formvar is needed: and this automatically enables high resolution and magnification of any part of the field.
The water used to wash the grids after staining is also filtered rigorously.