User Notes - C010 & C012 Osmium tetroxide

This chemical is the most commonly used E.M. fixative for biological specimens. As it is a very toxic substance, safe handling procedures and adequate facilities should be available to all users.  

IMPORTANT:  Basic safety considerations before opening product 

Work with Osmium Tetroxide only under a fume hood to prevent inhalation and contact by OsO₄ vapour, particularly of the eyes which are very sensitive to exposure. Prevent contact with skin by wearing gloves. Never pipette osmium by mouth!

Preparation of a stock solution

1. Commercial OsO₄ is supplied in sealed glass ampoules. Handle osmium only under a fume hood, with the sash lowered to protect the operator's face and improve the draw. Plastic or rubber gloves are recommended. 

2. Remove label and soak the vial in water to remove any glue. The solution is conveniently prepared in a wide mouth, preferably brown, glass stoppered bottle. 

3. Place the open bottle on a sheet of paper and open the vial above the paper also. Unscored vials may be scored with a diamond pen or a triangular file and then opened. Scored vials may be opened with a pull and bend motion with both thumbs behind the score line. Both parts of the opened vial are dropped into the jar. Alternatively, a hot glass rod, pressed against the score-line, produces a crack around the ampoule. Any crystals spilt onto the paper should be added to the bottle. 4. To make a 2% solution of OsO₄: add 50ml of distilled water to 1g of Osmium Tetroxide crystals. Crystals dissolve within about two days at refrigerator temperature. Five minutes of ultrasound breaks up crystals and aids solution.

Preparation of fixative: 

The aqueous osmium stock solution may be mixed 1:1 with double strength buffer. Alternatively, OsO₄ may be prepared complete with the desired buffer. However, some chemicals (e.g. sucrose) cause rapid reduction. Preparation of double strength aqueous osmium and double strength buffer solutions gives greater flexibility for the selection of buffers, pH and other variables. For special requirements OsO₄can be made up in water. It also dissolves rapidly in organic solvents. 

Storage: 

Osmium tetroxide ampoules should be stored in a cool, dark place. OsO₄ solutions are quickly reduced at room temperature and will turn from clear/yellowish to brown and then black. Brown or black solutions are exhausted and should not be used as a fixative. Stock of a working solution should be refrigerated. Alternatively, small volumes may be dispensed in glass vials (check that these will not crack with water during freezing) and store in a freezer. 

Physical Properties: 

Osmium Tetroxide (Osmic Acid) is a translucent, yellowish, crystalline substance. The melting point is 40°C. It has a high vapour pressure and pungent odour. The chemical is a powerful oxidising agent and is rapidly reduced at room temperature. Solubility in water is poor. Threshold Limit Value as Osmium in the work place is 0.0002ppm; this concentration is too low for our sense of smell. Excessive exposure results in lung congestion, skin and eye damage.

Disposal: 

The small quantities used in laboratories can be flushed down the sink in a fume hood. It is dangerous to stockpile old OsO₄ solutions in the refrigerator.

Spill control: 

A major spill could require the evacuation of a building, whereas a spill inside a fume hood could be almost inconsequential. When heaped over spilled Osmium Tetroxide, full-cream powdered milk will exhaust crystals, solution and vapour. Before re-entering a room after a spillage, ventilate the room well; even for smaller spills, gas-tight eye goggles are needed.

Neutralization:

 A 2% solution of Osmium Tetroxide can be fully neutralized by twice the volume of oil (corn oil is preferred because of its high percentage of unsaturated bonds).¹ That is to say, for every 10ml of 2% Osmium Solution, 20ml of corn oil is required.

First Aid: