FIXATION OF SUSPENDED UNICELLULAR ORGANISMS FOR TRANSMISSION ELECTRON MICROSCOPY
A. Preparation of tissue
- Obtain culture of unicellular organisms. These usually require mild centrifugation to concentrate the sample. The suspended is then mixed 1:1 with 4% low temperature agarose. (Agarose is made with distilled water heated to 60° C and mixed thoroughly until it dissolves. This is allowed to cool to RT° before use.)
- Cool the material until it gels and then cut the material into 1 mm2 blocks (or smaller). These blocks are then handled similar to higher plant or animal tissues.
- Transfer tissue to a vial of 3% glutaraldehyde in 0.1 M cacodylate at pH 7.4. Fix 1 hour to overnight, depending on block density and scheduling. As with all tissue fixed by immersion, it must sink for best results! After one hour, transfer material to refrigerator at 4° C.
- Tissue may be processed immediately or held up to several months in a holding solution of 0.1 M cacodylate buffer at pH of fixation at 4° C.
- Rinse tissue with three changes of cold buffer, 10 minutes per change.
- Postfix in cold 2% osmium tetroxide (mixed 1:1 4% osmium with the 2X buffer) for 30 minutes to 2 hours on ice or in the refrigerator. Work with uncapped osmium solutions only under the fume hood.
- Rinse tissue with cold buffer for 10 minutes.
- Dehydrate the tissue using the following concentrations of ethanol in a cold graded ethanol series (10 minutes per change): 30%, 50%, 70%, 80%, 90%, 100%. If you have to stop at any point in the dehydration process 70% ethanol is best.
- Continue dehydration with two more changes of cold 100% ethanol (10 minutes per change).
- Follow cold 100% ethanol with one 10 minute change of cold (1:1) 100% ethanol : propylene oxide. Use propylene oxide in the fume hood.
- Complete dehydration with three changes of propylene oxide in the refrigerator, 10 minutes per change. Bring the tissue slowly to room temperature.
- Infiltrate tissue using the following concentrations of Spurr's resin, approximately 2-4 hours per change or longer: (1:2) Spurr's resin : prop. oxide, (2:1) Spurr's resin : prop. oxide. Refractory material may take up to one day per change.
- Use two changes of 100% Spurr's resin (2 hours or more per change).
- Embed in fresh Spurr's resin (less than one day old). Polymerize for 6 to 8 hours at 70° C.