A. Preparation of tissue
  1. 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.)
  2. 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.
B. Fixation
  1. 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.
  2. 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.
  3. Rinse tissue with three changes of cold buffer, 10 minutes per change.
  4. 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.
  5. Rinse tissue with cold buffer for 10 minutes.
C. Dehydration
  1. 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.
  2. Continue dehydration with two more changes of cold 100% ethanol (10 minutes per change).
  3. Follow cold 100% ethanol with one 10 minute change of cold (1:1) 100% ethanol : propylene oxide. Use propylene oxide in the fume hood.
  4. Complete dehydration with three changes of propylene oxide in the refrigerator, 10 minutes per change. Bring the tissue slowly to room temperature.
D. Infiltration
  1. 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.
  2. Use two changes of 100% Spurr's resin (2 hours or more per change).
  3. Embed in fresh Spurr's resin (less than one day old). Polymerize for 6 to 8 hours at 70° C.