Spurr's Embedding Kit (Low Viscosity Embedding Media)

Spurr's Low Viscosity embedding mixture is recommended because of its excellent penetration qualities, which provide good and rapid infiltration of tissues. It is easy to prepare and mixes rapidly by shaking and swirling. The hardness is adjusted by changing the amount of the flexibilizer, DER 732; the blocks have good trimming and sectioning qualities and the sections are tough under the electron beam. (Grids, without supporting membranes can be used.)

INGREDIENTS:

ERL-4221 is a Cycloaliphatic Epoxide Resin.
Specific Gravity (H2O=1): 1.173 @ 20°C/20°C
Flash Point – Closed Cup: 118°C
Boiling Point (760mm/Hg):>250°C

DER 732 - diglycidyl ether of polypropylene glycol, a flexibilizer to control the hardness of the polymerized block. It was selected because of its low viscosity:

30-60 cP at 25°C.

It has a M.W of 380 and an epoxy equivalent of 175-205. There is little difference between DER 732 and DER 736. The latter is used elsewhere but is not available in Australia

NSA - nonenyl succinic anhydride, a hardener with a relatively low viscosity of 102.8cP at 25°C, and a M.W. of 227. A minimum exposure to air is recommended to avoid hydrolysis.

DMAE - dimethylaminoethanol (S-1), an accelerator, used because of its low viscosity and results in blocks with less colour. In addition, it induces rapid cure when the temperature is elevated to 70°C. It is effective in a very low concentration. (less than 1.0%) The optimum concentration for colour transparency is 0.7-0.75%.

MIXING INSTRUCTIONS:

Add each component in turn to a disposable plastic beaker. An exact weight is recommended, and care must be used in dispensing the final amounts of each component so that no excess is added.

The catalyst (DMAE) should be added last, after gently mixing the three other components.

The complete formula should be mixed thoroughly.

The complete mixture with the hardener can be used immediately for infiltration, and then for embedding. Although the mixture can be stored in a disposable syringe, well capped and with no air, in a freezer for several months, it is highly recommended that freshly prepared embedding medium always be used. If you choose to store the mixture, it is imperative that you warm it thoroughly prior to use.

DEHYDRATION - INFILTRATION AND POLYMERIZATION:

This embedding media is compatible with all dehydrating agents: acetone, dioxane, ethanol, hexyleneglycol, isopropyl alcohol, propylene oxide, tert-butyl alcohol. The schedule and concentration can be established by the investigator. Dehydration is generally done at room temperature. All dehydrating agent must totally be removed during infiltration due to the fact that it will affect curing.

The embedding media is completely compatible with ethanol. Thus, it is not mandatory to have a change to propylene oxide prior to infiltration, as is true for other epoxy resin mixtures. If working with plant cells it is recommended to use propylene oxide.

The infiltration (one should employ a specimen rotator) can be started by adding the embedding media to an equal quantity (1:1) of the dehydrating fluid left in the vial with the tissue. Swirl the mixture and allow it to stand for 30 minutes to 2 hours.

Replace with a 1:3 dehydrating agent/embedding medium, swirl, and allow it to stand for another 30 minutes to 2 hours. Pour and drain the mixture and add fresh embedding media. For small specimens leave 4-6hours; for large specimens leave 4-6 hours followed by overnight. Curing takes 16-24 hours at 60°C. The mixture can be left in an oven overnight.

LOW VISCOSITY EMBEDDING MEDIA KIT CONSISTS OF:

C056, ERL 4206 - vinyl cyclohexene dioxide - 225ml
C047, DER 732 - diglycidyl ether polypropylene glycol - 200ml
C059, NSA - nonenyl succinic anhydride - 500ml
C050, DMAE - dimethylaminoethanol - 25ml

REFERENCES:
Spurr, A.R.(1969), J. Ultrastruct. Res. 26,31.