Ester Wax


Diethylene glycol


Stearic acid

Ester wax is an embedding medium which is based largely on diethylene glycol distearate mixed with a few other ingredients to modify its characteristics. On its own diethylene glycol distearate is a very hard and brittle, waxy material melting at about 50°C. It is not suitable as a support medium for tissues of the size usually seen in light microscopy and which are sectioned on ordinary microtomes, although it has been used for semi-thin sections in electron microscopy with ultramicrotomes. Steedman tried mixing this compound with several others to counteract its inherent hardness and brittleness and to permit ribboning, so that reasonably sized pieces of tissue could be prepared. The first mixture was recommended in 1947, and modified formulas were recommended in 1960, including a variant for laboratories with a high ambient temperature. The 1960 formula is generally considered superior to the 1947 one. in addition, Chesterton and Leech suggested a formula which improved on the original. Diethylene glycol has also been recommended as a suitable wax for producing semi-thin sections using an ultramicrotome (and possibly an intermediate microtome), usually as an adjunct to electron microscopy, although the sections could presumably be used for light microscopy in their own right as an alternative to methacrylate embedding. Ester wax is best purchased already formulated and, unless stated to be otherwise, will almost certainly be Steedman's 1960 variant.

Ester Wax Variants
Steedman 1947

Diethylene glycol distearate
Ethyl cellulose
Stearic acid
Castor oil
Diethylene glycol monostearate
 

73 g
4 g
5 g
8 g
10 g
Steedman 1960

Diethylene glycol distearate
Glyceryl monostearate
300 polyethylene glycol distearate
 

60 g
30 g
10 g
Chesterman and Leech 1956

Diethylene glycol monostearate
Diethylene glycol distearate
Castor oil
Note: Both diethylene glycol esters
must be of a purified grade

 

100 g
100 g
5 g
Steedman 1960 Tropical Ester wax

Diethylene glycol distearate
Glyceryl monostearate
Triethylene glycol monostearate
 

60 g
30 g
10 g
Taleporos, 1973

Diethylene glycol distearate alone
A hard translucent wax, M.P 47-52°C

Note: This was recommended for producing semi-thin sections for light microscopy from freeze dried pieces of tissue 1 mm thick as an adjunct to electron microscopy. A water filled trough is essential for 0.5-1 µ sections.
Graham, 1982

Diethylene glycol distearate
Cellulose caprate

Note: Recommended as suitable for semi-thin sections using an ultramicrotome and Ralph knives with water filled troughs on ethanol dehydrated, xylene cleared blocks. Suitable for sections 1-3 µ thick. MP 50°C.

 
80 g
20 g

All of the ester wax variants by Steedman and Chersterman and Leach have similar properties. They are translucent or opaque, cream coloured, hard, waxy materials. All can be sectioned in ribbons but require a sharp knife and generally a slower sectioning speed than paraffin waxes due to their hardness. They all melt at around 48-50°C, give or take a degree of two, and all can tolerate a degree of moisture. Generally sections can be cut from about 2µ to 20µ at a room temperature of 20°C, and Steedman stated that 1µ sections were possible. The waxes are miscible with 95% and absolute ethanol and many other alcohols such as butanols and propanols, hydrocarbons such as xylene and toluene, chlorinated hydrocarbons, ketones, 2-ethoxy-ethanol (cellosolve) and similar compounds, various esters, and natural oils, such as clove and cedarwood oils. Most of these can be used as the antemedium immediately prior to wax infiltration with appropriate adjustments to the processing schedules. Steedman particularly recommended 2-ethoxy-ethanol and xylene. Sections may be flattened with water at 45-50°C. and will successfully bake onto a clean, grease free slide at 40-45°C for 1-2 hours.

The water tolerance of ester waxes means that dehydration does not have to be done so thoroughly as with paraffin wax, although it is still a necessary part of the preparation as the waxes are water tolerant, not water soluble. It also means that sections may be stained from aqueous solutions without removing the wax. This can be done by floating the sections on one or more dye solutions before baking on to a slide, then simply dewaxing and coverslipping.

The simplest processing is accomplished using ethanol as antemedium:

  1. Fix and wash the tissue as wished.
  2. Bring to 95% ethanol using graded ethanols.
  3. Place into an equal parts mixture of 95% ethanol and molten ester wax at about 40°C
  4. Place into pure ester wax
  5. Cast using Leuckhart's moulds, Fill any depression that forms while cooling.

Chesterton and Leech proposed two processing schedules, and Steedman two more in addition to the simplest one given above. The details are given below. These schedules could typically be used with the first four ester wax variants, and possibly the last two (the Taleporos and Graham variants) as well, although those wishing to investigate any of these processing systems are advised to consult the original papers.

Alternate Processing Schedules
Traditional Processing
Chesterman & Leach

  1. Fix as desired. Wash if necessary.
  2. Ethanol, 70% for 12 hours
  3. Ethanol, 90% for 12 hours
  4. Ethanol, 95% for 12 hours
  5. Ethanol, absolute for 12 hours, changing once
  6. Ethanol, absolute for 12 hours
  7. Ethanol and xylene mixed 1:1, overnight, changing twice
  8. Xylene for 2 hours or until cleared
  9. Ester wax at 53°C overnight or up to 24 hours. Change after 1-2 hours and repeat. Use fresh, unused wax.
  10. Cast using Leuckhart's moulds, Fill any depression that forms while cooling.
Non-Traditional Cellosolve Processing
Chesterman & Leach

  1. Fix as desired. Wash if necessary.
  2. Cellosolve, absolute for 24 hours, changing a minimum of 4 times.
  3. Ester wax at 53°C overnight or up to 24 hours. Change after 1-2 hours and repeat. Use fresh, unused wax.
  4. Cast using Leuckhart's moulds, Fill any depression that forms while cooling.
Non-traditional cellosolve processing
Steedman

  1. Fix as desired. Wash if necessary.
  2. Bring to 70% ethanol.
  3. Place into equal parts mixture of 70% ethanol and cellosolve.
  4. Place into cellosolve, 100%.
  5. Place into an equal parts mixture of cellosolve and ester wax.
  6. Place into pure ester wax, 3 baths. Use fresh, unused wax.
  7. Cast using Leuckhart's moulds, Fill any depression that forms while cooling.
Non-traditional monochlorisothymol processing
Steedman

  1. Fix as desired. Wash if necessary.
  2. Bring to 70% ethanol.
  3. Bring to 90% ethanol.
  4. Place into a mixture of:
      95% ethanol – 1 part
      monochlorisothymol – 1 part
      molten ester wax – 2 parts.
  5. Place into pure ester wax, 4 baths. Use fresh, unused wax.
  6. Cast using Leuckhart's moulds, Fill any depression that forms while cooling
Note: Monochlorisothymol is removed by diffusion, not evaporation. The extra bath in ester wax is to facilitate this, as does stirring.


Ester waxes are quite hard and sectioning has to be adjusted to allow for this. A sharp knife is essential and a higher angle of tilt than usual is likely to be required. Thick sections tend to break and crumble, so trim and rough down the block carefully using thinner sections than with paraffin.

Leuckhart's molds are recommended for casting the block. They should be adjusted to give an area larger than required, then filled with molten wax. When the wax begins to solidify at the edges, the tissue should be placed eccentrically. As the block cools there is a tendency for the center to contract down, so placing the tissue on the edges reduces the likelihood of it being exposed above the wax. The surface of the block should be melted with a hot spatula during hardening so that the block solidifies from the bottom up and melted wax fills in any depression that forms. Do not submerge the block to harden. A cold plate or block of ice may be used if it is necessary to increase the speed of hardening.

Wikipedia has articles about diethylene glycol and stearic acid.

References
Steedman, H.F., (1947)
Ester Wax: A New Embedding Medium
Quarterly Journal of Microscopical Science, v. 88, pp. 123-133

Steedman, H.F., (1960)
Ester Wax 1960: A Histological Embedding Medium
Quarterly Journal of Microscopical Science, v. 101, pp. 459-462

Steedman, H.F., (1960)
Section cutting in microscopy
Blackwell Scientific Publications, Oxford, UK.

Chesterman, W. and Leech, E.H., (1956)
A modified ester wax for embedding tissue
Quarterly Journal of Microscopical Science, v. 97, pp. 593-597

Taleporos, P., (1974)
Diethylene glycol distearate as an embedding medium for high resolution light microscopy
Journal of Histochemistry and Cytochemistry, v. 22, pp. 29-34

Graham, E. T., (1982)
Improved diethylene glycol distearate embedding wax
Biotechnic & Histochemistry, v. 57, pp. 39-43

 


 

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