Freeze-substitution and low-temperature embedding of dairy products for transmission electron microscopy

Dairy products are comprised largely of fat, air and water, which makes it difficult to preserve their ultrastructure for electron microscopy. Keeping the samples frozen throughout fixation and embedding protects the structure and distribution of the components of emulsions and foams. Therefore, dairy products were freeze-substituted and embedded at low temperature (-20 degrees C) to prepare them for transmission electron microscopy. Whipped cream, ice cream mix and dairy/non-dairy mixed systems were frozen by plunging in propane, at its boiling point (-187 degrees C). Ice cream, because it is already frozen, was fractured into 1-mm3 pieces in liquid nitrogen and then added to frozen fixative (-196 degrees C). Fixative solution consisted of glutaraldehyde, osmium tetroxide and uranyl acetate dissolved in either methanol or acetone. When material was to be stained after sectioning the fixative was limited to glutaraldehyde in methanol. The temperature was increased step-wise from -80 to -20 degrees C. Solvent was replaced with resin; the polar resin Lowicryl HM4, the non-polar resin Lowicryl HM20, LR White and LR Gold were tested. Samples were embedded and polymerized at -20 degrees C using ultraviolet light to cross-link the resin. Methanol proved to be the most effective solvent for substituting the ice; the hydrophobic resin Lowicryl HM20 was the most effective resin for retaining fat structure following osmium fixation.