Ultrastructure and energy-dispersive x-ray microanalysis of cartilage after rapid freezing, low temperature freeze drying, and embedding in Spurr's resin.

In order to undertake meaningful high-resolution x-ray microanalysis of tissues, methods should be used that minimize the introduction of artefacts produced by loss or translocation of ions. The most ideal method is rapid freezing but the subsequent sectioning of frozen tissues is technically difficult. An alternative method is to freeze dry the tissues at a low temperature, and then embed them in resin. This facilitates the rapid production of reproducible thin sections. With freeze-dried, embedded hypertrophic cartilage, the morphology was similar to that seen using aqueous fixatives even when no additional electron density is introduced by the use of osmium vapor. Energy-dispersive analysis of specific areas show that little or no loss or migration of ions occurs from structures such as mitochondria. Mitochondrial granules consisting of calcium and phosphorus precipitates were not observed except where the cells were damaged as a result of the freezing process. This may suggest that these granules only appear when tissue is damaged because of inadequate preservation.

An inexpensive device for freeze drying and plastic embedding tissues at low temperatures.

The preparation of biological tissues for electron microscopy by rapid freezing retains the original localization of ions and molecules. A reproducible freezing regime was established by quenching tissues in liquid propane according to the method of Elder et al. (1981). Tissue was thereafter freeze dried in a custom built freeze drying device with a liquid nitrogen cooled stage to prevent ice recrystallization during drying. The device was also designed to allow the vacuum embedding of tissue in low temperature resin such as Lowicryl and polymerization in situ. This paper describes the design of the device and an example of its use in the freeze drying of cartilage. The results show that minimal ice damage occurs to the chondrocytes and that intracellular organelles are clearly visible. The regime described may prove a useful and pragmatic alternative to cutting tissue in the frozen state. Translocation of elements is unlikely except perhaps in the case of very labile elements such as Na and K, but this remains to be fully elucidated.

An ultrastructural investigation of the filamentous surface appendages of suspensions of plaque bacteria as revealed by negative staining.

The filamentous surface appendages of freshly-collected aqueous suspensions of plaque bacteria, obtained from 1 day old supragingival plaque have been examined in the electron microscope by the technique of negative staining with methylamine tungstate. Approximately half of the bacteria revealed surface appendages as either fimbriae (45%), flagella (13%) or both (3%). The appendages were distributed either polarly, intermittently or peri-trichously around the bacteria and varied in length from 0.2 micron to more than 20 micron. It was not possible from these observations alone to determine either the topology of the appendages or their role as they existed originally in dental plaque.