Klaus Neumann

Cryofixation Without Pretreatment at Ambient Pressure

Almost a century ago biological or medical objects were used in their frozen state in order to accelerate pathological diagnosis (immediate section) and to better maintain their chemical constitution (Altmann 1890). This alternative to chemical fixation gained in importance during the first half of this century due to the availability of liquefied air used as coolant (“cryogen”) as well as to the fact that effective cooling systems for cryostats had been developed (see e.g. Gersh 1932; Simpson 1941; Eranko 1954; Kulenkampff 1955; Neumann 1958). Attempts to freeze wet objects for electron microscopy revealed that at normal atmospheric pressure and under the most favourable conditions only an approx. 30- μm border zone can be perfectly frozen (see e.g. Sitte 1979; Plattner and Bachmann 1982; Robards and Sleytr 1985). At greater depths the mixed plasmatic phases segregate. The size of these segregation compartments formed by the growing ice crystals within the specimen increases so rapidly that deeper layers cannot be used for electron microscopy. The depth of the well-preserved border zone can be increased without chemical pretreatment to, at the most, 300 μm by applying high pressures of about 2100 bar (Muller and Moor 1984). These limits can be considerably extended by the use of anti-freezing agents (“cryoprotectants”): glycerol has proved to be an effective anti-freeze for the freeze-fracture/freeze-etch method (Moor and Muhlethaler 1963) and saccharose in cryoultramicrotomy (see e.g. Bernhard and Leduc 1967; Tokuyasu 1973; Griffiths et al. 1984).

Cooling rate and ice-crystal measurement in biological specimens plunged into liquid ethane, propane, and Freon 22

Specimens sandwiched between copper planchettes were plunged up to a depth of 430 mm into coolants used for cryofixation. Hydrated gelatin containing a miniature thermocouple was used to mimic the behaviour of tissue during freezing. Gelatin and red blood cells were used for ice‐crystal analysis. Ethane produced the fastest cooling rates and the smallest ice‐crystal profiles, and Freon 22 produced the slowest cooling rates and the largest crystal profiles. Smaller crystal profiles were often seen in the centre of the specimens than in subsurface zones. The results show that ethane, rather than propane, should be used for freezing metal‐sandwiched freeze‐fracture specimens by the plunging method, and probably also in the jet‐cooling method. They further suggest that good cryofixation could occur at the centre of thin specimens rather than only at their surfaces. Comparison between theoretical and experimental ice‐crystal sizes was satisfactory, indicating that where the experimental parameters can be defined then realistic predictions can be made regarding cryofixation results.

Fast freezing device

A fast freezing device for cryofixation of biological material, without cryoprotection, has been developed based on metal freezing (Van Harreveld et al., 1964, 1974) combined with preceding sectioning (Bernard & Krigman, 1974; Van Harreveld & Fifkova, 1975) as well as freeze sectioning and freeze‐etch replication (Dempsey & Bullivant, 1976).

Safety Rules for Cryopreparation

As is often the case in every day life, the main risk in the routine use of cryopreparation methods is frequently due to inadequate knowledge or underestimation of the hazards. Vapourization of larger quantities of nitrogen in badly ventilated, small rooms or cool rooms may be lethal (Sect. 2). The same applies to explosive propane/air mixtures (Sect. 3). Eyes and skin may be severely damaged by secondary cryogen splashing, e.g. liquid propane, ethane or halogenated hydrocarbons (Sect. 4). The problems presented by primary cryogen splashing, e.g. liquid nitrogen (LN2) or liquid helium (LHe) are completely different (Sect. 5). Finally, precautions should be taken when working with inflammable, secondary cryogens and when using glass containers or vessels of materials which become brittle at cryogenic temperatures (Sects. 6 and 7). Transport and removal of cryogens require particular safety measures (Sect. 8).

Examination of cell suspensions prepared by cryobiological techniques

Ultrathin frozen sections have been obtained from single cells both in the fixed and native state. For fixed material a rapid method is described which yields excellent structural preservation. The preparation is based on brief treatment with glutaraldehyde, polyvinylpyrrolidone and ‘encapsulation’ in gelatin. Ultra‐thin frozen sections prepared after this technique seem to be especially suitable for cytochemical work. Unfixed testis has been also cut with a dry knife. The contrast in this completely untreated section is sufficient to discern the different parts of spermatozoa.

Gefrier-Ultramikrotomie des Knochenmarkes

ZusammenfassungKnochenmarkpunktate können nach kurzer Fixierung mit Glutaraldehyd und nachfolgendem Einschluß in Gelatine mit Zusatz von Polyvinylpyrrolidon als Gefrierschutzmittel mit dem Gefrier-Ultramikrotom (C. Reichert-Wien) ultradünn geschnitten werden. Die Präparationszeit wird dadurch auf 20 bis 30 Minuten verkürzt. Die beschriebene Methode führt zu sehr guten Resultaten und kann als Schnellverfahren für die Elektronenmikroskopie verwendet werden. Auch histochemische Untersuchungen an den ultradünnen Gefrierschnitten sind möglich. Dabei erbrachte die Nachweisreaktion der Peroxidase gut reproduzierbare Ergebnisse.SummaryUltrathin frozen sections of human bone marrow have been obtained with a cryoultramicrotome (Reichert/Austria) after short fixation with glutaraldehyde and inclusion in gelatine with polyvinylpyrrolidone as freeze protecting agent. The whole preparation up to the electron microscopical observation took not more than 20 to 30 min. The rapid method described in this paper gave excellent morphological preservation of cells. Moreover, histochemical investigations could be carried out on these sections. Especially peroxidase reaction was well reproducible.

Detection of a plant virus in cryosections of its host plant

Abstract The technique of cryo-ultramicrotomy was used for the detection of a plant reovirus in the tissue of its host plant. The observations made on cryosections, compared to those made on conventional sections, show that virus and related structures are well preserved, and that this technique can be usefully employed in plant virology.