High‐pressure freezing of cell suspensions in cellulose capillary tubes

Abstract

Summary A procedure for efficient cryoimmobilization of large volumes of cell suspensions or micro‐organisms by high‐pressure freezing is described. This procedure uses transparent, porous cellulose capillary tubes with an inner diameter of 200 μm, into which the suspensions are drawn by capillary action. The tubes are processed by high‐pressure freezing and freeze‐substitution as if they were tissue samples. Centrifugation of suspensions at low temperatures is no longer necessary and cryopreparation is greatly facilitated. A very high yield of adequately frozen specimens is obtained due to the constant, defined sample geometry. This approach can also be used to process suspensions by conventional chemical fixation, eliminating the need to embed pellets in low‐melting‐point agarose, for example, prior to chemical fixation. The preparation procedure is demonstrated with suspensions of nematodes, paramecia and bacteria.

Taxonomic heterogeneity within the Planctomycetales as derived by DNA–DNA hybridization, description of Rhodopirellula baltica gen. nov., sp. nov., transfer of Pirellula marina to the genus Blastopirellula gen. nov. as Blastopirellula marina comb. nov. and emended description of the genus Pirellula

“…Morphological studies involving phase-contrast microscopy and electron microscopy were performed as described previously Gade et al, 2004). For cryofixation and cryosubstitution, bacteria were fixed using high-pressure freezing (Hohenberg et al, 1994). Samples in 1 % (w/v) osmium tetroxide in acetone were dehydrated in a freeze-substitution unit (AFS Leica) at temperatures of 290, 260 and 230 uC (for 8 h each).…”

Listeria monocytogenesl‐forms respond to cell wall deficiency by modifying gene expression and the mode of division

“…The specimen carriers were placed in the holder of a high-pressure freezer (HPM010, BAL-TEC) and shock frozen as described (Hohenberg et al, 1994). Freeze substitution with Osmium tetroxide, embedding in Epoxy resin, and thin sectioning with a cryo-ultramicrotom were performed as described elsewhere (Dahl and Staehelin, 1989;El-Kest and Marth, 1992;Hohenberg et al, 1994). Electron microscopy of the frozen-hydrated ultrathin sections was performed in a Zeiss EM 912 microscope at a temperature of 100 K (-173°C).…”

“…Parental Scott A from a broth culture were soaked into cellulose capillary tubes (BAL-TEC), immediately submerged in 1-hexadecene (Sigma-Aldrich, Germany) at room temperature and placed on an aluminium specimen carrier (BAL-TEC). The specimen carriers were placed in the holder of a high-pressure freezer (HPM010, BAL-TEC) and shock frozen as described (Hohenberg et al, 1994). Freeze substitution with Osmium tetroxide, embedding in Epoxy resin, and thin sectioning with a cryo-ultramicrotom were performed as described elsewhere (Dahl and Staehelin, 1989;El-Kest and Marth, 1992;Hohenberg et al, 1994).…”

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes

“…To study the structure of the MxA͞N complexes, we performed EM of infected MxA cells, using the cellulose capillary tube technique (24). After virus infection, stacks of filamentous bundles were detected in MxAexpressing cells (Fig.…”

“…The cells were trypsinized, collected by centrifugation, resuspended in DMEM, and drawn into cellulose capillary tubes (24). Cells were fixed in 1.75% glutaraldehyde, postfixed with 1% OsO4, and followed by staining in 1% uranyl acetate.…”