Wolf K V, Stockem W, Wohlfarth-Bottermann K E
Eur J Cell Biol. 1980 Oct;22(2):667-77.
The influence of different fixatives (glutaraldehyde, osmium, osmium/glutaraldehyde, and osmium/mercuric chloride) and freeze-protecting agents (glycerol and sucrose) on the fine-structural preservation of micro- and macroplasmodia of the acellular slime mold Physarum polycephalum was investigated in both freeze-substituted and freeze-fractured material. Glutaraldehyde fixation and subsequent infiltration with glycerol or sucrose caused severe destruction in the morphology of plasmodial strands and protoplasmic drops, whereas osmium- or osmium/mercuric chloride fixation prevented the formation of normal fracture planes running through the hydrophobic core of the plasma membranes. A short prefixation in a mixture of osmium/glutaraldehyde followed by postfixation in glutaraldehyde delivered the most satisfactory results in the preservation of the fine structure. For comparison, the distribution of integrated membrane particles (IMP) was analysed in freeze-fracture replicas of unfixed controls as well as glutaraldehyde- and osmium/glutaraldehyde-fixed specimens by evaluating the number of IMP per 1 micrometer 2 in two different plasmodial regions; in the peripheral plasmalemma and in the central plasmalemmal invaginations. In controls not receiving chemical pretreatment and in specimens fixed with osmium/glutaraldehyde, the central plasmalemmal invaginations showed a clearly reduced total amount of IMP (exoplasmic + protoplasmic fracture face: about 3100) as compared with the peripheral plasma membrane (about 3700). In addition both membrane systems were characterized by an asymmetrical distribution of IMP between the protoplasmic fracture face (PF) and the exoplasmic fracture face (EF): the PF:EF ratio (particle partition coefficient) in the peripheral plasma membrane is the same in controls and in osmium/glutaraldehyde-fixed specimens (2.4:1 and 2.5:1, respectively), whereas the PF:EF ratio in the central plasmalemmal invaginations is 1.5:1 in controls and 3.5:1 in fixed specimens. This shows that the membrane of the central plasmalemmal invaginations is more sensitive to chemical fixation than the peripheral plasmalemma. The results point to differences in the physiological properties and functions between the plasmalemma of the cell periphery and the plasmalemma of the invagination system.
研究了不同固定剂（戊二醛、锇、锇/戊二醛和锇/氯化汞）和冷冻保护剂（甘油和蔗糖）对无细胞黏菌多头绒泡菌微观和宏观原质团精细结构保存的影响，研究对象包括冷冻替代和冷冻断裂材料。戊二醛固定并随后用甘油或蔗糖浸润导致原质团丝和原生质滴的形态严重破坏，而锇或锇/氯化汞固定则阻止了贯穿质膜疏水核心的正常断裂面的形成。在精细结构保存方面，先用锇/戊二醛混合物进行短时间预固定，然后用戊二醛进行后固定能得到最满意的结果。为作比较，通过评估两个不同原质团区域（外周质膜和中央质膜内陷）每1平方微米的整合膜颗粒（IMP）数量，分析了未固定对照以及戊二醛和锇/戊二醛固定标本的冷冻断裂复制品中IMP的分布。在未接受化学预处理的对照和用锇/戊二醛固定的标本中，与外周质膜（约3700）相比，中央质膜内陷处的IMP总量明显减少（胞外 + 胞质断裂面：约3100）。此外，两个膜系统的特征还在于IMP在胞质断裂面（PF）和胞外断裂面（EF）之间的不对称分布：对照和锇/戊二醛固定标本中外周质膜的PF:EF比值（颗粒分配系数）相同（分别为2.4:1和2.5:1），而中央质膜内陷处对照的PF:EF比值为1.5:1，固定标本中为3.5:1。这表明中央质膜内陷处的膜比外周质膜对化学固定更敏感。结果表明细胞外周质膜和内陷系统质膜在生理特性和功能上存在差异。
