Fixation and Acid Fuchsin Staining for Light Microscope Observations of Whole Mitochondria

The fine structure of mitochondria and mitochondrial nucleoids in exponentially growing Physarum polycephulum was studied at various periods throughout the mitochondrial division cycle by light and electron microscopy. The mitochondrial nucleoid elongates longitudinally while the mitochondrion increases in size. When the nucleoid reaches a length of approximately 1.5 ~m the mitochondrial membrane invaginates at the center of the mitochondrion and separates the mitochondrial contents. However, the nudeoid does not divide even when the mitochondrial sections are connected by a very narrow bridge. Just before division of the mitochondrion, the nucleoid divides by constriction of the limiting membrane of the dividing mitochondrion. After division, one end of the nucleoid appears to be associated with the inner mitochondrial membrane. The nucleoid then again becomes situated in the center of the mitochondrion before repeating these same processes. It has been reported that in the plasmodium of Physarum polycephalum morphological changes in the mitochondria occur throughout the mitochondrial division cycle (15) (Fig. 1). The mitochondrion contains a large, rodlike nucleoid situated in the center of the inner matrix (6, 7, 9, 16, 21) which is composed of a large amount of DNA (10, 11), RNA (9), and protein (9, 11, 14). Little information is available about the behavior of the mitochondrial nucleoid during the mitochondrial division cycle. The object of the present study was to clarify the morphological steps in the division of the mitochondrial nucleoid. The morphology of the nucleoid at various phases of the mitochondriai division cycle was studied by light microscopy using acid fuchsin and thionine staining techniques and by electron microscopy. MATERIALS AND METHODS Culture of Plasmodia Mitotically synchronized plasmodia of Physarum polycephalum were prepared by fusion of microplasmodia with the methods reviewed by Guttes and Guttes (5). Surface plasmodia from the second postfusion mitosis (MII) to the third postfusion mitosis (Mill) were used in these experiments. Identification of Mitotic Cycle The length of each portion of the mitotic cycle after fusion was determined by removing small explants from TeE JOURNAL OF CELL BIOLOGY" VOLUME 72, 1977" pages 687-694 687 on O cber 0, 2017 jcb.rress.org D ow nladed fom the plasmodium and examining smears of these pieces stained with azure B stain by a procedure described previously (9). Fixation and Acid Fuchsin Staining for Light Microscope Observations of Whole Mitochondria After MII, small explants of plasmodia were harvested at hourly intervals. They were fixed in ice-cold Champy's fluid (4) for 24 h, dehydrated in a graded series of water and water-soluble resin, glycol methacrylate (Oken Shoji Co., Tokyo, Japan) (15), and then embedded in glycol methacrylate. Thin sections (approximately 2 /xm) were cut on a Porter-Blum ultramicrotome (DuPont Instruments, SorvaU Operations, Newtown, Conn.) with a glass knife, mounted on glass slides, and dried gently with an alcohol lamp. The sections were covered with a small drop of acid fuchsin containing 1 g of acid fuchsin in 10 ml of aniline water (4), air dried on the slide, and stored at room temperature until analysis. Just before examination, any excess acid fuchsin on the sections was washed out with tap water, and then a drop of glycerin and a cover slip were placed on the sections. The stained sections were examined by oil immersion microscopy. Mitochondria and nuclei were stained brilliant red. Fixation and Thionine Staining for Light Microscope Observations o f Mitochondrial Nucleoids After MII, small explants of plasmodia were harvested at hourly intervals. They were fixed in ice-cold 1% glutaraldehyde solution (buffered with phosphate to pH 6.8) for 5 rain, hydrolyzed for 10 rain in 1 N HC1 at 45~ and again fixed in 6% glutaraldehyde solution (buffered with phosphate to pH 6.8) for 3 h. Then they were washed in cold distilled water, dehydrated in a graded series of water and glycol methacrylate, and finally embedded in glycol methacrylate. Thin sections were obtained as described above. The samples were stained with thionine by the method of Schaecher (17). Mitochondrial nucleoids were stained dark blue. The lengths of a mitochondrion and mitochondrial nucleoid and the constriction ratio of a mitochondrion were examined by the method described previously (4). The constriction ratio of a mitochondrion was defined as a/b, where a and b are the minimum and the maximum lengths of the minor axis of a mitochondrion, respectively (Fig. 3). The lengths of a mitochondrion and mitochondrial nucleoid and the constriction ratio of a mitochondrion were determined by examining more than 20 figures. Fixation for Electron Microscope Observations Small explants of plasmodium at various times after MII were fixed for 3 h in ice-cold 6% glutaraldehyde buffered with acetate to pH 6.8, washed in acetate buffer, pH 6.8 for 1 h, and postfixed in 1% OsO, for 12 h. They were then dehydrated in a graded series of ethanol and propylene oxide (30 min at each step) and embedded in Epon 812 (9). Ultrathin sections were cut on a Sorvall Porter-Blum ultramicrotome with a glass knife, and the sections were mounted on grids which were coated with Formvar. Thin sections were stained with saturated uranyl acetate for 1 h and, after observation of the degree of uranyl staining, poststained with lead citrate for 5 min. These sections were examined with a Hitachi l l E electron microscope operated at 80 kV.