Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha.

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorphia has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop growth on or methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H2O2 dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H2O2 dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H2O2 was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H2O2 for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.     

CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.     

Cytochemical Localization of Polyphenol Oxidase Activity in K2‐Bodies of Saprolegnia ferax Secondary Zoospores

Zoospores of the oomycete Saprolegnia ferax release adhesive material from K‐bodies at the onset of attachment to substrates. To understand more fully how K‐bodies function in adhesion, enzyme activity was investigated cytochemically in secondary zoospores. Presence of catalase, a marker enzyme for microbodies, was explored in the diaminobenzidine (DAB) reaction. Although pH 9.2 DAB‐staining characteristic of catalase activity was detected in the granular matrix regions of K‐bodies, reaction controls indicated that the reaction was due to oxidative enzyme activity other than catalase. Because polyphenol oxidase (PPO) is another metal‐containing enzyme capable of oxidizing DAB, activity of this enzyme was tested with a more specific substrate, dihydroxyphenylalanine (DOPA). In the DOPA procedure, reaction product was exclusively localized within K‐bodies, indicating the presence of PPO. Results with three methods of reaction controls (elimination of substrate, addition of a PPO enzyme inhibitor, and heat‐inactivation of enzymes) all supported the presence of PPO in K‐bodies. This study highlights potential roles for K‐body PPO in stabilization of adhesion bodies by: cross‐linking matrix phenolic proteins or glycoproteins as K‐bodies discharge adhesives onto substrates, or polymerizing phenolics protective against microbial attacks of the adhesion pad.     

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorpha has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop during growth on methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H₂O₂ dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H₂O₂ dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H₂O₂ was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H₂O₂ for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.     

Diaminobenzidine reactions in control and treated amoeba proteus

Summary Cytochrome oxidase activity was demonstrated in Amoeba proteus by diaminobenzidine (DAB) cytochemistry. Deposition of the reaction product occurred on the inner mitochondrial membranes and the cristae. The reaction was abolished by cyanide incubations. Positive reactions were produced with both unfixed and fixed cells: although staining potential was destroyed by any prefixatives which included glutaraldehyde. Cells prefixed with 4% formaldehyde, to raise structural preservation, retained staining ability.Amoebae subjected to prolonged anaerobiosis or to treatment with the carcinogen N-methyl-N-nitrosourethane (MNU) displayed a reduction in DAB reactivity. A positive reaction was only produced in incubations of unfixed cells and even in these the intensity of cristal staining was depleted. The possible use of DAB reactions where lesions in mitochondrial functioning have occurred is considered.     

Effect of Nitrate on Peroxisome Ultrastructure and Catalase Activity in Nodules of Lupinus albus L. cv. Multolupa

This paper reports on an ultrastructural study of peroxisomesin infected nodule cells of Lupinus albus L cv Multolupa plantsgrown with and without nitrate (in short-term experiments) Cytochemicallocalization of catalase and peroxidase was performed applyingthe diamino-benzidine (DAB) technique in these tissues The infected cells presented a mean of seven peroxisomes, themaximum being 16 in some cells Peroxisome shapes proved to befairly round or egg-shaped, with maximum and minimum diametermeans of 0-35 and 0 18µm, respectively They were preferentiallypositioned on the cell periphery. The intense osmiophilic stainingobtained by applying the DAB technique indicates a strong catalaseactivity reaction in these microbodies The addition of nitrate (20 mol m^–3) to the growing plantsexerted a negative effect on nitrogenase activity, which diminishedby 31 6% after 5 d of treatment Severe alterations in the ultrastructureof microbodies, bacteroids, and penbacteroidal membranes wereobserved Cytochemical data show a reduction in catalase localizationin peroxisomes and an increased peroxidase activity in the cytosol Finally, leghaemoglobin (Lb) localization was studied in nitrate-grownplants, confirming our previous observation of a decrease inthis protein Discussion focuses on the involvement of theseresults in tissue senescence of the nodules following nitrateapplication Key words: Peroxisomes, diamino-benzidine, nitrate, catalase, leghaemoglobin     

A comparative histochemical study of peroxidase activity in the submandibular glands of five mammalian species including man

Summary A light microscopic histochemical investigation of endogenous peroxidase activity in specimens of the submandibular salivary glands of man, hamster, rabbit, dog and guinea pig was carried out. A modification of the original Graham and Karnovsky diaminobenzidine (DAB)-hydrogen peroxide method was employed at different pH's.At all pH's (6.0, 7.6, and 9.0) a positive DAB reaction was found: in serous acinar cells in four of seven human submandibular glands, in convoluted tubule cells of the hamster, in acinar tissue, in secretory granular tubule cells and in the saliva of the guinea pig. This staining pattern was not markedly affected by KCN or 2,4-dichlorophenol (DCP). Furthermore, small cytoplasmic granules in collecting ducts of the dog displayed positive, KCN- and DCP-resistant DAB staining at all pH's tested. No reaction was observed in the acinar cells of the dog and rabbit glands.Mitochondrial oxidation of DAB in the striated duct cells occurred in all of the glands examined. Optimal staining of these cells was obtained at pH 6.0, but there was also strong positive staining at pH 7.6. At pH 9.0, however, the staining of the striated duct cells was very faint. The positive reaction in the striated duct cells was completely abolished by KCN.     

Cytochemical studies on peroxisomes in rat and guinea pig myocardium

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.     

Cytochemical localization of peroxidase activity in the mitochondria of Hymenolepis diminuta.

A cytochemical study of mitochondria of Hymenolepis diminuta indicates the presence of a mitochondrial peroxidase. Utilizing a 3,3'-diaminobenzidine (DAB) medium at pH 9.7, the reaction product is localized in the intracristal space, and between the inner and outer membranes of the mitochondria. No inhibitory effects are exerted on the peroxidase reaction by cyanide, azide, or aminotriazole. In addition, the mitochondria appear to have an enzyme which is cytochemically similar to vertebrate cytochrome c-oxidase. The possible physiological significance of the peroxidase is discussed.     

Ultrastructural localization of photosynthetic activity in thylakoids during chloroplast development in maize

The localization of photosynthetic activity in developing maize (Zea mays L.) chloroplasts was studied in situ by two electron-microscopic-cytochemical methods. The activity of photosystem I was detected by photooxidation of 3,3-diaminobenzidine (DAB) and the activity of the photosystem II by photoreduction of thiocarbamyl nitrotetrazolium blue (TCNBT). During the transformation of proplastids into chloroplasts, at the base of the leaf blade the DAB reaction appeared before the TCNBT reaction. A positive DAB reaction was observed in the single thylakoids of plastids in cells located only about 0.5 mm above the base. Dark, osmiophilic DAB polymers accumulated in the lumina of the thylakoids. Plastid envelopes and tubules of the prolamellar bodies in immature chloroplasts were DAB-negative. In fully differentiated leaf tissue the DAB reaction was intense in the thylakoids of bundle-sheath chloroplasts, as well as in the stroma thylakoids and the peripheral grana thylakoids of mesophyll chloroplats. The photoreduction of TCNBT started in leaf tissue about 1 mm above the base. Dark granular material of reduced TCNBT appeared mostly in the partitions of grana, i.e. interthylakoidally, but some granules were also attached to the stroma thylakoids. The membranes of plastid envelopes and the tubules of prolamellar bodies showed a negative TCNBT reaction. Young bundle-sheath chloroplasts contained some reduced TCNBT in their grana; these deposits largely disappeared in the course of further differentiation. In mature leaf tissue the photoreduction of TCNBT was conspicuous in the grana of mesophyll chloroplasts, but very weak in the single thylakoids and in the granal rudiments of bundle-sheath chloroplasts.Abbreviations DAB 3,3-diaminobenzidine·4 HCl - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS(I,II) photosystem (I,II) - TCNBT thiocarbamyl nitrotetrazolium blue chloride     

Evidence for two classes of microbodies in meiocytes of the red algaPalmaria palmata

Summary Microbodies, usually spherical and about 0.2 m in diameter, were found to be associated with prophase nuclei in vegetative cells and meiocytes of the red algaPalmaria palmata. Nucleus-associated microbodies in meiocytes were numerous, but they did not react to the DAB cytochemical test for catalase and peroxidase activity. Microbodies not associated with nuclei in the same cells were intensely DAB-positive. Neither aminotriazole nor potassium cyanide inhibited the DAB reaction.     

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells

The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope. The presence of catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.     

Fine structural and cytochemical analysis of phragmosomes (microbodies) during cytokinesis inAllium root tip cells

Summary The ontogeny and distribution of phragmosomes (microbodies) during cytokinesis inAllium sativum root tip cells have been studied and complemented with a cytochemical analysis of reactivity with diaminobenzidine (DAB). Incubation in different DAB media revealed the presence of catalase but not peroxidase in these organelles, identifying them as a type of microbody associated with the forming cell plate. Only vacuoles, segments of endoplasmic reticulum and portions of the mature walls stained positively with DAB for peroxidase activity. Microbodies begin to appear in the region of the future cell plate as cells enter late anaphase. They exhibit a moderately electron-opaque anucleoid matrix and are continuous with segments of endoplasmic reticulum (ER). Certain observations have led us to consider that certain aspects of plate formation inAllium require the participation of microbodies: (a) their pronounced numerical increase at the onset of plate formation, (b) their intimate association with regions of the plate where vesicle fusion is in progress, and (c) their rapid numerical decline following vesicular fusion and concomitant cell plate formation. The characteristic spatial association observed between microbodies and the plate-forming vesicles may well reflect their mutual involvement in the metabolism of carbohydrates comprising the middle lamella, being coordinated by metabolic activities in the cytosol, mitochondria and dictyosomes.This study was supported in part by NIH training grant HD 174 to Dr.Hewson Swift and the Marquette University Committee on Research Grants 5641 and 5532.     

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells

Summary The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope.The presence of catalase (E.C. 1.11. 1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.Abbreviations used CAT catalase - CYT-OX cytochrome oxidase - DAB diaminobenzidine - ER endoplasmic reticulum - f filaments - HVEM high voltage electron microscopy - M mitochondrion - MT microtubule - P peroxisome - PB protein body - PER peroxidase - Pl plastid - Pg plastoglobuli - RER rough endoplasmic reticulum - RuBPcase ribulose-1,5-bisphosphate carboxylase - S starch - T tubule - V vacuole Scientific Article No. A3997, Contribution No. 6981, of the Maryland Agricultural Experiment StationThe scale bar on each micrograph is 0.1 , unless indicated otherwise     

Cytochemical localization of catalase in leaf microbodies (peroxisomes)

Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3''-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide. Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles. The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide. Reaction of the microbodies with DAB was completely inhibited by 0.02 M 3-amino-1,2,4-triazole and was considerably reduced by 0.01 M potassium cyanide. These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction. Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates. A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme. This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases.     

Seasonal rhythmics of the leucoplast structure in bulb scales of Scilla sibirica L

A study was made of seasonal changes in plastids of ground tissue cells of bulb scales in early-spring ephemeroid Scilla sibirica L. In summer, plastids are represented by typical amyloplasts, with their main volume (97.0 +/- 4.3%) being occupied by one large starch grain. The volume fraction of plastid stroma is at its minimum. The stroma contains small plastoglobuli and no thylakoids. The same structure is characteristic of plastids in October. However, no starch is found in December, when some thylakoids are seen at the plastid periphery. In the early spring (March), when leaves still remain below the ground, the volume fraction of starch grains is 53.0 +/- 2.2%. In the stroma some structures superficially similar to those of microtubuli are revealed. The thylakoid system is fairly well developed, some of thylakoids being concentrically arranged. Some electron-opaque material is seen in the thylakoid lumen. Many plastids are sheathed with elements of the smooth endoplasmic reticulum. Based on the analysis of these and literature data, a conclusion is made that plastids of bulb scales not only store starch, but also seemingly participate in phytohormone biosynthesis.     

Microbodies und Diaminobenzidin-Reaktion in den Acetat-Flagellaten Polytomella caeca und Chlorogonium elongatum

Summary In two forms of acetate flagellates, the colourless Volvocale Polytomella caeca and the green Volvocale Chlorogonium elongatum, cell organelles can be demonstrated which are ultrastructurally similar to microbodies of higher organisms. The organelles do not have a close association with the endoplasmic reticulum and are located in the peripheral cytoplasm between the elongated mitochondria. In Polytomella they exhibit more or less spherical profiles in section and have a maximum diameter of approximately 0.2–0.25 . In Chlorogonium the organelles occasionally have an elongated shape and are larger than in Polytomella. Employing the electron microscopic cytochemical reagent diaminobenzidine (DAB)/H₂O₂ to localize the microbodial marker enzyme catalase in these organelles, it was found that no accumulation of the electron-opaque product occurs in the microbodies either at alkaline or neutral pH or at room temperature or 37° C. Only the cristae of mitochondria are stained with the DAB reaction caused by cytochrome oxidase and possibly by a cytochrome peroxidase.Organelles of Polytomella caeca containing catalase or cytochrome oxidase can be separated by rate centrifugation of a crude particulate fraction on a sucrose gradient (Gerhardt, 1971). The particles isolated from the peak of catalase activity show the same fine structural characteristics as the microbodies in situ do. But again, there is no detectable staining of these organelles by the DAB/H₂O₂ reaction.The identity of the microbody-like particles in Polytomella caeca and Chlorogonium elongatum with microbodies in general is deduced despite the negative results in cytochemical localization of catalase in these organelles.     

Mitochondrial morphology in Basidiobolus haptosporus

Young hyphal cells of the potentially zoopathogenic fungus Basidiobolus haptosporus characteristically exhibit unusual proportions of annulate views of mitochondria in the two-dimensional perspective of thin sections. Such views exhibit a central space containing cytoplasmic ground substance and often profiles of other cytoplasmic organelles (lipid bodies, other mitochondrial forms, and especially crystalloid-containing microbodies). Three-dimensional projections are presented to suggest that these mitochondria have assumed the form of a goblet-shaped enclosure, and that the various annulate views are the consequence of plane of section viewed by electron microscopy. Their frequent occurrence and consistent morphology argues against their being random expressions of mitochondrial plasticity, but rather for close spatial associations amongst cytoplasmic organelles of young hyphae. When the fungus is grown on xanthine or its catabolites as sole sources of nitrogen, there is a proliferation of crystalloid-containing microbodies, double-membraned vesicles, and ovate to ellipsoidal mitochondria. Annulate views of mitochondria then are no longer observed, but microbodies again frequently appear in close association with mitochondria and at times in intimate contact with the mitochondrial outer membrane.     

Staining of xylem parenchyma mitochondria with photo-oxidized 3,3'-diaminobenzidine

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.