Phase Contrast Optics Research Articles

Studies on the structure and hatching of the eggs of Schistosoma mansoni.

The structure and function of the vacuoles in the eggs of Schistosoma mansoni is unknown. In this study the eggs have been immersed in serum albumin and examined by phase-contrast optics. Under these conditions the vacuoles exhibit a definite internal structure, suggesting that they may be or may have been cellular. Histochemical staining techniques to detect the nature of the vacuolar contents revealed that the vacuoles stained heavily with iodine and gave a positive PAS reaction. This suggested the presence of carbohydrate. Stains for nucleic acids, protein and lipid were not taken up by the vacuoles. The egg shell stained with o–toluidine blue and with basic fuchsin uniformly, except for a thin irregular band which might indicate a possible line of weakness. The vacuolar membranes seem to be similar to the vitelline membrane in permeability to hydroxyl ions and it is possible that these membranes have a common origin. Plasmolysis studies with salts, urea, glycerol and sucrose showed that the vacuolar membranes have about the same permeability properties to these compounds as the other egg membranes. Glycerol treatment of the eggs until equilibrium is attained, followed by transfer of the eggs to water or saline results in a hatching of the eggs. In this, a dead mira-cidium is extruded from the shell. Similar treatment of eggs equilibrated with sucrose does not result in this hatching process. It is postulated that glycerol solubilizes a factor which aids the hatching process.I should like to thank the following: Dr S. A. Ibrahim, in whose department this work was carried out; Dr S. Dawood of the Stack Laboratory, Khartoum, who kindly made his microscope available for use; Mr J. R. Lauder for valuable discussions and suggestions; and many young patients in Khartoum Civil Hospital for stool samples and smiles.

Cytoplasmic fibres associated with streaming and saltatory-particle movement in Heracleum mantegazzianum

The giant cells of the petiolar hairs of Heracleum mantegazzianum contain cytoplasmic fibres that are readily visible with phase contrast or interference contrast optics. These fibres are associated with saltatory particle movement and protoplasmic streaming in this cell and are believed to be the living image of similar fibres reported in electron micrographs of a variety of cell types.

The structure of the major cell processes of isolated BHK21 fibroblasts

Abstract The structure of the major cell processes of BHK21/C13 fibroblasts was examined by phase contrast, differential interference and polarization optics and by the electron microscopy of thin sections and whole cell replicas. In living cells, the optical studies revealed a birefringent streak extending along the length of the processes. In fixed cells, in addition to endoplasmic reticulum and filamentous mitochondria, microtubules and smaller tubular filaments were seen within the cytoplasm and bundles of microfilaments just under the cell membrane. The latter structures could be correlated with oriented, fibrous material seen in the whole cell replicas. The microtubules and filaments were frequently associated with each other. Possible functions for these filamentous structures in producing and maintaining cell processes and in intra- and extracellular motility are proposed and the relationship of these structures to the birefringence seen in living cells is discussed.

Microelectrophoresis apparatus employing palladium electrodes

Abstract Microelectrophoresis apparatus for the electrokinetic study of suspended particles has been improved by the use of a novel palladium electrode system. These electrodes, suitably charged with atomic hydrogen by cathodic electrolysis, permit high current densities without evolving gas, and under prescribed operating conditions do not contaminate the suspensions under study with heavy metal ions or other electrode reaction products. Other features of the apparatus described are a precision-built, laterally oriented, flat observation chamber, phase contrast optics, a constant-current power supply, and a water bath for rapid dissipation of ohmic heat and maintenance of constant temperature. The simplicity of the electrodes makes them easily adaptable to any type of observation chamber.

Changes Associated with Parasitism in Nematodes. IV. Cytochemical Studies on the Ampulla of the Dorsal Esophageal Gland of Meloidogyne javanica and on Exudations from the Buccal Stylet

The ampulla of the dorsal esophageal gland and exudations from the buccal stylet were examined in both living and fixed nematodes by various cytological techniques. A pronounced ampulla is found only in females. It is packed with granules which are approximately 0.4 to 0.6 u in diameter. By means of histochemical tests and examination with a UV microspectrograph they have been shown to consist predominantly of protein. Living females, when dissected from roots and placed in distilled water, produce a stylet exudate within a few hours. The use of a perfusion slide enabled this precipitate to be tested with various chemical reagents including stains and enzymes and it was shown to consist of basic proteins containing carbohydrates. Neither lipids, nucleic acids nor enzymes were detected. The possible role of these basic proteins in the host-parasite relationship is discussed. Previous papers in this series (Bird, 1967; Bird and Saurer, 1967; Bird, 1968) have been concerned with changes taking place in the subventral esophageal glands during the initial stages of parasitic development. No changes were detected in the dorsal esophageal gland or its ampulla near the base of the spear during this transition. It is clear, however, from the work of earlier investigators (Linford, 1937; Rhoades and Linford, 1961; Hechler, 1962; Anderson, 1964; Doncaster, 1966; and Nickle, 1967) that the ampulla of the dorsal esophageal gland is not only closely associated with feeding in stylet bearing nematodes but that its granular contents appear to be morphologically similar among widely divergent genera. As yet there is little information on the chemical nature of either the material in the ampulla or the oral precipitate which exudes from the stylets of plant parasitic nematodes. However, in Meloidogyne javanica, Bird (1964) has shown that these oral exudates are antigenic and are thought to consist of a number of proteins. Further information on the chemical nature of these structures is required if their role in the establishment, development and maintenance of the tumorlike giant cells on which these nematodes feed (Bird, 1961; 1962) is to be understood. In this paper the chemical compositions of the contents of the ampulla of the dorsal esophReceived for publication 14 May 1968. ageal gland and the stylet exudate are examined by various techniques in an effort to clarify at least some aspects of M. javanica's complex host-parasite relationship. MATERIALS AND METHODS Preparasitic and parasitic larvae were obtained by methods described previously (Bird, 1967). Females were dissected from galls in tomato roots using a stereo microscope and males were collected in tubes by sedimentation after they had migrated from tomato roots in a thermally controlled mist chamber. Living larvae and males were photographed at high resolution under anesthetic (Bird 1967). However, living females were not anesthetized because of their relative immobility but were examined with the coverslip raised slightly by means of a fine brass ring to prevent crushing. All specimens were photographed with the aid of electronic flash using either oil or water immersion objectives with normal transmitted light optics or phase contrast. Observations on both the material exuded from the tip of the buccal stylet in living females and the influence of various chemicals on these precipitates were made possible by the use of a perfusion chamber (Paul, 1957) and phase contrast optics incorporating a long-focal-length condenser. Pepsin (Calbiochem) was made up as a 0.1% solution in 0.1 N hydrochloric acid, trypsin (both BDH and Mann Research Labs) as a 0.1% solution in phosphate buffer (both pH 7.3 and 8.0) and papain (Calbiochem) as a 0.1% solution in phosphate buffer (pH 6.0). After fixation in 4% buffered formaldehyde (Bird and Saurer, 1967) both larvae and adults were examined and photographed under a Leitz microspectrograph, the spectra being recorded and scanned as previously described (Bird and Saurer, 1967).

Responses of a psychrophilic marine bacterium to changes in its ionic environment.

A psychrophilic marine bacterium was stable in cold seawater or in a solution containing 0.1 M Mg++ and 0.5 M NaCl. In water and in a number of monovalent salts, including up to 3.0 M NaCl, turbidity of suspensions fell and some intracellular material was released into the external medium. The cells first became swollen and pale under phase contrast optics and then disintegrated into amorphous aggregates. Divalent salts alone, some in very low concentrations, could maintain cell structure. Effectiveness was in the order Cu++ > Zn++ > Ni++ > Ca++ > Mn++ > Mg++. The polyamine, spermine, was ineffective. In the presence of 0.1 M Mg++, NaCl and, less effectively, LiCl and RbCl could maintain structure, but KCl, NH4Cl, CsCl, glucose, and sucrose could not. Sodium phosphate, pH 7.0, caused lysis but other sodium salts maintained structure in the presence of Mg++. For growth, Mg++, NaCl, and traces of other ions were required.Phosphatidases attacking cells at 25 °C and higher required salts for maximum activity. The turbidity of water-lysed cells or of envelopes prepared from mechanically broken cells fell at 25 °C and higher; this fall was also dependent on the presence of salts. If cells were lysed in distilled water, or broken mechanically in the presence of salts, more than half their lipid phosphorus and hexosamine was released from material sedimenting after 30 min at 15 000 × g but these materials were not degraded into other substances. The released material seemed bound to smaller particles, since most of it sedimented after 1 hour at 100 000 × g. The lipid composition of smaller and larger particles, and of whole cells, was the same.

Time-Lapse Cinemicrography of Tissue Cultures Utilizing Phase-Contrast Optics: Design and Construction Considerations

The problems of time-lapse cinemicrography of cell cultures differ greatly from those of still photomicrography of fixed preparations. The cost of commercial equipment for a complete setup is too high for the small laboratory. Utilizing existing equipment, some purchased components and some equipment constructed by the engineering department of the Isaac Albert Research Institute, a time-lapse unit was assembled. Available components and the specially designed repetitive electronic flash unit, incubator and pump are discussed.

Ultrastructural studies on the spermatozoa of two primitive crustaceans Hutchinsoniella macracantha and Derocheilocaris typicus.

Spermatozoa of two primitive crustaceans have been examined with phase contrast and electron optics. The organisms are Hutchinsoniella macracantha of the subclass Cephalocarida and Derocheilocaris typicus of the subclass Mystacocarida. The H. macracantha sperm consists of a compound acrosome, a nucleus and a rod-like structure that extends from the base of the acrosome, through a nuclear canal to project posteriorly. Although this posterior projection seems to contain fibrils, it clearly lacks the characteristic “9 doublets + 2” microtubular structure of sperm flagella. No mitochondria or centrioles were found and sperm motility was not observed. The D. typicus sperm consists of a trough-shaped acrosome, a nucleus, mitochondria and a flagellum. The flagellum and two lateral rows of mitochondria, as well as the nucleus, arise at the base of the acrosome. These relationships impart a bilateral symmetry to the sperm. Mature D. typicus spermatozoa are stored in spermatophores in the vas deferens. These spermatophores are tubes each containing two spermatozoa. The spermatozoa in these tubes are oriented with a high order of precision. The ends of the spermatophores are closed by plugs with complex ultrastructure. No indication has been found as to the site or mechanism of enclosing the spermatozoa in the spermatophores. The sperm morphology is discussed in terms of crustacean phylogeny.

Development of acoustic ganglia in tissue cultures of embryonic chick otocysts

Abstract Otocysts with the associated acoustic ganglion of 4-day-old chick embryos were cultivated in Rose's multipurpose culture chambers under a dialysis membrane. The ganglion cells underwent maturation which was characterized by the nucleus becoming centrally located and by the development of Nissl substance which was distributed throughout the cytoplasm. As the ganglion cells matured the nerve fibers became enveloped in Schwann cells, thickened, and subsequently myelin-developed. Myelin was demonstrated as a refractile sheath with phase contrast optics, as birefringent with polarizing optics and could be stained with Sudan black B. Myelinated axons had well formed nodes of Ranvier and incisures of Schmidt-Lantermann. A phase of degeneration affecting a few ganglion cells occurred when a majority of the neurons reached maturity. Most of the cells were restored to health and stability but a few cells degenerated completely. The factors involved are discussed. Results obtained with four different media show that the best of these supported cytodifferentiation, myelination and maintenance. Other media supported only cytodifferentiation and maintenance but did not allow adequate myelin formation.

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance. In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as “nucleolar shadow”, overlaid by a few small irregular bodies of higher electron density than other nuclear contents. The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas. In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed. The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.

Structure of Trichomonas gallinae (Rivolta)

Morphology of Trichomonas gallinae is described from three strains maintained in axenic cultures. The employment of protargol for staining of permanent preparations and of phase-contrast optics in the observations of living organisms was found most helpful in interpretation of many hitherto inadequately understood structural details. The first of the aforementioned methods was particularly valuable in the study of the parabasal apparatus and of the anterior part of the axostyle; the second proved most useful in the study of structural details and motion of living organisms. Trichomonas gallinae (Rivolta) occurs in the upper digestive tract and various organs of different avian groups, but it is particularly common in columbids. The morphology of the flagellate has been described by several investigators; however, the most significant contributions to the understanding of its structure and biology were made by Stabler (1941, 1947, 1954), whose papers include, in addition to significant original findings, reviews of the older works and lists of the important references to the literature. In their morphological account of Trichomonas tenax (0. F. Miller), the oral flagellate of man, Honigberg and Lee (1959) pointed out the need for investigation, with the aid of protargol, of the two remaining species in the genus Trichomonas Donn6: T. vaginalis Donn6 and T. gallinae (Rivolta). A report by Honigberg and King (1964) on T. vaginalis has already been published. This latter investigation indicated the need for further implementing the study of small trichomonads through the extensive employment of phase-contrast microscopy in observing the living organisms. The present paper deals with the morphology of T. gallinae as revealed with the aid of the two aforementioned methods in addition to iron hematoxylin staining used by many previous writers. As anticipated, the employment of protargol technique and of phase-contrast microscopy elucidated several imperfectly understood structural details and helped in finding additional valid criteria for differentiation among the species of the genus Trichomonas. MATERIALS AND METHODS Three strains of Trichomonas gallinae were used: Jones' Barn (JB), Lahore (YG), and Kupferberg (TG). The two former strains were isolated originally by Dr. Robert M. Stabler and the latter by Dr. A. B. Kupferberg (for detailed histories of all strains, see Honigberg, 1961). All strains were maintained at 37.5 C in axenic cultures either on simplified trypticase (Kupferberg, Johnson, and Sprince, 1948) or on BBL fluid thioglycollate medium, both adjusted to pH 7.0 to 7.5 and containing 5% normal horse serum. Inasmuch as only single isolates of YG and TG strains were available, these were employed in making permanent preparations as well as in the observations of living material. August 1956 and July 1958 isolates of the JB strain were used for most of the permanent preparations, and the November 1962 isolate served in the study of living organisms. In addition to being examined in permanent films derived directly from cultures, the flagellates were studied also in fixed and stained smears of the contents of 14-day subcutaneous lesions resulting from inoculations of the cultures into C57iBL6J mice (Honigberg, 1961; Frost and Honigberg, 1962). All observations of living organisms were made with the aid of darkand bright-phase contrast. In taking photomicrographs of such organisms, dark-phase contrast and electronic flash (300 watt seconds) were employed. Permanent preparations were stained with iron hematoxylin or iron hematein (method of De Freitas, 1936, modified by Honigberg, 1947) after fixation in weak Flemming's or Bouin's fluids. Fixation in Bouin's or Hollande's fluids always preReceived for publication 29 May 1964. * This investigation was supported by Research Grant AI-00742 from the National Institute of Allergy and Infectious Diseases, Public Health Service. t Permanent address: Department of Zoology, Osmania University, Hyderabad, India.

NUCLEOLAR ORGANELLES SHOWN BY LEAD PRECIPITATION IN UNFIXED CULTURED CELLS.

Unfixed coverslip cultures of HeLa cells or of mouse ascites tumor cells are placed in a 4 mM solution of lead acetate in Michaelis buffer, pH 5.5, for 30 min at 37 C. After a thorough water wash, brief treatment with 1% aqueous ammonium sulfide exhibits multiple (1-20) dark brown granules within each nucleolus. The remainder of the nucleolus is bright yellow, and other parts of the cell are unstained. The nucleolar granules thus visualized show strict correspondence in sue and distribution to the granules of ribonucleoprotein stained by toluidine blue-molybdate, and also to the clear spaces seen in the nucleolus of living cells under phase-contrast optics. Fixation, or heating to 90 C, prevents the staining by lead but not by toluidine blue-molybdate. The reaction to the lead salt is pH dependent; optimum about 5.5. These features suggest that the retention of lead in localized areas of the nucleolus is due to enzymatic reactions which produce inorganic phosphates from endogenous substrates.

Nuclear structure of human spermatozoa.

Van Duijn states that Shettles claims that discrete chromosomes can be demonstrated in dried unstained smears of human spermatozoa and that X and Y-bearing types can be morphologically distinguished are not true. He states that Shettles had misinterpreted a number of optical artefacts. Objects that are not completely flat exert a focusing action like tiny lenses so that an image can be produced of another object at some distance. In spherical particles there is not much distortion whereas in structures of cylindrical or irregular curvature distortion occurs. In phase-contrast studies a spurious image may be produced. Other factors of technique are also involved in producing the effects shown by Shettles. With properly adjusted phase-contrast optics there is no difficulty in observing dried unstained spermatozoa with oil-immersion objectives. Presence of 2 distinct types of different shape and size has been observed by others in abnormal ejaculates but it is considered unlikely that these should bear any ratio to sex relationship. Simple drying does not produce discrete chromosomes which can be demonstrated by electron microscopy. The Feulgen-positive material remaining after an extraction with concentrated acetic acid and special staining methods confirms that in the sperm nucleus the chromatin material is in a condensed karyosome form controverting Shettles statements. Shettles in a reply following states that in thin-dried smears of normal spermatozoa from more than 200 men of proved fertility the 2 populations have been seen as reported. Chromosomal patterns of the 2 types are illustrated. The chromosomes are dumbbell in outline and touch end-on like beads in a necklace. In some heads of flattened spermatozoa all 23 chromosomes have been seen. On breaking the thick membrane of the spermatozoan head the Feulgen-positive discrete separate dumbbell shaped chromosomes may be seen. An exact pattern of the Feulgen-positive chromosomes has been observed for each type of spermatozoan head.

Ultraviolet irradiation of small portions of nerve cell processes in tissue culture

Abstract The Uretz ultraviolet microbeam provides an effective and convenient probe for experiments on cultures of nervous tissue, because of its accuracy and the ease with which the size and shape of the beam may be varied. In cultures of adult Amblystoma spinal ganglia, 2-μ segments of small neurites were exposed to a microbeam for 10 to 60 seconds. Time-lapse motion pictures of subsequent changes were made, using medium dark, phase-contrast optics. Exact irradiation dosages were not determined because accurate measurements of the distance of the processes from the quartz cover slip could not be made, but when irradiation of adequate intensity reached the neurite, the portion distal to the target site disintegrated. When observation was continued beyond 24 hours the new termination resumed growth. Survival of the distal portion and repair of the break after complete interruption of the process, as observed by Levi and Meyer in chick embryo cultures, was never seen. In some experiments bombardment was followed by extreme thinning of the process, requiring good phase-contrast optics to observe the continuity of the fiber. Distal to the attenuated region the process developed irregular enlargements, which moved longitudinally but not in a continuous distalward flow. This tendency for motion to occur in either direction was also characteristic of beadlike enlargements of processes, both before and after irradiation. If such irregular to and fro activity has any correlation with the apparent distal movement of axoplasm reported by Weiss and Hiscoe after constricting nerve trunks, these experiments do not offer an explanation of its significance.

Observations on the cytoplasmic membranes of testicular cells, examined by phase contrast and electron microscopy.

In freshly isolated cells of the guinea pig germinal epithelium examined with phase contrast, dark contours are seen in the cytoplasm that appear to be optical sections of the cisternae of the endoplasmic reticulum. These increase in contrast, in number, and in linear extent with increasing time up to 4 hours after isolation of the cells from the testis. During this period, cisternae originally present in the cells are extended and new ones appear to be formed by coalescence of tubular and vesicular elements of the reticulum. The cisternae become associated in parallel array and ultimately form elaborate concentric systems resembling structures that have often been interpreted as intracellular "myelin figures." Until now our knowledge of the endoplasmic reticulum has been based largely upon electron micrographs. The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.

Action of ribonuclease on living cells in vitro and synthesis of deoxyribonucleic acid.

THE action of ribonuclease on living cells from various materials has recently been studied1,2. Chick fibroblasts and myoblasts in vitro are readily affected; growth of the cultures and the number of mitoses are considerably reduced2; cytoplasmic and nucleolar basophilia is diminished or suppressed and a typical modification of the chromatin, seen both with phase-contrast optics in living cells and on fixed preparations, is observed. Many nuclei (up to 95 per cent when the action of ribonuclease has been particularly intense) contain granular, or granular and filamentous, material or even are prophasic in appearance3. Even when many nuclei are showing this prophasic aspect, few—if any—other mitotic stages are encountered.

Electrophoretic Mobility of Red Cells and Their Ghosts as Observed with Improved Apparatus*

ABSTRACT This paper is concerned with the application of two new techniques, both of which depend essentially on improved instrumentation, to the study of the electrophoretic mobility of red cells and their ghosts. The first technique has been developed to overcome difficulties which are encountered in the measurement of the mobility of ghosts. If the ghosts are substantially haemoglobinized, they can be seen, although with some difficulty, with the ordinary microscope and their mobility can be measured in an electrophoresis cell such as that described by Abramson (1934); if they are relatively Hb-free, on the other hand, they are almost invisible under the ordinary microscope, and can be seen satisfactorily only with phase contrast. The great majority of existing measurements of the mobility of ghosts, accordingly, are measurements of the mobility of ghosts which contain relatively large amounts of Hb. The new vertical cell to be described uses phase-contrast optics, and is fitted with removable electrodes which have several advantages over those usually employed. The second technique is the result of the observation that the Antweiler electrophoresis apparatus with a Philpot-Svensson attachment, although designed for the separation and observation of the mobilities of protein fractions, can also be used for measuring the relative mobilities of red cells and their ghosts.

Mitochondria in Allomyces under experimental conditions

Abstract 1. 1. The vegetative hyphae of aquatic fungi are described as favorable material for the study of mitochondria in the living state, particularly when observed with phase contrast optics. 2. 2. Immobilization of mitochondria without morphological change was realized by treatment of hyphal cells with 1 per cent solution of OsO 4 . 3. 3. When hyphae were mounted in isotonic NaCl, the mitochondria, being as sensitive to mechanical shock as they were when the hyphae were in water, disintegrated under pressure. In hypertonic saline, cells were at first plasmolysed, but recovered. Mitochondria in plasmolysed cells changed shape, fragmented, became vesiculated, and disappeared after becoming pale and non-refractive. 4. 4. In cells treated with 0.88 M sucrose, which has been useful in isolating mitochondrial particles from animal tissues, the fungal mitochondria quickly fragmented into spheres about 1 micron in diameter. 5. 5. A solution of 10 −2 M KCN in 10 −2 M glutamic acid, at pH 7.0, caused cessation of movement of mitochondria and inhibited staining by Janus green B. No evidence of mitochondrial damage was apparent. 6. 6. Temperatures of 8 °–45 ° C. had no visible effect on mitochondria. At higher temperatures, the results were as follows: 50 ° C., mitochondria became slender, and almost invisible; at 65 ° C., all mitochondrial motion ceased; at 75 ° C., mitochondria broke up into beads which became hollow spheres and disappeared. 7. 7. Mitochondrial orientation is determined more by the direction of protoplasmic streaming than by direction of diffusion. 8. 8. Small granules, never positively identified as normal mitochondria, were “stained” red after 48 hours with triphenyl tetrazolium chloride when the salt was incorporated in the medium at a concentration of 0.005 per cent. Higher concentrations were lethal. The sites at which reduction of the tetrazolium was most active were in the fat droplets. 9. 9. Because of the diversity of behavior and function of the particles referred to as mitochondria, it is suggested that many cytoplasmic particles, even though they do accumulate Janus green, are scarcely comparable to one another.