A comparison of various procedures for fine grain development in electron microscopic radioautography.

Fine grain development for electron microscopic radioautography was investigated with two types of radioactive specimens: sections of tritiated methacrylate, which provide a homogeneously labeled source for quantitative evaluation of the radioautographic reaction, and sections of 125I-labeled thyroid. Radioautographs were prepared with Ilford L4, Sakura NR-H2, Agfa-Gevaert NUC 307 or Kodak NTE emulsions. The radioautographs were developed with one of several "solution physical" development procedures (Agfa-Gevaert, phenidone-ascorbic acid, p-phenylenediamine developers) or with arrested "direct" developments (D-19b, Elon-ascorbic acid developers). By arresting each development at an early stage of the reaction and at progressively longer time intervals, it was possible to examine the sequence of shapes in the growth of developed silver deposits for each emulsion-development combination. Thus, conditions which resulted in the development of small, round, compact silver deposits were defined for each emulsion. These developments were used in conjuction with gold latensification, a treatment which increases the sensitivity of the emulsions and thus compensates for the lowered sensitivity of fine grain development procedures. The location of the silver deposits in relation to the silver bromide crystals from which they derive was investigated. The emulsion gelatin surrounding the crystals was stained whereas the spaces, which remained after the crystals were dissolved in the photographic fixer, appeared transparent. This analysis permitted the selection of development procedures in which the single or multiple round silver deposits originating from a single crystal will remain within or on the boundary of this crystal. By this method, quantitation of radioautographic reactions composed of small, round silver deposits was studied by using the uniformly labeled 3H-methacrylate sections as a standard source of radiation. The conditions under which grain counting is feasible are discussed.     

ABSOLUTE SENSITIVITY OF ELECTRON MICROSCOPE RADIOAUTOGRAPHY

A calibration method is described for measuring absolute radioautographic sensitivities under various experimental conditions. Sensitivities to ³H and ³⁵S radiation, i.e. ratio of developed grains to radioactive decays in the specimen, were determined with Ilford L4 and Kodak NTE emulsions. The highest values obtained in monolayers of emulsion were ⅛ for ³H and 1/21 for ³⁵S. The influence of various experimental parameters on sensitivity is described, and the possibilities for quantitative electron microscope radioautography are discussed.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF THIN SECTIONS: THE GOLGI ZONE AS A SITE OF PROTEIN CONCENTRATION IN PANCREATIC ACINAR CELLS

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (~ 600 A) of OsO₄-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H³ injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.     

Autoradiography of Tracks from Beta-Particle Emitters in Tissues

Mounted paraffin sections, 2-4μ thick, ˙were stained, dehydrated, allowed to air dry, and given a thin coating of 1 % Plexi-glas solution in chloroform. The chloroform was allowed to evaporate completely in a dry atmosphere. An emulsion whose dried thickness was 100-150μ, was prepared from Ilford G5 type in gel form and glued to the section by means of a 15% solution of shellac in absolute alcohol. The surface of the emulsion was then cleaned with absolute ethyl alcohol, to remove the impermeable shellac layer. The exposure for radiation reaction was made at about 2°C and required, in the conditions of our experiment, about 24 hrs. The emulsions were processed by the “temperature-development method.” With the described procedure, autoradiographs have been obtained of various organs of albino rats, labeled with P³², S³⁵ and other radioisotopes, and very precise localizations of the origin of electron tracks was attempted. This technic has allowed the fixing and staining of the tissues by means of all the reagents commonly employed in histology, without any damage to the emulsion and the obtaining of good adhesion and minimum separation between specimen and emulsion, thus permitting reliable extrapolations of electron tracks. Due to the fact that the emulsion is fully sensitized when placed in contact with the preparation the limits of the exposure times were well defined. The uniform development at all depths of the emulsion achieved by the temperature-development method facilitated the work with fast electron tracks.     

Effect of oxygen and storage conditions on the metabolic activities of polychlorinated biphenyls dechlorinating microbial granules

The effect of oxygen and storage conditions on the metabolic activities, measured by volatile fatty acid (VFA) degradation and methane production, and by the dechlorinating activity of methanogenic polychlorinated biphenyl (PCB) granules, were studied. Incubation of the granules in air for different periods did not result in significant inhibition in volatile fatty acid degradation and methane production activities. The inhibitory effect of oxygen increased with increased length of exposure. The overall methanogenic activities, however, recovered after a 10-day incubation period in the absence of oxygen. Oxygen exposure did not cause any significant effect on the dechlorinating activity of the granules tested with a PCB mixture, Aroclor 1254. In 6 months, approximately 80% [based on the concentration (M) of chlorine removed] of the Aroclor 1254 was dechlorinated even by granules exposed to oxygen for 168 h. Granules stored at room temperature (20° C) appeared to be more active compared to the granules stored at 4° C C or –20° C. Similarly, granules stored with a nutrient mixture, containing methanol, glucose and yeast extract showed higher metabolic activities. Our results demonstrate that the effect of oxygen exposure was not significant and was reversible. PCB granules could be stored at room temperature with an auxiliary carbon source in the presence of PCB without significant loss of activity. These properties make methanogenic PCB granules suitable candidates for practical use in PCB dechlorination and biodetoxification.     

Assessment of resolution by half distance values for tritium and radioiodine in electron microscopic radioautographs using Ilford L4 emulsion developed by “Solution Physical” or D-19b methods

Summary Half distance values for electron microscopic (EM) radioautographs with the isotopes³H and¹²⁵I were determined using Ilford L4 emulsion processed with either fine grain, solution physical development, or filamentous grain, chemical development with D-19b.³H- and¹²⁵I-line sources, obtained by cutting perpendicular sections from sections of³H-labeled methacrylate or¹²⁵I-labeled thyroid glands, were processed for EM radioautography. The distribution of silver grains around a line source was determined by measuring their distance from the source in photographs of EM radioautographs. The number of silver grains per unit distance from the line source was plotted on graphs and half distance values were calculated. With solution physical development, the half distance value was 76 nm for³H and 80 nm for¹²⁵I; whereas with D-19 b development it was 187 nm for³H and 157 nm for¹²⁵I. Since solution physical development produced a reduction of about 50% in the half distance values for both isotopes, it is concluded that the production of fine grain by this method provides better resolution for EM radioautography than filamentous grain development with D-19 b. This work was the subject of a McGill University dissertation (Levine 1977)     

Characteristics of three nuclear emulsions for autoradiography at the electron microscope

Summary On account of their low grain size three commercial emulsions, Gevaert NUC 307, Ilford L4 and Kodak NTE have been investigated to assess their qualities for electron microscope microautoradiography. Grain size distribution curves were determined and a developer suitable for microautoradiography was selected after having tested different types of developers.In order to investigate the sensitivities of the three emulsions, monolayer preparations were irradiated in the electron microscope, using an energy of 5.7 keV corresponding to the mean -energy of tritium. After exposure the specimens were developed but left unfixed. The sensitivity may then be determined using the percentage of developed grains. For the formation of one latent image the Ilford L4 emulsion must be hit on the average by 1 – 1.4 electrons per AgBr grain; the corresponding figures for Gevaert NUC 307 and Kodak NTE are 2 – 3 and 4 – 5 respectively.The problem of resolution of point and plane sources of radioactivity is discussed.Future advances in microautoradiography will depend on the development of emulsions with lower grain sizes, but such improvement must not be at the expense of sensitivity.

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Zusammenfassung Drei handelsübliche Kernspuremulsionen, Gevaert NUC 307, Ilford L4 und Kodak NTE, wurden wegen ihrer geringen Korngröße auf ihre Eignung zur elektronenmikroskopischen Autoradiographie untersucht. Korngrößenverteilungskurven wurden aufgenommen und ein geeigneter Entwickler ausgesucht.Zur Bestimmung der Empfindlichkeit dieser drei Emulsionen wurden Einkornschichten im Elektronenmikroskop mit Elektronen einer Energie von 5,7 keV, der mittleren -Energie des Tritiums, bestrahlt. Anschließend wurden die Emulsionen entwickelt, aber nicht fixiert. Mit dem Anteil der entwickelten AgBr-Körner kann dann über Trefferkurven die Empfindlichkeit der Emulsionen bestimmt werden.Man benötigt zur Bildung eines latenten Bildkeimes für die Ilford L4-Emulsion 1 – 1,4 Elektronen pro AgBr-Korn, für die Gevaert NUC 307-Emulsion 2 – 3 und für die Kodak NTE-Emulsion 4 – 5 Elektronen pro AgBr-Korn.Folgerungen für das Auflösevermögen bei radioaktiven Punkt- und Flächenquellen werden diskutiert.Fortschritte in der Mikroautoradiographie werden von der Entwicklung feinkörniger Emulsionen abhängen, deren Empfindlichkeit bei etwa einem Elektron pro AgBr-Korn liegen sollte.

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Parts of the paper have been presented at the XI. International Congress of Radiology, Rome, September 1965.

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The authors express their gratitude to Professor Dr. B.Rajewsku for his support of this investigation. Thanks are also due to Dr. W.Lippert for valuable discussions, and to Miss W.Friese and Miss S.Unger for technical assistance.     

Biochemical and metabolic effects of a six-month exposure of small animals to a helium-oxygen atmosphere

Rats and mice were exposed for periods of up to six months and two successive generations of mice were raised in a ground-level chamber system filled with 80% helium –20% oxygen, at 24°C. A duplicate chamber for controls contained a comparable nitrogen-oxygen mixture, and in both the other environmental parameters were well-controlled and nearly identical. Animals adapted to helium showed no greater increase in oxygen consumption (P>0.05) when placed in helium-oxygen than did those raised in air. Growth rates were identical, but the helium mice consumed more food and water.Selected biochemical analyses were made on the parent and two successive generations of mice. These included blood indices; electrophoretically separated tissue protein patterns from liver, skeletal muscle, and cardiac muscle; quantitative determinations of LDH, MDH, and G6PDH from the same tissues; serum insulin; and semi-quantitative histochemical estimates of liver glycogen. No cases of statistically significant difference or consistent trends were seen between the experimental environmental groups. Additional analyses of liver nucleotides and redox-coenzymes also failed to show a significant difference.The relative weights of liver, heart, kidney, and diaphragm (wet and dry) were the same in both groups. Histopathological examination of kidney and adrenal tissue produced unremarkable findings and none that were attributable to the nature of the gaseous environment.It must be concluded that prolonged exposure to helium-oxygen, relative to air, does not produce detectable changes in several key subcellular factors which might be altered by serious metabolic disturbances, and therefore the helium exposure is well tolerated.Prepared under Contract No. NAS 2-3900 for Union Carbide Corporation, Linde Division Research Laboratory, Tonawanda, N.Y. for Ames Research Center.     

Tracing neuronal connections with radioisotopes applied extracellularly.

Methodological and technical problems of the autoradiographic-neuroanatomical tracing (ARNT) technique are discussed. The size of the labeled area after a tritiated amino acid injection varies directly with the volume of isotope, the rate of injection, and the length of exposure of the secretion to the emulsion. Frozen sections can be used for autoradiography if they are mounted on subbed slides, dehydrated in ethanol, defatted for 1 hour in xylene, rehydrated through ethanol and water, and dried before coating with emulsion. Brushes used for mounting frozen sections should be used only for this purpose and dipped in boiling distilled water before use to avoid chemoreduced streaks in the emulsion due to contamination from the brushes. Excessive dilution of Kodak emulsion can leave less than a monolayer of grains over certain types of sections; the emulsion thickness should be checked by exposing coated test sections to a brief flash of light and developing immediately. A high intensity safelight recommended for the ARNT darkroom is the Thomas Duplex Super monochromatic sodium vapor bulb safelight; for Kodak NTB-2, -3 and Ilford L4 emulsions the red-banded and yellow-banded filters are used. A useful stain combination for ARNT is Luxol fast blue stained before coating and cresyl violet stained after developing which demonstrates both neuronal cell bodies and myelinated tracts in the same section.     

Preliminary results in improving essential fatty acids enrichment of rotifer cultured in high density

High density cultured marine rotifer (Brachionusrotundiformis) fed on freshwater Chlorella wascultured secondarily with an emulsion of ethyl esters(65% DHA, 15% EPA) for EFA-enrichment, with orwithout freshwater Chlorella. Culture tanks (30 l, 20 lworking volume) set in a water bath (24 °C)were continuously supplied with air (2.50 lm^–1) or high-purity oxygen gas (1.25 lm^–1). Samples for fatty acid compositionanalysis were collected at 0, 6, 12, and 24 hoursafter the inoculation of rotifer, followed by themonitoring of rotifer density, DO, NH₄-N and pH.The n-3 HUFA content plateaued at 6 hours afterthe onset of the secondary culture and no significantdifferences were observed afterwards, regardless ofthe aeration methods. The HUFA content increased withdecreasing amounts of Chlorella added. Thehighest content (ca 3.5% on a dry basis) was notedin non-Chlorella feeding groups. When largeamounts of Chlorella were fed, the DO of culturewater drastically decreased and the NH₄-Nconcentration increased. The results from theexperiment indicate that the presence of Chlorella cells greatly affect the HUFA intake ofrotifers during the secondary culture process. Alsothe supply of high-purity oxygen gas was effective forpreventing a culture crash of rotifers during EFAenrichment.     

YdfH Identified as a Repressor of rspA by the Use of Reduced Genome Escherichia coli MGF-01

The effects of carnauba wax addition on the physical state of palm kernel oil-in-water emulsions were investigated. The oil-in-water emulsion (40 wt% oil + 60 wt% aqueous phase) kept the liquid state at 25°C irrespective of the presence or absence of carnauba wax in the oil phase. The emulsion containing the wax transformed from the liquid state to the solid state by shearing after storage for 20 h at 4°C, although the liquid-solid transition was not observed for the emulsion not containing the wax upon the same treatment. The viscoelasticity of the solid emulsions was demonstrated by small-deformation mechanical testing. Analysis of flow behavior of the emulsions showed that the change in physical properties of the emulsion containing the wax at 4°C was caused by the shearing at a low shear rate, around 50 s^?1–100 s^?1. According to the transition from the liquid state to the solid state of the emulsion containing the wax, the aggregation of oil droplets was found to occur to a large extent. The results of differential scanning calorimetry and surface pressure–surface area isotherms suggested that triglyceride molecules of palm kernel oil were more oriented at the oil–water interfaces in the emulsions after the wax addition. Based on these results, it is thought that carnauba wax is important in destabilization of palm kernel oil-in-water emulsions by modifying the physical state of the oil triglyceride molecules at the interfaces.     

A method for the use of Zenker-formol fixation and the periodic acid Schiff staining technique in light microscopic radioautography

Summary Although providing superior histological preservation, Zenker-formol fixation has not been utilized in radioautographic experiments, since some component of the fixative (presumably mercury) desensitizes the emulsion and thus prevents the appearance of a radioautographic reaction. Attempts to identify and eliminate the effects of this desensitizing agent led to experiments with Zenker-formol fixed tissues from rats injected with ³H-leucine or ³H-thymidine. Radioautographs of such tissues contained a brown-black precipitate and occasionally a heavy background fog, but no normal radioautographic reaction. Moreover, when the radioautographs were exposed to light, the emulsion was transparent over the tissues, indicating that a tissue-bound component had completely desensitized the emulsion. The two components of Zenker's fluid-mercuric chloride and potassium dichromate-plus the mercurous chloride which forms as a precipitate in the course of fixation, were then tested for their individual effects on the radioautographic emulsion. It was found that mercuric chloride was primarily responsible for the desensitization of the emulsion. Merourous chloride, which formed the dark precipitate in radioautographs, produced the background fog. While potassium dichromate caused some desensitization of the emulsion, in normal histological processing where tissues had a lengthy post-fixation wash, this substance was for the most part washed out.A number of attempts were made to remove the mercury by treating the Zenker-fixed tissues with solutions of iodine, sodium thiosulfate and cysteine. It was found that the most intense radioautographic reaction was obtained with the following procedure: prior to embedding, tissue blocks were treated with 0.5% iodine solution in 70% alcohol for 17 hours. Optionally, sections of this material could be given a second treatment with 0.5% alcoholic iodine for 10 minutes. Although simple iodination eliminated the mercurous chloride precipitate and the background fog, there was still no radioautographic reaction. However, subsequent treatment of sections of this iodinated material in 5% aqueous sodium thiosulfate for 5 minutes yielded an adequate radioautographic reaction. Therefore, the simultaneous removal of both mercurous chloride precipitate and tissue bound mercury required iodination followed by sodium thiosulfate. Using this procedure, a method was described for preparing tissues for radioautography using Zenker-formol fixation and the periodic acid-Schiff staining technique.This work was supported by a grant from the Medical Research Council of Canada to Dr. C. P. Leblond.     

An aminoglycoside biosensor incorporating free or immobilized bacterial cells

Summary Immobilized and free bacterial cells stored at 4° C in substrate free buffer were found to exhibit two phases of oxygen uptake on resuspension in an oxygen rich growth medium in the well of a standard polarographic Clarke electrode. A rapid oxygen uptake phase 1, followed by a stower, phase 2 associated with cellular division. The duration (mV) and rate of phase 1 oxygen uptake (mV min^-1) was a linear function of cell concentration. Phase 1 duration was sensitive to the presence of the aminoglycoside antibiotic gentamycin. For a fixed cell concentration the inhibition of phase 1 was a direct function of gentamycin concentration. Whole cells stored at 4° retained phase 1 activity for at least one month.     

Effects of storage temperature on oxygen depletion in intra-valve water and survival of the Mediterranean mussel Mytilus galloprovincialis Lmk

Mussels are commonly air‐stored during transportation and as a result suffer from anoxia. In this study, storage temperature effects on the viability and characteristics of the released intra‐valve water of mussels were examined. Mussels kept at 20°C released all of their intra‐valve water within approximately 60 h and died within 4 days; oxygen concentration in the intra‐valve water dropped below the detectable level. In contrast, mussels kept at 0 and 5°C released 8.8% and 12% of their intra‐valve water, respectively. The oxygen concentration in this water remained stable at about 3–4 mg L⁻¹ until hour 72 of exposure to air and all mussels survived (5°C). Mussels immersed in seawater over‐saturated with oxygen (35 mg L⁻¹) did not show any uptake of the surplus oxygen into their intra‐valve water.     

Local and systemic impacts of pleural oxygen exposure in thoracotomy

The pleural cavity is normally in a state of negative pressure and low oxygen tension. It is exposed to the atmosphere during thoracic surgery. However, no reports of pathophysiological investigation of the effects of pleural oxygen exposure involved in thoracotomy are available. In this study, the effects of pleural oxygen exposure on systemic and pleural inflammation were investigated. Male Wistar rats (9 weeks old) were placed on mechanical ventilation and underwent thoracotomy with lipopolysaccharide (LPS) administration, which simulates latent inflammatory condition. The pleural cavity was exposed to nitrogen (N(2) thoracotomy group), air (20% oxygen, air thoracotomy group), or 100% oxygen (O(2) thoracotomy group) under mechanical ventilation for 2 h. Animals were sacrificed 2 h or 8 h after LPS administration, and inflammatory indices (plasma tumor necrosis factor-alpha and interleukin-6, histology) were examined. For examination of inflammatory mediators, pleural effusion was added to cultured RAW264 cells, a murine macrophage cell line, and tumor necrosis factor-alpha levels in supernatant were measured. The capacity of pleural superoxide generation was investigated without LPS administration. Results showed increases in plasma interleukin-6 concentration and lung injury in the air and O(2) thoracotomy groups. Pleural oxygen exposure stimulated pleural superoxide generation, and increased pleural 4-hydroxy-2-nonenal and lung lipid peroxide concentrations. Tumor necrosis factor-alpha levels in cell culture supernatants were increased by the addition of pleural effusion from the air and O(2) thoracotomy groups. In conclusion, pleural oxygen exposure induced pleural oxidative injury and aggravated latent systemic inflammatory response.     

The relevance of glycosaminoglycan sulfates to Apo E induced lipid uptake by hepatocyte monolayers

The binding of Apolipoprotein E supplemented triglyceride emulsions to sulfated glycosaminoglycans demonstrated specificity for the carbohydrate polymers. Glucosamine containing glycosaminoglycans with relatively less sulfate had little affinity for the Apo E emulsion whereas those with more sulfate (i.e. heparin and sulfated heparans) effectively bound the emulsion. Galactosamine containing glycosaminoglycans (chondroitin 4 sulfate and dermatan sulfate) demonstrated no binding. The Apo E induced uptake of triglyceride emulsions by hepatocytes was inhibited by highly sulfated polysaccharides (i.e. heparin, dextran sulfate) but other glycosaminoglycans which did not bind the emulsion were ineffective in this inhibition. The same sulfated compounds which inhibited the hepatocyte Apo E emulsion interaction effectively released hepatic lipase from isolated heptic perfusions. Glycosaminoglycan sulfates which did not bind the Apo E supplemented emulsions and did not inhibit hepatocyte association were ineffective in releasing lipase. A heparan mixture isolated from human liver was much less effective in inhibiting Apo E induced association of emulsions with hepatocytes, than heparin. A highly sulfated octasaccharide fraction isolated from bovine liver heparin inhibited more effectively than the human heparans but less than the heparin. Inhibition of Apo E mediated hepatocyte emulsion association was produced by a one hour exposure of the cells to either heparinase or heparanase. The heparanase was more active than the heparinase and both were effective in the presence of protease inhibitors. Enzymes hydrolyzing chondroitin sulfates and hyaluronic acid were ineffective in inhibiting the Apo E induced association. The specific binding of human low density lipoprotein to the hepatocyte was much less effected by the heparanase exposure than the Apo E mediated binding.     

Effect of Various Gas Atmospheres on Destruction of Microorganisms in Dry Heat

The heat resistance of dry bacterial spores was tested in various gases at temperatures ranging from 121.1 to 160 C (250 to 320 F). Spores of Clostridium sporogenes (PA 3679) were heated in air, carbon dioxide, and helium; spores of Bacillus subtilis 5230 were heated in these gases and also in oxygen and in nitrogen. The surrounding gas influenced the heat resistance, but the differences among gases were small. D values were about 7 min at 148.9 C (300 F); z values were about 18.3 C (33 F) for B. subtilis, and about 21.7 C (39 F) for C. sporogenes. The resistance of B. subtilis in carbon dioxide was about the same as in air, but lower than in all other gases; resistance in helium and nitrogen was about the same, and was higher than in all other gases. C. sporogenes had the least resistance in air; the resistance was about the same in carbon dioxide and helium. For B. subtilis, the gases in order of increasing heat resistance were carbon dioxide, air, oxygen, helium, and nitrogen, and for C. sporogenes, air, carbon dioxide, and helium. Neither oxygen content nor molecular weight of the gas appeared to have a marked influence on dry-heat resistance of the spores, whereas the more inert gases seemed to yield larger D values.     

Preservation of functional integrity during long term storage of a biological membrane

Sarcoplasmic reticulum vesicles freeze-dried in the presence of trehalose retain most of their original biological activity for short periods. When the dry vesicles are stored for longer periods in air, Ca²⁺-transport becomes uncoupled from ATPase activity within a few days. However, when they are stored under vacuum, ATPase activity, Ca²⁺ transport, and coupling between Ca²⁺ transport and ATP utilization are maintained essentially intact for at least 110 days.     

Temperature and oxygen in an Everglades alligator pond

The Everglades is a large subtropical marsh ecosystem. Ponds, maintained through the activity of alligators, are abundant and ecologically important components of this system. Temperature in a 18 x 12 m study pond was controlled primarily by ambient air temperature, with significant temporal lags because of thermal resistance caused by plant growth. Temperature changed seasonally being 10° to 15°C lower in summer than winter. Diurnal temperatures fluctuated 7°C in summer and 4°C in winter. Maximum temperature exceeded 34°C. Dissolved oxygen fluctuated between 50 to 85% saturation in winter to supersaturation in summer. During the dry season water levels fell and oxygen fluctuated markedly, for example from 4 to 200% saturation within a diurnal cycle.The relation of water temperature to air temperature was similar to other reported ponds but continued fluctuation in winter differed from some results from other ponds. The distinct seasonality has important effects on biological components. Fluctuation of dissolved oxygen can result from high animal density and plankton in the dry season and can lead to massive mortality of aquatic animals. The physio-chemical conditions of Everglades alligator ponds are controlled by a combination of seasonal temperature and insolation, water level changes and biologic factors such as fish density, bird predation and alligator activity.