A comparison of in situ and simulated in situ methods for estimating oceanic primary production

Primary production data measured by in situ (IS) and ‘simulated’in situ (SIS) incubations were compared. To minimize differencesbetween the two types of incubations, SIS experiments were conductedin temperature-controlled incubators in which the spectral distributionand irradiance were adjusted to approximate IS conditions. ISavailable irradiance (I_Is) was computed from vertical attenuationof integrated surface irradiance. Vertical attenuation was estimatedusing a spectral irradiance model, validated by measured profilesof the vertical attenuation coefficient. IS incubations werecarried out using two methods. The first involved deploymentof bottles on a drifting array for whole-day (dawn to dusk)incubations. The second method employed an autonomous submersibleincubation device that performed short term (<1 h) incubationsat multiple depths. Differences between whole-day IS and SISincubation estimates were attributed partially to differencesbetween I_IS and SIS-available irradiance (I_SIS). Photosynthesis-irradiance(P-I) properties of IS and SIS populations from the whole-dayincubations were not significantly different. P-I propertiesof the short-term IS and SIS populations were significantlydifferent, although estimates of P^B (mg C mg Chl^–1 h^–1)from contemporaneous IS and SIS incubations did not differ by>40%. Integrated water-column primary production (IPP) estimatedusing P-I models derived from SIS data were within 15% of ISestimates of IPP.     

Light conditions affect the measurement of oceanic bacterial production via leucine uptake

The effect of irradiance in the range of 400 to 700 nm or photosynthetically active radiation (PAR) on bacterial heterotrophic production estimated by the incorporation of 3H-leucine (referred to herein as Leu) was investigated in the northwestern Mediterranean Sea and in a coastal North Atlantic site, with Leu uptake rates ranging over 3 orders of magnitude. We performed in situ incubations under natural irradiance levels of Mediterranean samples taken from five depths around solar noon and compared them to incubations in the dark. In two of the three stations large differences were found between light and dark uptake rates for the surface most samples, with dark values being on average 133 and 109% higher than in situ ones. Data obtained in coastal North Atlantic waters confirmed that dark enclosure may increase Leu uptake rates more than threefold. To explain these differences, on-board experiments of Leu uptake versus irradiance were performed with Mediterranean samples from depths of 5 and 40 m. Incubations under a gradient of 12 to 1,731 micromol of photons m(-2) x s(-1) evidenced a significant increase in incorporation rates with increasing PAR in most of the experiments, with dark-incubated samples departing from this pattern. These results were not attributed to inhibition of Leu uptake in the light but to enhanced bacterial response when transferred to dark conditions. The ratio of dark to light uptake rates increased as dissolved inorganic nitrogen concentrations decreased, suggesting that bacterial nutrient deficiency was overcome by some process occurring only in the dark bottles.     

An accurate method for measurement of aldosterone production

A method is described for isolation of the acid-hydrolysable metabolite of aldosterone in sufficient purity to measure accurately the daily production rate. Values obtained with six hospital patients were 84-131mug./day on a daily intake of 100m-equiv. of Na(+) and 227-464mug./day on a daily intake of 10m-equiv. of Na(+). Corresponding values for aldosterone excretion were also recorded, but these are a poor index of production rate since they represent from 1.6 to 9.8% of the total daily output of aldosterone.     

In situ primary production in young Antarctic sea ice

An in situ incubation technique used successfully to measure the photosynthetic carbon assimilation of internal algal assemblages within thick multiyear Arctic ice was developed and improved to measure the photosynthetic carbon assimilation within young sea ice only 50 cm thick (Eastern Weddell Sea, Antarctica). The light transmission was improved by the construction of a cylindrical frame instead of using a transparent acrylic-glass barrel. The new device enabled some of the first precise measurements of in situ photosynthetic carbon assimilation in newly formed Antarctic sea ice, which is an important component in the sea ice ecosystem of the Antarctic Ocean. The rates of carbon assimilation of the interior algal assemblage (top to 5 cm from bottom) was 0.25 mg C m^–2 d^–1 whereas the bottom algal community (lowest 5 cm) attained only 0.02 mg C m^–2 d^–1. Chl a specific production rates (P^Chl) for bottom algae (0.020 – 0.056 g C g chl a ^–1 h^–1) revealed strong light limitation, whereas the interior algae (P^Chl = 0.7 – 1.2 g C g chl a ^–1 h^–1) were probably more limited by low temperatures (< –5 °C) and high brine salinities.     

A simple method for the production of bacterial controls for immunohistochemistry and fluorescent in situ hybridization

Immunohistochemical and fluorescent in situ hybridization (FISH) assays are useful diagnostic methods for the identification of bacteria on formalin fixed paraffin embedded histological sections. To validate an anti-bacterial antibody or an oligonucleotide probe and to ensure fidelity during subsequent analyses, suitable positive and negative controls are necessary. Suspensions of fixed bacteria are often used, but ideally, these controls should be fixed, embedded and processed in the same way of tissue samples under analysis. Herein, we describe a simple method for the production of bacterial histological control samples: the sandwich. The sandwich is composed of two external layers of equine lung parenchyma and a central layer of the target bacterium. We prepared sandwiches containing Escherichia coli, Campylobacter jejuni, and Arcanobacterium pyogenes and tested them with appropriate antibodies and Eub338 FISH probe. The sandwich is an effective and simple method to prepare bacterial histological controls fixed and processed in the same way as the diagnostic tissues.     

A simple method to preserve oceanic phytoplankton for flow cytometric analyses

A simple method was developed to preserve marine phytoplankton populations so that delayed flow cytometric analyses could be performed. The method consisted of immediate fixation with 1% glutaraldehyde (final concentration) followed by storage in liquid nitrogen. The method was tested on individual algal species and on natural samples from both coastal and pelagic waters. In most cases, it caused little cell loss and preserved well both forward angle light scatter and chlorophyll fluorescence, but phycoerythrin fluorescence sometimes was significantly increased. The technique performed best for the small-sized picoplankton (below 2 microns) such as Synechococcus cyanobacteria or the newly discovered oceanic prochlorophytes. For larger-sized cells it had to be applied on a case by case basis as some fragile species, particularly dinoflagellates and cryptophytes, were poorly preserved.     

In situ measurements of net primary production in a Colorado mountain stream

Six stations were established on a Colorado mountain stream, and net primary productivity was measured in situ during all seasons. For 24-hour periods the dissolved oxygen and temperature of the water were electronically monitored over an undisturbed 1 x 1 m section of rubble bottom enclosed by a large plastic dome tightly fitted to the substrate. A submerged pump maintained a current within the dome, and the whole apparatus was submerged below the stream level. The bottom community net metabolism varied between heterotrophy and autotrophy with no correlations with altitude, season, light, water chemistry, and temperature. Readings were all very low and ranged from -27.38 to 35.59 grams of carbon fixed per square meter per year. There were no correlations between biomass of the bottom fauna and net community productivity.Contribution No. 71, University of Colorado Limnology Laboratory     

Biodiesel production by in situ transesterification of municipal primary and secondary sludges

The potential of using municipal wastewater sludges as a lipid feedstock for biodiesel production was investigated. Primary and secondary sludge samples obtained from a municipal wastewater treatment plant in Tuscaloosa, AL were freeze-dried and subjected to an acid-catalyzed insitu transesterification process. Experiments were conducted to determine the effects of temperature, sulfuric acid concentration, and mass ratio of methanol to sludge on the yield of fatty acid methyl esters (FAMEs). Results indicated a significant interactive effect between temperature, acid concentration, and methanol to sludge mass ratio on the FAME yield for the insitu transesterification of primary sludge, while the FAME yield for secondary sludge was significantly affected by the independent effects of the three factors investigated. The maximum FAME yields were obtained at 75 degrees C, 5% (v/v) H(2)SO(4), and 12:1 methanol to sludge mass ratio and were 14.5% and 2.5% for primary and secondary sludge, respectively. Gas chromatography (GC) analysis of the FAMEs revealed a similar fatty acid composition for both primary and secondary sludge. An economic analysis estimated the cost of $3.23/gallon for a neat biodiesel obtained from this process at an assumed yield of 10% FAMEs/dry weight of sludge.     

In situ phage screening. A method for identification of subnanogram tissue components in situ

We have established a novel method, in situ phage screening (ISPS), to identify proteins in tissue microstructures. The method is based on the selection of repertoires of phage-displayed antibody fragments with small samples of tissues microdissected using a laser. Using a human muscle frozen section with an area of 4800 microm2 as a model target, we successfully selected monoclonal antibody fragments directed against three major (myosin heavy chain, actin, and tropomyosin-alpha) and one minor (alpha-actinin 2) muscle constituent proteins. These proteins were present in the sample in amounts less than one nanogram, and the antibodies were used to visualize the proteins in situ. This shows that the use of ISPS can obtain monoclonal antibodies for histochemical and biochemical purposes against minute amounts of proteins from microstructures with no requirement for large amounts of samples or biochemical efforts.     

A portable chamber for measuring algal primary production in streams

This paper describes a portable chamber that measures net primary production of stream periphyton using a ¹⁴C uptake method. The unique feature is that substrates are moved through water at a velocity of 20 cm s ⁻¹ rather than moving water over substrates. The chamber consists of a plexiglass cylinder that is 9 cm in height and 15 cm in diameter. On the top of the cylinder is a DC gearmotor powered by a 12 volt, deep cycle, marine battery. The motor turns a shaft that rotates a 13.3 cm plexiglass plate at a velocity of 20 cm s ⁻¹ . Small tiles (3.2 cm × 3.2 cm × 0.5 cm) that have natural algal assemblages are mounted on the rotating plate. After adding 500 ml of filtered stream water and 185 kBq (5 μCi) NaH¹⁴CO₃ to the chamber, the chambers are placed along a stream margin for 5 h. Measurements of ¹⁴C uptake by algae on the tiles provide estimates of net primary production (NPP). To assess the sensitivity and practicality of the chamber, algal primary production was measured in open and closed canopy sections of Kingsley Creek, Randallsville, New York. In autumn, primary production was higher in the open than closed canopy section and NPP was lower in spring in both sections probably because of scouring of algae due to snowmelt.     

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.     

A semi-automatic method for measurement of seedling length

A method for the measurement of seedling length is presented here. Hand-drawn images of seedlings are converted into numerical files and processed further by the computer to obtain length approximations based on intercept measurements. Error assessment is performed based on measurement replications. The numerical results obtained on examples show that the errors are rather small compared to the biological length variation of seedlings. The high speed of measurement, the simple construction of this system and its fidelity make it very attractive for the acquisition of this type of data.     

A new method for protease activity measurement.

A new method for protease activity measurement is described. In the presence of excess leucine aminopeptidase from Aspergillus japonica, action of protease on succinyl-casein results in the production of l-amino acids and their amino acids are simultaneously determined by l-amino acid oxidase-peroxidase system. Our proposed method is less time consuming and has a much higher sensitivity than the casein-Folin method. The present method is suggested to be suitable for the assay of neutral or alkaline proteases from animals and microorganisms.     

A method for examining the endothelial cytoskeleton in situ using immunofluorescence

A simple technique is described for producing en face preparations of endothelial cells (EC) from large blood vessels fixed in situ that are suitable for studying the distribution of specific antigens in EC by immunofluorescence. This method has permitted us to examine the distribution of various components of the cytoskeleton, including microtubules (MT), centrioles, and microfilaments (MF) in EC in vivo.