Radiometric Method for Detection of Bacteremia

A study was performed with simulated blood cultures to evaluate the production of (14)CO(2) as an index of bacterial growth. With a range of inoculum sizes from 4 to 4,250 colony-forming units, it was not possible to detect (14)CO(2) within 6 hr after inoculation in 59 separate experiments. In a limited trial with patients' blood cultures, the radiometric method failed to provide any earlier evidence of bacteremia than did routine broth cultures.     

Radiometric Method for the Detection of Coliform Organisms in Water

A new radiometric method for the detection of coliform bacteria in water has been described. The method is based on the release of ¹⁴CO₂ from [¹⁴C]lactose by bacteria suspended in growth medium and incubated at 37 C. The evolved ¹⁴CO₂ is trapped by hyamine hydroxide and counted in a liquid scintillation spectrometer. The method permits the detection of 1 to 10 organisms within 6 h of incubation. Coliform bacteria suspended in water for several days recover from starvation and may be quantitated by the proposed method. Bacteria from water samples may also be concentrated by filtration through membrane filters and detected by the radiometric assay.     

A Rapid Method for the Measurement of Rates of Photosynthesis using 14CO2

An apparatus for measuring the rates of photosynthesis of leavesis described. In principle, the method consisted of interruptingthe flow of air over a leaf for 15sec, during which time aircontaining ¹⁴C dioxide was passed over it. The amount of¹⁴Cassimilated by the leaf was then measured. The results werecompared with those obtained using an infra-red gas analyser.The principal cause of the discrepancies between the resultsappeared to be preferential fixation of ¹²C by the leaves andloss of respiratory ¹²C at low light intensities. There wasa linear relation between the results obtained by the two methodsand thus, using the ¹⁴C method, the rates of net photosynthesiscould be estimated. By fitting a set of neutral-density filtersover the chamber that enclosed the leaf, the rates of net photosynthesiswere determined for several light intensities simultaneously.Examples are given of curves showing the relation between lightintensity and net photosynthesis for leaves of Beta and Phaseolusplants.     

Visual Reading Method for Detection of Bacterial Tannase

Tannase activity of bacteria capable of degrading tannin-protein complexes was determined by a newly developed visual reading method. The method is based on two phenomena: (i) the ability of tannase to hydrolyze methyl gallate to release free gallic acid and (ii) the green to brown coloration of gallic acid after prolonged exposure to oxygen in an alkaline condition. The method has been successfully used to detect the presence of tannase in the cultures of bacteria capable of degrading tannin-protein complexes.     

A device for the liberation and determination of 14CO2

A simple closed system for the serial determination of 14CO2 in small volumes of fluid samples, is described. The device consists of commercially available scintillation vials and silicone tube seals. 14CO2 is selectively liberated by citric acid and absorbed in a scintillation vial by Hyamine. Experiments on the effect of dichloroacetate on pyruvate dehydrogenase activity in rat hindlimbs perfused with [1-14C]pyruvate demonstrate the applicability of the method.     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

Radiometric Detection of Bacteremia in Neonates

The predicted prevalence of false positive blood cultures due to hyperactive neonatal blood cells in a radiometric detection system was confirmed. Suppression of this blood background radioactivity in the system was achieved by using a hypertonic medium containing 10% sucrose. The radiometric system produced accurate results as fast as the conventional blood culturing method, saved labor and minimized the recovery of extraneous contaminants.     

Radiometric Detection of Some Food-Borne Bacteria

Studies on detection of bacteria by radiometric techniques have been concerned primarily with aerobic species in clinical specimens. The data presented here are related to detection of aerobic and anaerobic species that are of significance in foods, by measurement of (14)CO(2) evolved from the metabolism of (14)C-glucose. Salmonella typhimurium and Staphylococcus aureus were inoculated into tryptic soy broth containing 0.0139 muCi of (14)C glucose/ml of medium. Detection times ranged from 10 to 3 hr for inocula of 10(0) to 10(4) cells/ml of broth. Heat-shocked spores of Clostridium sporogenes or C. botulinum were incubated in tryptic soy broth supplemented with Thiotone and NaHCO(3). The medium was rendered anaerobic with N(2). Spores were detected when 0.0833 muCi of labeled glucose was available/ml of medium but not when 0.0139 muCi of glucose was present/ml. The spores required 3 to 4 hr longer for detection than did comparable numbers of aerobic vegetative cells. The results demonstrate the importance of availability of sufficient label in the media and the potential of the application of this technique for sterility testing of foods.     

An Integrated Flow Cytometry-Based System for Real-Time,High Sensitivity Bacterial Detection and Identification

Foodborne illnesses occur in both industrialized and developing countries, and may be increasing due to rapidly evolving food production practices. Yet some primary tools used to assess food safety are decades, if not centuries, old. To improve the time to result for food safety assessment a sensitive flow cytometer based system to detect microbial contamination was developed. By eliminating background fluorescence and improving signal to noise the assays accurately measure bacterial load or specifically identify pathogens. These assays provide results in minutes or, if sensitivity to one cell in a complex matrix is required, after several hours enrichment. Conventional assessments of food safety require 48 to 56 hours. The assays described within are linear over 5 orders of magnitude with results identical to culture plates, and report live and dead microorganisms. This system offers a powerful approach to real-time assessment of food safety, useful for industry self-monitoring and regulatory inspection.     

DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, ³²P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 × 10⁴ cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-μg DNA sample.     

A 14CO2 ratios method for detecting pyruvate carboxylation

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state 14CO2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14CO2 production from two isotope pairs, [2-14C]pyruvate:[3-14C]pyruvate and [1-14C]acetate:[2-14C]acetate. These ratios are defined as the "pyruvate 14CO2 ratio" and the "acetate 14CO2 ratio," respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14CO2 ratio will be less than the acetate 14CO2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14CO2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.     

Intrapulmonary receptors in the bullfrog: Sensitivity to CO2

Summary Single-unit vagal afferent recordings were made on 55 intrapulmonary receptors in 15 anesthetized or decerebrate bullfrogs. Intrapulmonary CO₂ concentration and intrapulmonary pressure were controlled independently by unidirectionally ventilating the lungs. No CO₂ receptors (insensitive to stretch of the lung) of the kind reported in birds and reptiles were found; all 55 receptors were mechano-sensitive. Of these mechanoreceptors, 39 adapted slowly to inflation of the lung and 16 adapted rapidly. Thirtythree of the slowly-adapting receptors and 15 of the rapidly adapting receptors decreased their discharge frequency as intrapulmonary CO₂ concentration was increased. Inflating the lung enhanced CO₂ sensitivity. The results indicate that the frog possesses CO₂-sensitive pulmonary mechanoreceptors similar to those of mammals and reptiles.Abbreviations P _ip intrapulmonary pressure - fractional inspired concentration of CO₂ The authors wish to thank Dalyn Wilson for his help in gathering the experimental animals and for his technical assistance. The study was supported in part by a grant-in-aid from the American Heart Association, Kansas Affiliate, Inc. Contribution No. 78-185-J Department of Anatomy and Physiology, KAES, Kansas State University, Manhattan, Kansas 66506, USA.     

A nondestructive method for the measurement of radioactive 14CO2 in blood

A method for estimation of 14CO2 present in blood and tissular samples is described. It is basically based on the introduction of large amounts of a gas mixture (95% O2, 5% CO2) in the samples which serves to remove the CO2 label by gas dilution. The gas phase is later captured in scintillation vials containing an organic-soluble base that retains the carbon label. The results obtained by means of this methodology show much better recoveries for blood samples than those obtained when the classic acid-diffusion method is used. In addition, it is a very fast procedure which does not alter the pH or protein integrity of the biological sample.     

Products of CO2 fixation and 14C labelling pattern of alanine in Methanobacterium thermoautotrophicum pulse-labelled with 14CO2

The incorporation of ¹⁴CO₂ by an exponentially growing culture of the autotrophic bacterium Methanobacterium thermoautotrophicum has been studied. The distribution of radioactivity during 2s–120s incubation periods has been analyzed by chromatography and radioautography. After a 2 s incubation most of the radioactivity of the ethanolsoluble fraction was present in the amino acids alanine, glutamate, glutamine and aspartate, whereas phosphorylated compounds were only weakly labelled. The percentage of the total radioactivity fixed, which was contained in the principal early labelled amino acid alanine, increased in the first 20 s and only then decreased, indicating that alanine is derived from primary products of CO₂ fixation.The labelling patterns of alanine produced during various incubation times have been determined by degradation. After a 2 s ¹⁴CO₂ pulse, 61% of the radioactivity was located in C-1, 23% in C-2, and 16% in C-3. The results are consistent with the operation of a previously proposed autotrophic CO₂ assimilation pathway which involves the formation of acetyl CoA from 2 CO₂ via one-carbon unit intermediates, followed by the reductive carboxylation of acetyl CoA to pyruvate.