A modified m-CP medium for enumerating Clostridium perfringens from water samples

Medium m-CP, designed for the isolation of Clostridium perfringens from water samples, contains indoxyl beta-D-glucoside, an expensive chemical that is present at a high concentration in this medium. The use of m-CP with three concentrations of indoxyl beta-D-glucoside was tested at 0, 60, and 600 mg/L. Lowering the amount of indoxyl beta-D-glucoside to 60 mg/L (1/10 the recommended concentration) reduced the cost of this medium without affecting its sensitivity.     

Oxygen cost and oxygen uptake dynamics and recovery with 1 min of exercise in children and adults.

To test the hypothesis that O2 uptake (VO2) dynamics are different in adults and children, we examined the response to and recovery from short bursts of exercise in 10 children (7-11 yr) and 13 adults (26-42 yr). Each subject performed 1 min of cycle ergometer exercise at 50% of the anaerobic threshold (AT), 80% AT, and 50% of the difference between the AT and the maximal O2 uptake (VO2max) and 100 and 125% VO2max. Gas exchange was measured breath by breath. The cumulative O2 cost [the integral of VO2 (over baseline) through exercise and 10 min of recovery (ml O2/J)] was independent of work intensity in both children and adults. In above-AT exercise, O2 cost was significantly higher in children [0.25 +/- 0.05 (SD) ml/J] than in adults (0.18 +/- 0.02 ml/J, P less than 0.01). Recovery dynamics of VO2 in above-AT exercise [measured as the time constant (tau VO2) of the best-fit single exponential] were independent of work intensity in children and adults. Recovery tau VO2 was the same in both groups except at 125% VO2max, where tau VO2 was significantly smaller in children (35.5 +/- 5.9 s) than in adults (46.3 +/- 4 s, P less than 0.001). VO2 responses (i.e., time course, kinetics) to short bursts of exercise are, surprisingly, largely independent of work rate (power output) in both adults and children. In children, certain features of the VO2 response to high-intensity exercise are, to a small but significant degree, different from those in adults, indicating an underlying process of physiological maturation.     

Assembly of the 26 S complex that degrades proteins ligated to ubiquitin is accompanied by the formation of ATPase activity

In the ubiquitin pathway for intracellular protein breakdown, proteins ligated to ubiquitin are degraded by a large (26 S) ATP-dependent protease complex. It was found previously that the 26 S complex is assembled from three different enzyme components by a process that requires MgATP. In addition, MgATP is also required for the continued action of the 26 S complex in the breakdown of ubiquitin-protein conjugates. In the present study we have tried to gain some insight into the mode of action of ATP by following ATP hydrolysis by the 26 S complex and its three components. It was found that none of the three unassembled components had significant ATPase activity, but such activity appeared following their entry into the 26 S complex. The presence of all three components and of MgATP was required for the formation of complex-associated ATPase activity. GTP and UTP cannot replace ATP for complex assembly, but these nucleotides can substitute for ATP in the stimulation of the conjugate-degrading activity of the 26 S complex. Unlabeled GTP and UTP inhibit the hydrolysis of [gamma-32P] ATP by complex-associated ATPase, indicating that this activity is related to the latter site of ATP action in this system.     

Oral [13C]bicarbonate measurement of CO2 stores and dynamics in children and adults

During exercise, less additional CO2 is stored per kilogram body weight in children than in adults, suggesting that children have a smaller capacity to store metabolically produced CO2. To examine this, tracer doses of [13C]bicarbonate were administered orally to 10 children (8-12 yr) and 12 adults (25-40 yr) at rest. Washout of 13CO2 in breath was analyzed to estimate recovery of tracer, mean residence time (MRT), and size of CO2 stores. CO2 production (VCO2) was also measured breath by breath using gas exchange techniques. Recovery did not differ significantly between children [73 +/- 13% (SD)] and adults (71 +/- 9%). MRT was shorter in children (42 +/- 7 min) compared with adults (66 +/- 15 min, P less than 0.001). VCO2 per kilogram was higher in the children (5.4 +/- 0.9 ml.min-1.kg-1) compared with adults (3.1 +/- 0.5, P less than 0.0001). Tracer estimate of CO2 production was correlated to VCO2 (r = 0.86, P less than 0.0001) and when corrected for mean recovery accurately predicted the VCO2 to within 3 +/- 14%. There was no difference in the estimate of resting CO2 stores between children (222 +/- 52 ml CO2/kg) and adults (203 +/- 42 ml CO2/kg). We conclude that orally administered [13C]bicarbonate can be used to assess CO2 transport dynamics. The data do not support the hypothesis of lower CO2 stores under resting conditions in children.     

A simple, rapid and sensitive presence/absence detection test for bacteriophage in drinking water

R. ARMON AND Y. KOTT. 1993. A rapid, simple and sensitive direct bacteriophage presence detection method for 500 ml drinking water samples has been developed. The method includes a glass device consisting of a jar containing the water sample and an immersible probe filled with solidified soft agar containing bacterial host cells. Host bacteria in logarithmic phase were added to the experimental volume and the probe was submerged. The entire device was incubated in a water bath at 36C.
Plaques of somatic bacteriophage infecting Escherichia coli strain CN₁₃, could be detected within 3 h. Male-specific bacteriophages infecting E. coli F+ amp were detected within 6 h. Bacteriophage infecting the anaerobe Bacteroides fragilis subsp. fragilis HSP40 were detected after 8 h. Application of this device and the associated technique, enabled a one-step detection of 1 pfu of E. coli or Bact. fragilis specific bacteriophage in 500 ml drinking water samples.     

Cell Squeezing as a Robust,Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.     

Adsorption of flavobacterium breve and pseudomonas fluorescens p17 on different metals: Electrochemical polarization effect

The electrochemical polarization effect on early adsorption of Flavobacterium breve and Pseudomonas fluorescens P17 to platinum, titanium, stainless steel, copper, aluminum alloy and mild steel was studied. A well‐defined peak characterized the bacterial adsorption dependence on externally applied potential. Maximal adsorption occurred in the potential range of ‐0.5 to 0.5 V (SCE) for all tested metals. A shift of applied potential towards both a positive and a negative direction from the maximal adsorption potential (Emax,ad) was accompanied by a gradual decrease in bacterial adsorption. The extent of bacterial adsorption strongly depended on the nature of the metallic substratum and decreased accordingly as follows: platinum > titanium > stainless steel > aluminum alloy > carbon steel > copper. Adsorption on all tested metals was approximately two orders of magnitude higher with the relatively more hydrophobic F. breve compared to the less hydrophobic P. fluorescens P17. The effect of electrochemical polarization on the initial stages of bacterial adsorption onto metallic substrata is further discussed.     

Bacteriophage ecology in a small community sewer system related to their indicative role in sewage pollution of drinking water

In view of various studies looking for the merit of coliphages as indicators of water pollution with viruses originating from faecal material, a small agricultural community (population of approximately 1500 inhabitants of all ages, 2-3 km from Haifa) was selected in order to understand these bacteriophage ecology (F-RNA and somatic coliphages) in its sewer and oxidation pond system. Along the sewer lines, it was possible to isolate constantly both bacteriophage types (F-RNA and somatic coliphages) at 10(2)-10(4) plaque-forming units (pfu) ml(-1). The average numbers of somatic and F-RNA phages isolated from oxidation pond were 10(3)-10(4) pfu ml(-1); however, somatic coliphages were undetectable for several months (April-August). Significant high correlation (0.944 < R(2) < 0.99) was found between increased anionic detergent concentrations and F-RNA coliphage numbers. Infants less than 1 year old excreted both phage types and few only F-RNA coliphages (at high numbers > 10(5) pfu g(-1)) for up to 1 year. The excretion of F-RNA coliphages was highly linked to Escherichia coli F(+) harborage in the intestinal track as found in their faecal content. Finally, three bacterial hosts E. coli F(+), F(-) and CN(13) tested for survivability in sewage filtrate revealed that E. coli F(+) had the highest survivability under these conditions. Presence of somatic and F male-specific phages in sewer lines of a small community are influenced by several factors such as: anionic detergents, nutrients, temperature, source (mainly infants), shedding and survival capability of the host strain. Better understanding of coliphages ecology in sewer systems can enhance our evaluation of these proposed indicator/index microorganisms used in tracking environmental pollution of water, soil and crop contamination with faecal material containing enteric viruses.     

Denitrification by a mixture of bacterial strains derived from an upflow sludge blanket reactor, following entrapment in sol-gel glass

Whole cell extracts of a bacterial mixture grown in an upflow sludge blanket reactor used for drinking water denitrification, were successfully entrapped into sol-gel glass, and afterwards air dried. The sol-gel complex was able to reduce sodium nitrate and nitrite solutions. Compared with free whole cell extract, the sol-gel complex required twice as much time for nitrate and nitrite reduction. This study is the first successful attempt to entrap denitrifier reductases for experimental denitrification into sol-gel glass. Denitrifying enzymes entrapped into sol-gel glass matrix, can be used several times without significant loss in specificity, but gradual loss in reactivity occurs, probably due to electron donor depletion.