The extraction and purification of DNA labelled with [methyl-<sup>3</sup>H]thymidine in aquatic bacterial production studies

Wicks, Richard J.; Robarts, Richard D.

doi:10.1093/plankt/9.6.1159

Cited by 111 publications

(81 citation statements)

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“…At the end of incucation, 5 mL of NaOH 5N was added ; after 10 minutes, 28 mL of cold trichloroacetic acid (TCA) were added ; after 10 minutes at 0 °C, the sample was filtered through a 0.2 fi m pore size nitrate cellulose membrane (Sartorius) and rinsed with 5 mL of a phenol-chloroform (50% w/v) mixture and then with 5 mL of ice-cold 80 % (v/v) ethanol. This biochemical procédure has been proposed by WICKS and ROBARTS (1987) in order to eliminate radioactivity incorporated in macromolecules other than DNA. The value of incorporation in a blank sample has been substracted ; it consists in a sample initially treated by 1% (final concentration) of a saturated solution of HgCI 2 in order to stop biological processes.…”

Section: Biomass and Activity Of Suspended Bacteriamentioning

confidence: 99%

Studies of BDOC and bacterial dynamics in the drinking water distribution system of the Northern Parisian suburbs

Servais¹,

Billen²,

Praly³

et al. 2005

rseau

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The deterioration of water quality in distribution systems due to bacterial regrowth is, at the present time, a major concern of drinking water producers. In this context, a good knowledge of the factors controlling bacterial development is required; the aim of the present study is to understand the rote of biodegradable dissolved organic carbon (BDOC) in the bacterial dynamics of the distribution system. This paper discusses the results obtained in a study carried out in order to assess the dynamics of biodegradable dissolved organic carbon and suspended bacteria in the water distribution system of the Northern Parisian suburbs lad by the Méry-sur-Oise treatment plant. The results show clearly that a significant decrease in BDOC occurs within the smallest pipes, when the BDOC level in the finished water is higher than about 0.20 mgC.L-1. However, no decrease in BDOC is observed when the BDOC in the finished water is lower than 0.16 mgC.L-1. The bacterial abundance in the distribution system is primarily linked to the absence of free chicane. Temperature and BDOC concentration in the finished water are also major controlling factors of bacterial numbers. Bacterial growth rates are in the range 0.005 to 0.1 h-1 in the absence of free chlorine, the highest of these values are in the same range as the growth rates measured for bacteria in natural aquatic ecosystems. Fixed biomass to the inner pipes surface are in the range 0.25 to 0.65 µgC.cm-2 and the average growth rate of fixed bacteria seems to be roughly in the same order of magnitude as the average growth rate of the suspended bacteria. A model of the dynamics of BDOC and bacteria in distribution network, incorporating the knowledge gained from this and previous studies concerning the control of bacterial activity by dissolved organic matter, is presented. It involves a mathematical representation of the kinetics of bacterial adsorption-desorption processes, bacterial attachment, bacterial utilization of biodegradable dissolved organic matter and impact of chlorine on free and fixed bacteria. It allows simulation of the impact of reducing the BDOC in the finished water on processes associated with bacterial regrowth in the distribution network..

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Section: Biomass and Activity Of Suspended Bacteriamentioning

confidence: 99%

Studies of BDOC and bacterial dynamics in the drinking water distribution system of the Northern Parisian suburbs

Servais¹,

Billen²,

Praly³

et al. 2005

rseau

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“…3H-methyl thymidine (Amersham, specific activity 84 Ci mmol-l, final concentration 5 nM) was added to subsamples in sterile tissue culture tubes and incubated for 1 h. Two replicates (10 cm3) were made along with a formalin-killed blank. Additional time course experiments were occasionally performed and thymidine incorporation was always found to be linear over at least 2 h. Thymidine incorporation was halted by the addition of formaldehyde (0.5 cm3, 1 % final concentration) and within 12 h macromolecular material was precipitated by adding 1 cm3 of ice-cold 20% TCA and leaving the samples to stand on ice for 15 min (Wicks & Robarts 1987). The precipitates were then collected on 0.2 pm polycarbonate filters (Ducklow et al 1992;Poretics Inc.), washed twice with 1 cm3 of ice-cold 5 % TCA and once with 5 cm3 of ice-cold 80% ethanol to remove unincorporated label.…”

Section: Jg35'mentioning

confidence: 99%

Phasing of autotrophic and heterotrophic plankton metabolism in a temperate coastal ecosystem

Blight¹,

Bentley²,

Lefèvre³

et al. 1995

Mar. Ecol. Prog. Ser.

101

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Plankton abundances, bacterial production, and the size distribution of oxygen rnetabolism and chlorophyll a concentration were determined through 3 seasonal cycles in the Menai Strait [North Wales, UK). Spring blooms were comprised of a diatom to Phaeocystis succession. Meso-and microphytoplankton dominated phytoplankton production and biomass during diatom blooms, and nanophytoplankton predominated during summer, when activity and biomass were low. Correlation analysis showed temperature to be the best predictor for chlorophyll a-specific gross colnmunity production. Bacterioplankton were implied to be the major resplrers Consequently the phasing of respiration in relation to photosynthesis was strongly influenced by bacterloplankton metabolism and abundance changes. The respiration maximum occurred 1 to 2 wk after the Phaeocystis abundance maximum. An explanation for this temporal lag was sought by considering the time scales of flow of organic material between the phytoplankton and the bacter~oplankton. The observations were consistent with routes via a slo\vly cycling pool, such as polymeric organic material. This pool would function as a reservoir and result in microheterotrophic respiration persisting after the decline of photosynthesis, and causing a positive to negative temporal sequence in net community production.

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“…The rate of Tdr incorporation into bacterial DNA was measured by extracting and purifying the labeled DNA according to the method of Wicks and Robarts (1987). After incubation, the reactions were stopped by adding 0.5 ml of 5 N NaOH.…”

Section: Notesmentioning

confidence: 99%

“…After 15 min, precipitated macromolecules were collected on cellulose nitrate filters, washed with 5 ml of phenol-chloroform (50% w/v) to remove labeled proteins followed by 5 ml of icecold 80% v/v ethanol. DNA was the only labeled macromolecule remaining on the filters after this treatment (Wicks and Robarts 1987). Filters were processed for radioactive content as described above.…”

Section: Notesmentioning

confidence: 99%

[Methyl‐3H]thymidine macromolecular incorporation and lipid labeling: Their significance to DNA labeling during measurements of aquatic bacterial growth rate

Robarts¹,

Wicks²

1989

Limnology & Oceanography

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It is essential during measurements of aquatic bacterial production with [methyl#x2010;3H]thymidine (Tdr) that only labeled DNA is measured. We found in 12 freshwater and marine systems that DNA labeling represented a variable proportion of total macromolecular labeling. Up to 87% of label appearing in precipitated labeled macromolecules from acid#x2010;base hydrolysis treatments was soluble in ethanol. Reverse#x2010;phase, high#x2010;pressure liquid chromatography showed that the composition of labeled molecules in the ethanol was 78–88% [3H]Tdr. The rate of labeling of the ethanol#x2010;soluble fraction was significantly correlated with the rate of total macromolecular labeling (r = 0.88, n = 40, P < 0.001) and less strongly with the DNA labeling rate (r = 0.49, n = 28, P = 0.005). Experiments in which bacterial cells were labeled with [3H]Tdr or32PO43− showed that above a total macromolecular labeling rate of ∼1 pmol Tdr liter−1 h−1, bacterial cells bind Tdr but do not incorporate it into phospholipids in the cell envelope.

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The extraction and purification of DNA labelled with [methyl-³H]thymidine in aquatic bacterial production studies

Cited by 111 publications

References 0 publications

Studies of BDOC and bacterial dynamics in the drinking water distribution system of the Northern Parisian suburbs

Studies of BDOC and bacterial dynamics in the drinking water distribution system of the Northern Parisian suburbs

Phasing of autotrophic and heterotrophic plankton metabolism in a temperate coastal ecosystem

[Methyl‐3H]thymidine macromolecular incorporation and lipid labeling: Their significance to DNA labeling during measurements of aquatic bacterial growth rate

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The extraction and purification of DNA labelled with [methyl-3H]thymidine in aquatic bacterial production studies

Cited by 111 publications

References 0 publications

Studies of BDOC and bacterial dynamics in the drinking water distribution system of the Northern Parisian suburbs

Studies of BDOC and bacterial dynamics in the drinking water distribution system of the Northern Parisian suburbs

Phasing of autotrophic and heterotrophic plankton metabolism in a temperate coastal ecosystem

[Methyl‐3H]thymidine macromolecular incorporation and lipid labeling: Their significance to DNA labeling during measurements of aquatic bacterial growth rate

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The extraction and purification of DNA labelled with [methyl-³H]thymidine in aquatic bacterial production studies