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C R Fricker

Publications and source records attributed to C R Fricker.

At least 19 recordsLinked to original sources
PubMed2003

Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.

Agrobacterium tumefaciens↗
PubMed2003

A comparison of the International Standards Organisation reference method for the detection of coliforms and Escherichia coli in water with a defined substrate procedure.

AIMS: This study investigated the use of the International Standards Organisation (ISO) procedure for the comparison of microbiological methods. Using this procedure the ISO reference procedure for the detection of coliforms and Escherichia coli in water was compared with a defined substrate method (ColilertTM). METHODS AND RESULTS: A total of 20 laboratories from 13 European countries compared the use of Colilert/Quanti-TrayTM, a quantitative defined substrate test (DST) for the presence of coliforms and E. coli with the ISO reference procedure, which utilizes tergitol-TTC medium. Results of the study showed that DST detected significantly more coliforms and E. coli than did the reference procedure. In the case of E. coli the recoveries were also higher when using DST and the difference seen was statistically significant. The confirmation rate obtained when using the DST product suggested that no confirmation of wells positive for E. coli was necessary during routine use. CONCLUSIONS: Colilert is a suitable alternative to the ISO reference procedure for the detection of coliforms and E. coli in water. The methods used during the comparison study indicated that confirmation of all colonies/positive wells led to the most accurate information and it is recommended that for future comparison studies this should become standard practice. Confirmation of a small proportion of colonies led to misleading conclusions and should be avoided when comparing microbiological methods. SIGNIFICANCE AND IMPACT OF STUDY: It has been demonstrated that the ISO reference procedure fails to detect a significant proportion of coliforms and E. coli in drinking water. Colilert/QuantiTrayTM is a more suitable alternative.

Bacteriological Techniques↗
PubMed2000

Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts.

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.

Animals↗
PubMed1999

Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water.

A species-specific Peptide Nucleic Acid (PNA) oligonucleotide probe directed against the V(1)region of the 16S rRNA molecule was synthesized for the detection of Escherichia coli in water. The specificity of the probe was tested in dot blot hybridizations against a number of environmental isolates including those from the genera Escherichia, Klebsiella, Enterobacter and Citrobacter. In situ hybridization experiments were performed with biotinylated PNA oligonucleotide probes with subsequent detection of the biotin label using a combination of Streptavidin-Horseradish Peroxidase and a tyramide signal amplification system. The results obtained enabled the specific detection of E. coli in under 3 h. Hybridizations were also performed on cells which were treated with chlorine (1.5 mg l(-1)) for up to 30 min. Escherichia coli cells were still detected after storage for 14 days at room temperature. No cells were detected by conventional plate count or the < > assay, a method currently used for the routine detection of E. coli and coliforms in the water industry. Cell viability was assessed by the ability of cells to reduce 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to highly fluorescent formazan crystals through bacterial respiration. Only cells that had not been chlorinated were detected. These results confirm that ribosomal RNA exists within the cell long after cell death has occurred and that rRNA cannot be used to assess the viability of individual cells. However rRNA probes in combination with viability markers should enable the specific detection of viable cells in situ. Hybridization experiments were also performed successfully on seeded tap water samples. The number of fluorescent cells detected correlated well with those obtained by plate count analysis. This represents the first reported use of PNA oligonucleotides for in situ detection of micro-organisms and offers a fast efficient alternative to conventional DNA approaches.

Chlorine↗
PubMed1999

Application of laser scanning for the rapid and automated detection of bacteria in water samples.

It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using 'viability markers' may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z-test, P < 0.05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 degrees C for 7 d was only 25.2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5-cyano-2,3,4-tolyl-tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z-test, P < 0.05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro-organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently-labelled antibodies or nucleic acid probes.

Bacteria↗
PubMed1999

Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data.

When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was developed for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (S.D. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique was found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.

Animals↗
PubMed1999

In situ reverse transcription for the specific detection of bacteria and protozoa.

In situ hybridization experiments with oligonucleotide probes directed against the 16S and 18S rRNA molecules have been used successfully to identify specific organisms in mixed microbial populations. However, there are limitations in applying these techniques to environmental samples. In the present study we have examined the possibility of using in situ reverse transcription as an alternative to hybridization methods for the rapid detection of Escherichia coli and the waterborne parasite Cryptosporidium parvum. Following fixation and permeabilization of the cells, extension reactions were performed with species-specific primers, AMV reverse transcriptase and either cy3-AP3-dUTP or fluorescein-11-dUTP at 45 degrees C for 45 min. The cells or oocysts were then filtered onto Costar metallic membrane filters and images captured with a CCD camera. The results have shown that this technique can successfully detect E. coli cells and C. parvum oocysts in under 2 h.

Animals↗
PubMed1998

Water-borne cryptosporidiosis: detection methods and treatment options.

Since the infamous outbreak in Milwaukee, WI, USA, of water-borne cryptosporidiosis affecting over 400,000 people, there have been at least 20 smaller outbreaks associated with this parasite in the UK and North America. These events have led to an explosion of interest in and research on the nature of cryptosporidiosis as a dangerous water-borne pathogen, particularly patients with acquired immune deficiency syndrome (AIDS). In addition, several major environmental laws and proposed regulations specifically address the control of this parasite. The possible ramifications of these laws include billions of dollars of modifications to water-treatment facilities in the USA. Unfortunately, the methods used to gather the information on which these laws are based have serious deficiencies that could lead to gross underestimation of the magnitude of this problem. The present review considers gaps in our understanding of water-borne cryptosporidiosis, new methods under investigation that could improve our ability to monitor water for the presence of this organism, and treatment and control strategies to limit the threat to our water supplies.

Animals↗
PubMed1998

An evaluation of the Gelman Envirochek capsule for the simultaneous concentration of Cryptosporidium and Giardia from water.

The Gelman Envirochek capsule is a membrane device for the simultaneous concentration of Cryptosporidium oocysts and Giardia cysts from water. Samples are filtered through a Supor polyethersulphone membrane with a 1 micron absolute pore size. (Oo)cysts are mechanically eluted from the membrane fibre using a wrist action shaker and a nonionic detergent and concentrated by centrifugation. The concentrate can be further processed using any separation technique to separate the target organisms from other debris. This method enables multiple samples to be processed within 1 h. Recoveries from seeded tap and source water samples were in excess of 70% for Cryptosporidium and 80% for Giardia.

Animals↗
PubMed1998

Immunomagnetic separation of Cryptosporidium parvum from source water samples of various turbidities.

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts.

Animals↗
PubMed1998

A simple method for the comparison of commercially available ATP hygiene-monitoring systems.

The purpose of this study was to evaluate a methodology which could easily be used in any test laboratory in a uniform and consistent way for determining the sensitivity and reproducibility of results obtained with three ATP hygiene-monitoring systems. The test protocol discussed here allows such comparison to be made, thereby establishing a method of benchmarking both new systems and developments of existing systems. The sensitivity of the LUMINOMETER K, PocketSwab (Charm Sciences) was found to be between 0.4 and 4.0 nmol of ATP with poor reproducibility at the 40.0 nmol level (CV, 35%). The sensitivity of the IDEXX LIGHTING system and the Biotrace UNILITE Xcel were both between 0.04 and 0.4 nmol with coefficients of variation (CVs) of between 9% at 0.04 nmol and 10% at 0.4 nmol for the IDEXX system and 17% at 0.04 nmol and 21% at 0.4 nmol for the Biotrace system. The three systems were tested with a range of dilutions of different food residues: orange juice, raw milk, and ground beef slurry. All three test systems allowed detection of orange juice and raw milk at dilutions of 1:1,000, although the CV of results from the Charm system (54 and 74% respectively) was poor at this dilution for both residues. The sensitivity of the test systems was poorer for ground beef slurry than it was for orange juice and raw milk. Both the Biotrace and IDEXX systems were able to detect a 1:100 dilution of beef slurry (with CVs of 17 and 10% respectively), whilst at this dilution results from the Charm system had a CV of 55%. It was possible by using the method described in this paper to rank in order of sensitivity and reproducibility the three single-shot ATP hygiene-monitoring systems investigated, with the IDEXX LIGHTNING being the best, followed by the Biotrace UNILITE Xcel, and then the charm LUMINOMETER K, PocketSwab.

Adenosine Triphosphate↗
PubMed1997

The detection of Escherichia coli DNA in the ancient remains of Lindow Man using the polymerase chain reaction.

The polymerase chain reaction has been applied to the detection of Escherichia coli DNA in the upper gut contents of Lindow Man, an Iron Age bog body dated to ca 300 BC. With sets of primers from the uidA and lacZ genes, E. coli DNA could be detected reproducibly. Initial attempts at detecting DNA from freshly voided faeces from a healthy volunteer were unsuccessful due to inhibition of the reaction. Development of a method, based on guanidine thiocyanate and silica extraction and purification of the DNA fragments, facilitated the detection of the E. coli DNA in both freshly voided faeces and the upper gut contents of Lindow Man. These findings indicate that it may be possible to study the existence of infectious diseases in ancient civilizations and to learn more about the evolution of microbes.

Animals↗
PubMed1995

Detection of Escherichia coli in potable water using direct impedance technology.

Direct impedance measurement utilizing a medium previously described as being specific for Escherichia coli and which contains trimethylamine N-oxide (TMAO) and glucuronic acid was used to detect E. coli in water samples. The system was compared with the Colilert presence/absence test and the United Kingdom standard membrane filtration technique using membrane lauryl sulphate broth. The impedance method correlated well with both the traditional membrane method (93%) and the Colilert method (93.95%) for a number of different water types. No interference from Citrobacter spp. (as reported in previous studies) was detected in this study although some Salmonella spp. did give false-positive results. The data presented here suggest that the use of direct impedance may offer an alternative to conventional methods for the detection of E. coli in water.

Bacteriological Techniques↗
PubMed1994

Application of the polymerase chain reaction to the identification of Escherichia coli and coliforms in water.

The polymerase chain reaction (PCR) has been applied to the identification of Escherichia coli and coliforms after overnight growth using two sets of primers described previously. The primer set for E. coli, which was derived from the uid A gene, correctly identified all E. coli strains tested. The sequence was also identified in five non-E. coli coliforms. The coliform primer set correctly identified approximately 70% of the coliforms tested. We conclude that PCR can be used for the rapid identification of E. coli using the primers described here but that further work is required accurately to define a new primer set for the coliform group.

Base Sequence↗
PubMed1993

A new method for the concentration of Cryptosporidium oocysts from water.

A novel method for the concentration of Cryptosporidium oocysts from water has been developed, based upon the precipitation of calcium carbonate. A 10 l water sample is treated by adding solutions of calcium chloride and sodium bicarbonate and raising the pH value to 10 with sodium hydroxide. Crystals of calcium carbonate form and enmesh particles in the Cryptosporidium oocyst size range. The crystals are allowed to settle, the supernatant fluid is discarded and the calcium carbonate precipitate dissolved in sulphamic acid. The sample can be concentrated further by centrifugation. Recoveries of oocysts from seeded samples of deionized, tap and river water were in excess of 68%.

Animals↗
PubMed1993

Routine monitoring of Cryptosporidium oocysts in water using flow cytometry.

A flow cytometric method for the routine analysis of environmental water samples for the presence of Cryptosporidium oocysts has been developed. It uses a Coulter Epics Elite flow cytometer to examine water samples and to separate oocysts from contaminating debris by cell sorting. The sorted particles are then rapidly screened by microscopy. The method has been evaluated and compared with direct epifluorescence microscopy on 325 river, reservoir and drinking water samples. The technique was found to be more sensitive, faster and easier to perform than conventional epifluorescent microscopy for the routine examination of water samples for Cryptosporidium.

Animals↗
PubMed1990

Study of haemolytic activity of some Campylobacter spp. on blood agar plates.

A total of 152 strains of Campylobacter jejuni, C. coli, C. laridis and C. fetus subsp. fetus were tested for haemolysis on blood agar plates. Distinct haemolysis was detected in 92.3% (96/104) of strains of C. jejuni and 21.7% (5/23) of strains of C. coli on sheep blood heart infusion agar after incubation for 4 d microaerobically at 42 degrees C. Haemolysis was also detected on horse blood heart infusion agar. Haemolysis was not detected at 37 degrees C except with one of 50 strains of C. jejuni tested at this temperature, which was weakly positive. Campylobacter laridis was not haemolytic; C. fetus subsp. fetus, which does not grow at 42 degrees C, showed no haemolysis at 37 degrees C. Blood agar (Oxoid, BA Base No. 2) was not suitable for testing for haemolysis by these organisms. A microaerobic gas mixture containing hydrogen is better than that containing nitrogen because the medium has a brighter colour, making haemolysis easier to detect. There was no synergistic haemolysis with Staphylococcus aureus or Streptococcus agalactiae. The plate haemolysis test as described here may aid differentiation within the thermophilic campylobacters.

Animals↗