Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 °C for 1 h or to light-induced oxidative stress by exposure to artificial light for up to 8 h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects

Rapid Bacterial Testing for Spacecraft Water

Evaluations of the fluorogenic stains and probes will continue. E. coli 0157:H7 will be used as the reference strain for optimizing protocols. We anticipate the continued use of the fluorescent antibodies (TRITC and FITC labeled) in conjunction with CTC, Rhl23, DiBAC4(3), DAPI and acridine orange. Chemunex, the manufacturer of the ChemScan analyzer system, also makes a fluorogenic probe, Chemchrome B, which will be incorporated into the suite of probes to evaluate once their system is on site. Regardless of the combination of stains and probes all will be evaluated on membrane filters. Development of a FISH protocol that will be applicable to our conditions will be continued. Complimentary 16s rRNA probes to Ps. aeruginosa and currently in our laboratory will be evaluated first. Once this protocol has been adequately optimized other probes will be ordered for u a select number of other species. Currently, protocols to evaluate the effects of disinfection and the resulting lethality, injury on stain and/or probe specificity and reliability are being developed. E. coli 0157:H7 is the reference strain and chlorine the disinfectant the reference protocol is being developed around. Upon completion of this work, the resulting protocol will be extended to other species and disinfectants (e.g., iodine). Similar disinfectant experiments will then be conducted on the same species after starvation to evaluate the effects of starvation on disinfection resistance and the applicability of the stains and probes. Development of the immunomagnetic separation system will continue. Combined with the rapid methods described above, with enumeration by the ChemScan, we anticipate that this will provide a highly sensitive technique for the detection of specific, active bacteria

Purification and properties of Streptococcus lactis β-galactosidase

Experiments were carried out to purify and characterize the galactosidase of the Streptococcus lactis 7962. Purification was accomplished using standard procedures; however the lability of the enzyme to numerous treatments limited the techniques that could be used and the amount of active enzyme recovered. Gel filtration revealed that the enzyme existed in two forms differing in size by a factor of two (molecular weights of 5 x 10⁵ and 10⁶); the smaller of the two forms was purified and characterized in detail. Chemical characteristics of the two forms were the same: pH optimum, 7.0; temperature optimum, 37 C; and Michaelis constant, 10⁻³ M ONPG. Ammonium sulfate exerted a stabilizing effect on the enzyme and caused an association of the smaller unit to form a larger molecular weight aggregate. Because of the size and the chemical properties of the two forms a subunit (monomer) - native enzyme (dimer) relationship was established. The observations that para-chloromecuribenzoate and ethylenediamine tetraacetic acid inactivated the enzyme and that ammonium sulfate prevented this inactivation suggested that essential sulfhydryl and metal ion groups became masked in the enzyme structure during salt stabilization. Comparison of the amount of change seen in the U.V. spectra of salt-stabilized and nonstabilized enzyme indicated that ammonium sulfate stabilized the structure of the enzyme. The salt also protected the enzyme from the inactivating effect of urea, but to a much lower extent than was seen with the sulfhydryl antagonist or metal chelator. Chemical analysis of the purified enzyme, when compared with ß- galactosidase from bacteria of the enteric group, showed similarities and differences in the amino acid composition. For example, alanine, aspartic acid, glutamic acid, glycine, isoleucine, phenylalanine, serine, threonine and valine were present in the same amounts. Incubation of the purified enzyme at 27 C caused a similar association (quaternization) as was observed in the presence of ammonium sulfate. This indicated that hydrophobic bonding is involved in holding the dimer form together as has been suggested for other polymeric proteins. The same relative amount of the two enzyme forms (monomer and dimer) observed following quaternization was also found in enzyme extracted from log phase culture; this suggested that an equilibrium between the two forms existed. When enzyme was obtained from bacteria in the late logarithimic phase of growth (12 hours) a disproportionately large amount of monomer was observed. This indicated that lactose was essential for association to occur in vivo since this carbohydrate was probably depleated at that time. No evidence for dissociation of the dimer form of enzyme was seen, however, prolonged incubation at either 27 C or 5 C caused an increase in the amount of monomer form of enzyme present. This effect apparently was caused by the activation of an inactive form of enzyme, possibly monomer, as has been suggested in other proteins

Inactivation combined with cell lysis of Pseudomonas putida using a low pressure carbon dioxide microbubble technology

BACKGROUND Inactivation processes can be classified into non-thermal inactivation methods such as ethylene oxide and γ-radiation, and thermal methods such as autoclaving. The ability of carbon dioxide enriched microbubbles to inactivate Pseudomonas putida suspended in physiological saline, as a non-thermal sterilisation method, was investigated in this study with many operational advantages over both traditional thermal and non-thermal sterilisation methods. RESULTS Introducing carbon dioxide enriched microbubbles can achieve ∼2-Log reduction in the bacterial population after 90 min of treatment, addition of ethanol to the inactivation solution further enhanced the inactivation process to achieve 3, 2.5 and 3.5-Log reduction for 2%, 5% and 10 %( v/v) ethanol, respectively. A range of morphological changes was observed on Pseudomonas cells after each treatment, and these changes extended from changing cell shape from rod shape to coccus shape to severe lesions and cell death. Pseudomonas putida KT 2440 was used as a model of gram-negative bacteria. CONCLUSION Using CO2 enriched microbubbles technology has many advantages such as efficient energy consumption (no heat source), avoidance of toxic and corrosive reagents, and in situ treatment. In addition, many findings from this study could apply to other gram-negative bacteria

Flow cytometry for microbial sensing in environmental sustainability applications: current status and future prospects

Practical and accurate microbial assessment of environmental systems is predicated on the detection and quantification of various microbial parameters in complex matrices. Traditional growth-based assays, considered to be both slow and biased, are increasingly being replaced by optical detection methods such as flow cytometry. Flow cytometry (FCM) offers high-speed multi-parametric data acquisition, compatibility with current molecular-based microbial detection technologies, and is a proven technology platform. The unique technical properties of flow cytometry have allowed the discrimination of bacteria based on nucleic acid staining, microbial identification based on genomic and immunologic characteristics, and determination of cell viability. For this technology to achieve the ultimate goal of monitoring the microbial ecology of distributed systems, it will be necessary to develop a fully functional, low cost, and networkable microsystem platform capable of rapid detection of multiple species of microorganisms simultaneously under realistic environmental conditions. One such microsystem, miniaturized and integrated in accordance with recent advances in micro-electro-mechanical systems technology, is named the Micro Integrated Flow Cytometer. This manuscript is a minireview of the current status and future prospects for environmental application of flow cytometry in general, and micro-flow cytometry in particular.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75610/1/j.femsec.2004.01.014.pd

Toxicity evaluation of metal plating wastewater employing the Microtox® assay: A comparison with cladocerans and fish

The relative sensitivity of the Microtox assay is closely related to the type of toxicant, and hence its utility in biomonitoring effluents is better evaluated on a case-by-case basis. The Microtox® assay, employing the marine bacterium Vibrio fischeri , was evaluated for its applicability in monitoring metal plating wastewater for toxicity. The results of the Microtox assay after 5, 15, and 30 min of exposure, were compared with data obtained from conventional whole effluent toxicity testing (WET) methods that employed Daphnia magna , Ceriodaphnia dubia , and the fathead minnow ( Pimephales promelas ). The Microtox assay produced notably comparable EC50 values to the LC50 values of the acute fathead minnow toxicity test (<0.5 order of difference). The Spearman's rank correlation analyses showed that the bacterial assay, regardless of exposure duration, correlated better with the acute fish than the daphnid results ( p <0.05). These observations were consistent to other studies conducted with inorganic contaminants. The relative sensitivity of the 30-min Microtox assay was within the range of the two frequently used acute daphnid/fish toxicity tests. In conclusion, the Microtox assay correlated well with the acute fathead minnow data and is well suited for toxicity monitoring for these types of industrial wastes. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 136–141, 2001Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35240/1/1017_ftp.pd

Methanotrophy, Methylotrophy, the Human Body and Disease

Methylotrophic Bacteria use one-carbon (C1) compounds as their carbon source. They have been known to be associated to the human body for almost 20 years as part of the normal flora and were identified as pathogens in the early 1990s in end-stage HIV patients and chemotherapy patients. In this chapter, I look at C1 compounds in the human body and exposure from the environment and then consider Methylobacterium spp. and Methylorubrum spp. in terms of infections, its role in breast and bowel cancers; Methylococcus capsulatus and its role in inflammatory bowel disease, and Brevibacterium casei and Hyphomicrobium sulfonivorans as part of the normal human flora. I also consider the abundance of methylotrophs from the Actinobacteria being identified in human studies and the potential bias of the ionic strength of culture media and the needs for future work. Within the scope of future work, I consider the need for the urgent assessment of the pathogenic, oncogenic, mutagenic and teratogenic potential of Methylobacterium spp. and Methylorubrum spp. and the need to handle them at higher containment levels until more data are available

Non-integumentary melanosomes can bias reconstructions of the colours of fossil vertebrates

The soft tissues of many fossil vertebrates preserve evidence of melanosomes-micron-scale organelles that inform on integumentary coloration and communication strategies. In extant vertebrates, however, melanosomes also occur in internal tissues. Hence, fossil melanosomes may not derive solely from the integument and its appendages. Here, by analyzing extant and fossil frogs, we show that non-integumentary melanosomes have high fossilization potential, vastly outnumber those from the skin, and potentially dominate the melanosome films preserved in some fossil vertebrates. Our decay experiments show that non-integumentary melanosomes usually remain in situ provided that carcasses are undisturbed. Micron-scale study of fossils, however, demonstrates that non-integumentary melanosomes can redistribute through parts of the body if carcasses are disturbed by currents. Collectively, these data indicate that fossil melanosomes do not always relate to integumentary coloration. Integumentary and non-integumentary melanosomes can be discriminated using melanosome geometry and distribution. This is essential to accurate reconstructions of the integumentary colours of fossil vertebrates