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Overview of Microbial Methods
Published in Joseph A. Cotruvo, Gunther F. Craun, Nancy Hearne, Providing Safe Drinking Water in Small Systems, 2019
Total coliform bacteria do not conform to a given taxonomic category. Traditionally several genera (Citrobacter, Enterobacter, Escherichia, and Klebsiella) of the family Enterobacteriaceae have been included in this group. The operational definition of a coliform is based upon the method used for detection. Lactose fermentation with the production of acid and/or gas has been considered a positive test for coliform bacteria when using carbohydrate-based media incubated at 35 to 37°C. Lauryl-tryptose broth (LT), with or without an acid indicator, and presence-absence broth (PA) are examples of lactose-based media used in these analyses [Eaton et al., 1995]. Chromogenic media have now been devised in which a positive test is based upon detecting the presence of the enzyme β-D-galactosidase. This enzyme is responsible for the cleavage of lactose to form glucose and galactose. The choromogenic media contain a specific chromogenic substrate which produces a color change due to hydrolysis by the enzyme. Three commonly used chromogenic substrates for β-D-galactosidase are ortho-nitrophenyl-β-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3 indolyl-β-D-galactopyranoside (XGAL), and chlorophenol red β-D-galactopyranoside (CPRG). When hydrolyzed by the enzyme β-D-galactosidase ONPG substrate changes from a clear to a yellow color, XGAL changes from a clear to a blue/green color, and CPRG changes from a yellow to a magenta color to indicate the presence of total coliform bacteria.
Polymer–Silver Nanocomposites: Preparation, Characterisation and Antibacterial Mechanism
Published in Huiliang Cao, Silver Nanoparticles for Antibacterial Devices, 2017
The inner membrane (cytoplasmic membrane) will be threatened where the outer membrane has been damaged. The enzyme β-galactosidase is present in the cytoplasm of Enterobacteriaceae (e.g. Escherichia coli). When β-galactosidase was released into the medium after cytoplasmic membrane disruptions, it can catalyse the hydrolysis of extracellular ortho-nitrophenyl-β-d-galactopyranoside (ONPG) to produce ortho-nitrophenol (ONP), which is determined by measuring the optical density value at 420 nm using UV–vis spectroscopy (Je and Kim 2006; Koepsel and Russell 2003; Vestro et al. 2000; Wu et al. 1999). Therefore, inner-membrane permeabilisation can be assayed by determining the release of β-galactosidase from the cytoplasm of E. coli into the culture medium using ONPG as the substrate. In this assay, the bacterial suspension is treated with ONPG and polymer–Ag nanocomposites in order and analysed. If the release of cytoplasmic β-galactosidase increases with an increase in nanocomposite concentration and reaction time, it indicates that the integrality of the inner membrane has been damaged, leading to leakage of the cytoplasm.
E. coli from drinking water
Published in Cara Gleeson, Nick Gray, The Coliform Index and Waterborne Disease, 1996
Total coliform detection is based on detection of ß-galactosidase. This enzyme catalyzes the breakdown of lactose into galactose and glucose and can be used for enumerating the coliform group within the Enterobacteriaceae family (Manafi, Kneifel and Bascomb, 1991). The assay is based on a biochemical reaction in which galactosidase cleaves the substrate o-nitro-phenyl-ß-D-galactopyranoside (ONPG) to produce yellow nitrophenol which absorbs light at 420 nm (Apte et al., 1995; Clark et al., 1991). Other substrates for ß-galactosidase which may also be used include p-nitrophenyl-ß-D-galactopyranoside (PNPG), indoxyl-ß-D-glucuronide (IBDG) and 5–4-chloro-3-indoxyl-ß-D-glucuronide (X-gluc) (Hofstra and Huis In’t Veld, 1989).
Bacilli as sources of agrobiotechnology: recent advances and future directions
Published in Green Chemistry Letters and Reviews, 2021
Zerihun T. Dame, Mahfuz Rahman, Tofazzal Islam
Some members of the microbial community might have the ability to secrete important degradative enzymes and growth factors, whereas others can exhibit the potential of biosurfactant production leading to the enhanced solubilization of hydrophobic hydrocarbons for their better utilization by microbes (249). Ubiquitous presence of cypermethrin as a contaminant in surface stream and soil necessitates developing potential bioremediation methods to degrade and eliminate this pollutant from the environment. B. subtilis has been found efficient in biodegradation and detoxification of cypermethrin (250). Enhanced degradation of cypermethrin by B. thuringiensis strain SG4 has also been reported (251). The β-glalactosidase generated by B. subtilis catalyzes the hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) to produce o-nitrophenol (ONP), which can be detected at 420 nm and used to evaluate acute biotoxicity of heavy metal ions that inhibit the activity of the enzyme (252). This B. subtilis-based bioassay is a sensitive, economically feasible, simple and promising alternative for acute biotoxicity assessment. Generally, remediation of heavy metal using microbes has several advantages. It is eco-friendly and considered to be a long-term solution compared to the physical and chemical methods. However, shortcomings like slowness of the process, difficulty in controlling the treatment, specificity of the microbes towards a particular metal contaminant, and problem with large-scale applications are the areas that need improvement.
A novel insight to screen the optimal spray-drying protectants and parameters for manufacturing lactic acid bacteria preparations
Published in Drying Technology, 2020
Zhuo Zhang, Sen Peng, Xiaoqi Sun, Yu Jie, Hongfei Zhao, Baoqing Zhu, Piotr Dziugan, Bolin Zhang
Spray-dried β-gal liposome powder (1 g) was reconstituted to 5 mL of sodium phosphate buffer (20 mM, pH 7.0), and 3 mL of 75% ethanol was added as the solvent to release the β-gal. The protein content of the β-gal liposome solution was determined using Coomassie brilliant blue G-250 dyeing method with a bovine serum albumin standard curve. The reaction of O-nitrophenyl-β-d-galactopyranoside (ONPG) and β-gal that produces O-nitrophenol (ONP) was used to measure of β-gal activity. The sample (1 mL) was added to the tube combined with 4 mL substrate solution of ONPG (20 mmol/L). After 30 min of incubation at 37 °C, the reaction was stopped by adding 5 mL (500 mmol/L) of sodium carbonate solution. The release of ONP was calculated using the concentration-absorbance standard curve of ONP at 420 nm wavelength. β-gal activity was expressed as μmol of o-nitrophenol liberated from ONPG per mg of sample weight per min. The β-gal specific activity of the sample equals β-gal activity divided by its protein content.
A β-galactosidase-expressing E. coli culture as an alternative test to identify skin sensitizers and non-sensitizers
Published in Journal of Toxicology and Environmental Health, Part A, 2018
Mahesh Raj Nepal, Youra Kang, Mi Jeong Kang, Doo Hyun Nam, Tae Cheon Jeong
When sensitizers are exposed to the bacterial cells during log (exponential) phase by IPTG induction, numerous macromolecules required for cell survival might be damaged or affected during the process of cell division where those macromolecules are rapidly expressing. Thereby cells may be more vulnerable to the toxic insults of chemicals in this unstable condition of rapid proliferation. Further, during IPTG-mediated induction of LacZ gene, a conformational change in the protein structure also makes cells more susceptible to external toxic stimuli. Thus, applying this mechanism, it was postulated that, when E. coli possessing LacZ gene was co-treated with IPTG and test chemicals of known skin sensitizers or non-sensitizers, the macromolecules under expression in bacterial cells might interact with these compounds producing alterations in biological and cellular functions, by mechanistically inhibiting or suppressing the β-galactosidase enzyme production. The β-galactosidase enzymes present in cells following incubation with chemicals were quantitatively analyzed by using ONPG as a substrate and measured colorimetrically.