Identification of Microorganisms

 
 

Reagents for Fast, Enzyme
Activity Based Identification

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To identify potential pathogenic and spoilage organisms in water for food and beverage production, MilliporeSigma maintains a wide range of products, including its Bactident® test kits. These kits help to identify colonies which have grown on the membrane after membrane filtration of the water sample and incubation.

The Bactident® Reagents use the activity of specific microbial enzymes to enable the quick and reliable identification of certain microorganisms, typically by color development. These reagents results are highly reliable and thus guarantee the safety and quality of finished food products and beverages

Identification of Microorganisms in Water


Identification of Microorganisms in Water

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Bactident® Oxidase

Bactident® Oxidase is used to support the identification of microorganisms on the basis of their cytochrome c oxidase activity. A positive test result normally means that the bacterium can use oxygen for energy production with an electron transfer chain. Among the medically relevant oxidase positive microorganisms are Aeromonas spp., Vibrio spp., Pseudomonas spp., Campylobacter spp. and Brucella spp.. Enterobacteriaceae, however, are mostly oxidase negative.

The cytochrome oxidase, an enzyme which is widespread in nature, oxidizes the reduced cytochrome c and is thereby itself transformed into a reduced and inactive form. Through transfer of the electrons to molecular oxygen, the reduced cytochrome oxidase is transformed back into the active form. In the presence of molecular oxygen the cytochrome oxidase/cytochrome c system can reduce a whole series of organic substances, among them the so-called NaDi reagent (1-naphthol dimethylparaphenylene diamine), leading to the formation of the condensation molecule indophenol blue. Bactident® Oxidase uses this reaction to identify microorganisms.

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Bactident® E. coli

Bactident® E. coli is used for the rapid identification of Escherichia coli. It is based on the detection of two enzymes found in most E. coli strains: β-D-glucuronidase and tryptophanase. β-D-glucuronidase activity is a specific marker for E. coli among the Enterobacteriaceae and is present in 94% of the species’s strains; apart from E. coli, the enzyme can only be detected in a few Salmonella and Shigella species. Tryptophanase activity (i.e. the ability to form indole from tryptophan) is present in 99% of all E. coli strains. Detection of both enzymes is a highly reliable indicator for the presence of E. coli.

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Bactident® Indole

Bactident® Indole is used to determine the ability of a microorganism to convert tryptophan into indole. Tryptophan is abundant particularly in tryptically digested peptone, setting free pyruvic acid, ammonia and indole. This reaction is performed by the enzyme system tryptophanase. The indole then reacts with 4-dimethylaminobenzaldehyde to form a dark red dye. As tryptophan, too, gives a color reaction with 4-dimethylaminobenzaldehyde, it must be separated from the indole beforehand. This is achieved by selectively extracting indole with butanol.

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Bactident® Catalase

Bactident® Catalase is a test reagent which is used for catalase detection. Catalase is an enzyme which is present in all cells with aerobic metabolisms, including for example Enterobacteriaceae and staphylococci. This enzyme contains iron protoporphyrin (haemin) as the coenzyme. Catalase cleaves the toxic hydrogen peroxide, a by-product of metabolic processes, into water and oxygen. The presence or absence of catalase activity is a taxonomically relevant property of microorganisms and can be used for their differentiation or identification.

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Bactident® Aminopeptidase

Bactident® Aminopeptidase is used for the detection of L-alanine aminopeptidase in microorganisms, an enzyme which is localized in the cell envelope of bacteria. Almost all aminopeptidase positive strains are Gram-negative. Exceptions are Bacteroides vulgatus, Bacteroides fragilis, Campylobacter species and Veillonella parvula. The enzyme splits off the amino acid L-alanine from various substrates. In the case of the Bactident® Aminopeptidase test strips, any L-alanine aminopeptidase present splits the substrate L-alanine-4-nitroanilide into 4-nitroaniline and the amino acid L-alanine. The resulting 4-nitroaniline is colored yellow, indicating the presence of L-alanine aminopeptidase. The results of the investigations performed so far indicate that there is a very good correlation between the aminopeptidase reaction and the Gram-behavior of the microorganisms.

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Bactident® Coagulase

Bactident® Coagulase is used for the detection of the enzyme coagulase, which almost all Staphylococcus aureus strains produce. The enzyme has the ability to coagulate blood plasma. Staphylococcus aureus forms two types of coagulase. The free coagulase is an extracellular enzyme; the bound coagulase is localized on the surface of the cell wall. The tube test detects both enzymes, whereas the slide test can detect only the bound coagulase.

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