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9th July 2019  Content supplied by: Merck Millipore

Using Peptidases for Identification and Confirmation of Microorganisms

All organisms possess peptidases which are needed to cleave proteins into smaller peptides, or single amino acid metabolites. In most cases the peptidase activity is used to cleave proteins for a range reasons including nutritive purposes, signal release, activation of proteins and decomposition of old damaged or unnecessary proteins. Each microorganism species has its own metabolism that is unique to the specific genus and strain and therefore characteristic peptidases are present. These specific peptidases are an excellent target for identification and confirmation of these specific microorganisms. Often these peptidases are also linked to the virulence of pathogens, or are involved in the spoiling process, which can give even more important information about the organisms.

Microorganism Detection Tests

The limitation of many microorganism detection tests is that additional confirmation or even further differentiation or identification is required. This confirmation step may involve another differential medium or method, such as a biochemical or immunological test. In most cases, it is important that the test be performed within a short time to obtain the results as soon as possible, and it should not be too costly.

To speed up the process, bacteria specific enzymes are therefore very useful as a detection target. Simple enzyme detection systems make use of chromogenic and fluorogenic substrates. For example, the aminopeptidase test is a widely accepted method to identify Gram-negative organisms.

Testing and Confirmation in Ten Minutes

We provide new test cards with chromogenic and fluorogenic substrates, where the reaction is visible to the naked eye, and the result is obtained within 10 minutes. To help you work smarter, our product range has been developed to confirm E. coli, Salmonella, Neisseria gonorrhoea, Staphylococcus aureus, enterococci, Group A streptococci, total coliform, fecal coliform and Gram-negative bacteria.

These tests each use a specific substrate which, when hydrolyzed by a specific enzyme of the target organism (during peptide hydrolysis), produces a blue/white fluorescence, or purple/blue color upon the addition of Reagent B (color developer) when applicable.

Each test set contains 12 test cards with 4 test spots on each card (sufficient for 48 tests) and a reagent A (buffer), and in some tests, a reagent B with color developer is provided.

  • All materials can be stored at room temperature (away from direct sunlight).
  • The shelf life of the test is more than 2 years.
  • Over 500 isolates were tested with these test cards, with a correlation of greater than 99% when compared to traditional biochemical testing.

Quick and simple bacterial confirmation—without expensive reagents and equipment

When using the test, it should be checked each time with positive and negative controls, using known stock strains of the target organism and another strain of bacteria that is not the target organism. Ideally, cultured bacteria should be between 14 and 18 hours old. After 24 hours of growth, most bacteria go into a stationary phase where reduced levels of enzymes are present, which may affect the results. (Peptidases are only produced during the exponential growth phase). For the test, a small quantity of colony material is taken and then smeared on the card. One drop of reagent is added to the control spot and to each sample spot. After 5-10 minutes the positive samples show a purple color or fluorescence.

Discover our full range on SigmaAldrich.com



Date Published: 9th July 2019

Source article link: Merck Millipore

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