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Microtiter plate assays for the measurement of phage adsorption and infection in Lactococcus and Enterococcus
M L Tortorello1, K M Trotter, S M Angelos
1Department of Microbiology, NYS College of Veterinary Medicine, Ithaca.
Abstract:
Three easy and rapid microtiter plate assays for determining phage sensitivity of lactococci and enterococci have been developed. In the microlysis assay, the degree of sensitivity was measured on the basis of the ability of the bacterial cells to grow in the presence of various concentrations of phage and to effect a color change of an acid-base indicator as a result of acid production. Two assays that specifically measure phage adsorption to bacterial cells have been developed on the basis of the enzyme-linked immunosorbent assay (ELISA) technique. In the direct phage adsorption ELISA, adsorption of phage particles to cells immobilized onto microtiter plate wells was measured using specific anti-phage antibody. In the competitive phage adsorption ELISA, phage adsorption was assayed by allowing phage to compete with specific antibody binding to the bacterial cell surface. All three assays were quantifiable photometrically.
Insights
Developed three rapid microtiter plate assays to determine phage sensitivity in lactococci and enterococci. These methods quantify phage adsorption and bacterial growth, aiding in phage resistance studies.
Area of Science:
- Microbiology
- Bacteriology
- Molecular Biology
Background:
- Phage sensitivity is crucial for dairy starter cultures like lactococci and enterococci.
- Accurate and rapid methods are needed to assess phage interactions with these bacteria.
Purpose of the Study:
- To develop and validate three simple, rapid microtiter plate assays for determining phage sensitivity.
- To quantify phage adsorption to bacterial cells and assess bacterial growth inhibition.
Main Methods:
- Microlysis assay: measures bacterial growth and acid production in the presence of phage.
- Direct phage adsorption ELISA: quantifies phage binding to immobilized bacterial cells using antibodies.
- Competitive phage adsorption ELISA: assesses phage competition with antibodies for bacterial cell surface binding.
Main Results:
- All three assays are easy to perform and provide rapid results.
- The assays are quantifiable using photometric measurements.
- Methods effectively determine phage sensitivity and adsorption in lactococci and enterococci.
Conclusions:
- The developed microtiter plate assays offer efficient tools for phage sensitivity testing.
- These assays facilitate research into phage-bacterial interactions in food microbiology.
- The methods are suitable for routine laboratory use in assessing phage resistance.