Overlay Technique
Overlay Technique - Related Videos
06:38
Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds
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Cited by 141 •
2017
We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.
04:33
Evaluation of a Liquid Overlay System for Viral Plaque Assays
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2026
Source: Baer, A. and Kehn-Hall, K. Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems. J. Vis. Exp. (2014).This video demonstrates the use of a liquid microcrystalline cellulose-based overlay in a plaque-based assay by comparing its effectiveness in viral plaque formation with that of a traditional semi-solid agarose overlay.
09:28
Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems
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Cited by 466 •
2014
Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.
03:17
Soft Agar Overlay Assay for Detecting Bacteriocin-Mediated Killing in Pseudomonas syringae
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2025
Source: Hockett, K. L., and Baltrus, D. A. Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds. J. Vis. Exp. (2017)This video demonstrates the procedure for detecting bacteriocin-mediated killing by spotting supernatant from an antibiotic-induced donor strain onto a soft agar overlay containing a test strain. Clear zones formed after incubation indicate bacterial inhibition.
08:38
Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
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Cited by 6 •
2011
Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.
FAQs
The soft agar layer keeps the bacteria and phage-containing sample in a localized region above the nutrient agar base. When viral replication occurs in susceptible bacteria, the resulting loss of bacterial growth appears as a clear plaque. This spatial pattern converts microscopic infection events into a visible assay outcome.
Clear plaques indicate that infectious phage particles encountered bacteria capable of supporting their replication under the assay conditions. Comparing plaque formation across different bacterial hosts can therefore reveal host range, meaning which hosts are susceptible. The result links phage activity to a specific microbial target rather than merely showing sample presence.
The layered format separates the nutrient-supporting base from the softer growth environment containing bacteria and sample. That arrangement supports a bacterial lawn in which plaques can be distinguished and compared. Consistent layering and visual scoring are important because the method's value depends on reproducible detection and measurement of infectious particles.
An assay begins by suspending susceptible bacteria together with a phage-containing sample in molten soft agar. This mixture is poured over a nutrient agar plate, allowed to form the overlay, and incubated. After incubation, clear plaques within the bacterial lawn provide the readout used to detect or measure viral activity.
To quantify infectious phage particles, investigators use the number or pattern of plaques as the assay's visible measurement, provided the plaques arise from replication in susceptible bacteria. The approach is consequently useful when the research question concerns infectious activity rather than only whether microbial material is present.
Host-range testing applies the same readout to multiple bacterial hosts. Plaque formation on one host and not another can distinguish differential susceptibility, helping researchers examine how a phage interacts with microbial targets. In immunology and infection studies, this information supports evaluation of phage-based approaches to pathogen control and antimicrobial strategies.