What is agar overlay assay?

The overlay assay is used as a rapid way to investigate the competitive dynamics between strains that may exist in vitro. To do so, a pneumococcal strain is pre-inoculated onto an agar plate and allowed to grow to achieve local bacterial concentrations sufficient for induction of the blp locus.

What is the purpose of top agar?

Top agar preparations contain lower concentrations of agar (7 g/L) than normal solutions used to prepare agar plates (15 g/L). The low agar concentration allows progeny phage from lysed cells to diffuse through the media and infect neighbouring bacterial cells.

What is the purpose of the agar overlay in the plaque assay?

Aim of the experiment: This technique is used to produce a homogeneous lawn of bacteria within a thin layer of agar across the surface of a plate. Number of plaque forming units per sample unit volume (pfu/mL) can be calculated.

When preparing cells for the overlay assay bacterial host cells in the phage top agar PTA need to be in the right phase of growth?

Preparation of cells for overlay assay: The cells need to be in the right phase of growth for a sufficient bacterial lawn formation. This is required for the formation of concise round plaques. → Set-up a 5 mL overnight culture of your cells in the media of interest.

How to make an agar overlay plaque assay?

Procedure 1. Melt 0.5 % Agarose NZCYM Medium until liquid. 2. Prepare 4 ml of liquid 0.5 % Agarose NZCYM Medium in a 15 ml falcon tubes for each plate. 3. Place 15 ml falcon tubes in the water bath at 48°C. 4. Prepare dilution row of phage solution. 5.

How much agar do you need for a double agar assay?

In this method a small volume of a dilution of phage suspension and a small quantity of host cells grown to high cell density, sufficient to give 107-108 CFU/ml, are mixed in about 2.5 ml of molten, ‘soft’ or ‘top’ agar at 46°-50°C.

How does the double overlay agar method work?

In the double overlay agar method, bacteria are mixed along with a dilution of phage in molten, low concentration agar which is then spread out over more solid, concentrated agar on a plate (3).

What is the significance of 0.7% soft agar in agar overlay?

Most journal stated that 0.7% of agar is used for the agar overlay assay to screen for antimicrobial substance. So basically the soft agar will be inoculated with indicator bacteria which will be overlaid on a prepared bacterial lawn. Will there be any effect if 1.7% agar used instead of 0.7% agar?