Agar overlay assay

To make up 2.5% Noble agar (Difco Labs.) add 25 g agar powder to 500 ml distilled water and make up to 1 1. Place the flask in a container of boiling water until the agar dissolves. Dispense 25 ml amounts into universal containers with metal caps and autoclave at 15 lb pressure for 15 min. Store at room temperature. 

To make the neutral red overlay medium first dissolve neutral red to 0.4% in distilled water (heat if necessary) and filter through Green s Filter Paper No. 904 (Appendix 3). Bottle in 20 ml amounts and sterilise by autoclaving at 15 lb pressure for 15 min. 

Foetal calf serum (for SV40) DEAE dextran (0.5%) 

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Fig. 4.2. Agar overlay assay for plastninogen activator. B16-F1 and -LS9 cells grown as isolated colonies have been overlaid with agar/milk (see text) in the presence (lower wells) or absence (upper wells) of plasminogen, to detect celt surface associated uPA activity. B16-LS9 cells clearly show a higher activity as compared to B16-F1, in which the activity is undetectable under this assay conditions (Rusciano et al., 1998a).

Figure 2. Agar-overlay assay used to screen for hydantoinase-producing microorganisms.

This test determines if a material itself has potential to release agents that may be cytotoxic and is suitable for biomaterials with large toxicity. The indirect contact test includes the agar overlay assay and filter diffusion, giving a qualitative assessment of cytotoxicity (Table 7.9). 

Figure 5.8 Quantification of bacterial virus by plaque assay using the agar overlay technique.

Figure 2-2 Bioluminescent Salmonella assay workflow. The luminescent histidine-dependent cells are exposed to tested compounds in agar overlay containing only traces of histidine in multiwell-plate format. The reverse-mutation events restore endogenous histidine synthesis resulting in luminescent colonies of histidine-independent cells that can be visualized via CCD camera. The fully automated instrument in conjunction with automated image analysis of plates enables the analysis of 100 plates in one run.

Figure 1.4. Diagram of a microscope slide assay. Agar drops containing a suspected antifouling compound are applied to a microscope slide and covered with an agar overlay. The microscope slide is then placed into a suspension of the test microbe, and after a predetermined incubation time, the slide is removed and the number of attached microorganisms counted by microscopy. Modified from Wahl et al. (1994).

For biocompatibility testing using cytotoxicity (ISO 10993-5), the samples are either tested directly, as in an agar overlay test, or are extracted, as in minimum essential medium elution. Extraction is a process in which the test material is typically subdivided, placed in an extraction vessel, and covered with the exttaction vehicle. Polar and nonpolar extraction vehicles are used separately. Examples of polar extraction vehicles are water, cell culture media, and physiological saline, that is, 0.9 % aqueous NaCl solution, which is usually the preferred polar vehicle for biological assays. Examples of nonpolar vehicles include cotton seed oil and sesame seed oil. 

In contrast to placing the testing material directly on a monolayer of cells as in the direct contact test, the agar diffusion assays use a monolayer of cells with a semisolid agar or agarose overlay (3-4imn) on which the material sample is placed (Fig. 7.23). Agarose is... 

Micrococcus luteus (ATCC 9341) Assay. Mueller-Hinton agar plates were overlayed with 0.7% Difco Bacto-agar containing approximately 2.5 x 10 cells of M. luteus/ml. When the agar is set, 15 yl of... 

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