Agar overlay

Plaques are essentially windows in the lawn of confluent cell growth. With bacterial viruses, plaques may be obtained when virus particles are mixed into a thin layer of host bacteria which is spread out as an agar overlay on the surface of an agar medium. During incubation of the culture, the bacteria grow and form a turbid layer which is visible... 

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

The soft agar overlays are prepared and maintained at 45°C prior to use. 

To 2.0 ml aliquots of soft agar overlay medium (0.6% and 0.5% sodium chloride in distilled water) containing a trace of histidine and excess biotin and maintained at 45° C in a dry block, add 100 pi at a solution of the test article. Use only one plate per dilution. 

Figure 5.10 Agar overlay screening procedure to screen microbial populations for hydantoinase activity (Morin, Hummel and Kula, 1986). A screen for dihydropyiidinase activity based on Schiff base formation with PDMB (upper panel) led to the identification of numerous strains with D-hydantoinase activity, which in combination with a D-carbamoylase is employed to produce D-amino acids.

A test-product was placed in each no. 6 well plate. Discs (6 mm dia.) prepared from porous fdter paper were placed in the test-product for a standard time (1 min) followed by transferring to a designated section of the agar/overlay plate. After 5 min, the plate was placed in an incubator (37°C) in the upside-down position. 

Figure I. Separation of PGM and AK electrophoresis was done for 22 hr at 6.5 V/cm at 4°C in a 1 mm, 14% starch gel prepared in JM Tris, EDTA, maleic acid, MgCl, = 7.4 tank buffer diluted 1 10. The PGM side of the gel was stained at 1-2 hours before the AK using an agar overlay technique at 37°C. The visualized bands are precipitated with fotmazan.

After adsorption add 5 ml agar overlay medium to each dish. This is made up as follows ... 

Biocompatibility at the Cell Culture Level. The evaluation of cytotoxicity on a cell culture agar overlay following the application of liquid MAP, liquid enzymatic crosslink catalyst, and extracts of crosslinked solids was done to de-... 

The mouse L-929 fibroblast line was cultivated in Eagle s Minimal Essential Medium (MEM) plus 10% calf serum. Cells were seeded in 100-mm-diameter cell culture plates at 4 x 106 cells per plate and allowed to become established for 24 hr prior to use. After the monolayer was washed, 10 mL of an agar overlay consisting of 2% Bacto-Agar and 2 x MEM was added to each plate and allowed to solidify. 

Biocompatibility at the Cell Culture Level. The liquid components, MAP and catechol oxidase, and extracts of two solid crosslinked materials were tested for cell culture toxicity and growth inhibition. The evaluation of cell culture cytotoxicity in an agar overlay method detects the impact on cells of any freely... 

MAP formulated with a crosslink catalyst is capable of bonding tissue to tissue as demonstrated with corneal stroma and bonding alloplastic materials to tissues, as shown in the bonding of a hydrogel to corneal stroma. Moreover, two adhesive formulations of MAP with catechol oxidase at different ratios were shown to be biocompatible in a cell culture agar overlay system. 

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 2-2 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.

Hydroxyquinoline plus blue RR salt 6-Bromo-2-naphthylcarbonaphthoxycholine iodide plus blue B salt Application of agar overlay containing a tetrazolium salt... 

Agar overlay with tetrazolium salt a-Naphthol plus dimethylpapraphenylenediamine a-Naphthylbutyrate plus diazo salt of fast blue RR Reduction of nitro blue tetrazolium salt Sodium a-naphthyl acid phosphate plus diazo salt of 5-chloro-o-toluidine... 

There are three basic methods which have been employed for evaluation of microbiological content on surfaces. These include ROD AC (Replicate Organism Detection and Counting) plates, swab testing, and agar overlay or rinse techniques. RODAC plates are the most commonly used of the surface monitoring methods. However, they are not suitable for irregular surfaces, in which case swab techniques are used. 

Figure 8 Compound 24 is a C3-isocyanide functional indole antibiotic that was isolated from an antibacterially active eDNA clone containing soil DNA. The active clone was identified in a top-agar overlay screen using Bacillus subtilis as the test organism. The isonitrile in this metabolite is biosynthesized in a single enzymatic step by IsnA using tryptophan and ribulose-...

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