Agar assay

Moved] Cranberry fruit of Early Black cultivar was fractionated chromatographically and fractions were analyzed for flavonoid content. The effects of the flavonoid fractions and ursolic acid, an abundant triterpenoid in cranberry peel, were assessed in two models of colon cancer and one model of breast cancer. Clonogenic soft agar assays were used to determine the effect of these compounds on tumor colony formation in HCT-116, HT-29 and MCF-7 cells. MTT and trypan blue assays were performed to assess their ability to inhibit tumor cell proliferation. TUNEL assays were performed to assess apop-totic response to the cranberry compounds. The proanthocyanidins inhibited tumor colony formation in HCT-116 and HT-29 cells in a dose-dependent manner, with greater effect on the HCT-116 cell line. Ursolic acid strongly inhibited tumor colony formation in both colon cell lines. These compounds also decreased proliferation in all three tumor cell lines with the HCT-116 cell line most strongly affected. (150 words)... 

Thierbach R, Steinberg P (2009) Automated soft agar assay for the high-throughput screening of anticancer compounds. Anal Biochem 387 318-320... 

Soft agar assay The soft agar assay measures the anchorage-independent growth of cells on a soft agar layer, and visualizes the colonies formed. 

The disadvantages of these assays are their relative long time frame (10-21 days) and, in case of the soft agar assay, poor solubility in the agar of many compounds. 

Passive. . Active (colony hybridization). . (continuous cultivation) (agar-assay, liquid assay) ... 

Cyclic Amide Amidohydrolase Agar Assay. Microbial strains obtained from the German Collection of Microorganisms were inoculated in recommended DSM medium supplemented with 1 g/1 hydantoin (Sigma Chemical Co., MO., USA). They were then streaked and further inoculated as single colonies on solid medium (2% agar pH 7.0) containing per litre 5 g... 

Species DSM number Agar assay (dihydrouracil) Liquid assay (U/ml) (hydantoin)... 

L. Reagor, J. Gusman, L. McCoy, E. Carino, J. P. Heggers (2002) The effectiveness of processed grapefruit-seed extract as an antibacterial agent I. An in vitro agar assay. J. Altem. Complement. Med 8, 325-332. 

Assay Methods. The primary assay for the streptovaricins is the microbiological assay using the agar diffusion method or a turbidimetric procedure (60). The streptovaricins can also be identified by paper (60,88) or thin-layer chromatography (3). 

Lowest twofold dilution giving zone of inhibition in agar-weU diffusion assay. 

The solution is placed in contaet with agar, which is already inoculated with the test organism and after ineubation zones of inhibition observed. The solution may be placed in a small eup sealed to the agar surfaee (a method used widely in anhbiotic assays) in a well cut from the agar with a sterile cork-borer, or applied in the form of an inpregnated disc of filter paper (Fig. 11.5 A). 

In the commonest form of microbiological assay used today, samples to be assayed are applied in some form of reservoir (porcelain cup, paper dise or well) to a thin layer of agar seeded with indicator organism. The drug diffuses into the medium and after incubation a zone of growth inhibition forms, in this case as a circle around the reservoir. All other factors being constant, the diameter of the zone of inhibition is, within limits, related to the concentration of antibiotic in the reservoir. 

Due to the inherent variability of these assays either by agar-plate diffusion measurement or turbidimetry measurement, the fiducial hmits are calculated according... 

Agar dif fu.sion assays were performed on medium YNB with 20 pg piA per filler disk. 

In our laboratories the following organisms have been selected as representative and qualitative antimicrobial assays are carried out using the agar well diffusion assay procedure (5) ... 

Figure 5.10. Protein complementation assay using murine DHFR. The F[l,2] and F[3] fragments are each fused to the homodimerizing GCN4 leucine zipper protein. A. Transformation of both Z-F[l,2] and Z-F[3] constructs results in reconstituted DHFR and growth of E. coh on agar plates containing trimethoprim. B. Transformation of Z-F[l,2] or Z-F[3] alone does not result in trimethoprim resistant E. coli cells. Figure adapted from Pelletier et al. (1998).

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

---