Agar format, screening

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.

Fig. 4.4 Phenotype of 4-hydroxybutyrate dehydrogenase-positive (A) and lipase/esterase-positive (B) E. coli clones. A, 4-Hydroxy-butyrate dehydrogenase-positive clones are marked by arrows. Tetrazolium indicator plates [37] containing 4-hydroxybutyrate as test substrate were employed for the screening procedure [9], Positive clones were identified by formation of a deep-red formazan inside the colonies. B, Lipase/esterase activity of the clones was detected on LB agar [32] containing tributyrin as test substrate [14]. Zones of clearance around the colonies were indicative for lipase/ esterase activity.

Semi-quantitative Screening in Agar-plate Formats 1161... 

Fig. 8.1. Screening for penicillin G acylase activity. A) Screening in agar plate formats using 6-nitro-3-(phenylacetamido)-benzoic acid (NIPAB) [106], Colonies secreting Penicillin G acylase activity stain a NIPAB-filter yellow. B) Screening in solution using phenylacetyl-MCA and periplasmic extracts without (open symbols) or with ) penicillin G acylases from Kluyvera citrophila, Proteus rettgeri and Escherichia coli respectively [65],...

A number of particular semi-quantitative screening methods in agar-plate formats depend on the secretion of (hydrolytic) enzymes. Clones that secrete active enzymes produce a so-called halo , i.e. a zone of clearing in turbid solid growth medium with suspended substrate. In many cases the size of the halo is a good measure for the amount of enzymatic activity released by the growing colony. 

Fig. 8.2. Stages of enzyme screening. Microorganisms are grown in flasks or deep-well microplates. Separation of single clones is achieved either by FACS or by plating on agar plates and subsequent colony picking into microplates. Finally, assays are carried out in microplate formats using whole bacteria or cell extracts. Alternatively, bacterial clones can be directly screened on the level of colonies on indicator agar plates or during FACS.

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