A novel and discernible plate assay method for the qualitative screening of bacterial keratinases

Abstract

Routinely, qualitative screening for keratinolytic activity exhibited by microorganisms is carried out on feather agar medium, containing keratin as the sole source of carbon and nitrogen. Formation of a clear zone of keratin hydrolysis may observed directly on feather agar medium or after flooding the plates with either 5% (w/v) trichloro acetic acid (TCA) or Commassie brilliant blue dye (CBB R-250) for 30 minutes followed by destaining. The main drawback of these methods is that the hydrolyzed area is not sharply discernible which makes judgment very difficult. Hence, an improved, novel and sharply discernible plate assay method is reported herein for the detection of extracellular keratinases production by bacteria and actinomycetes. In this method, feather agar plates are flooded with Gram’s iodine, which formed a bluish black complex with keratin but not with hydrolyzed keratin, giving sharp and distinct zones around the keratinase producing microbial colonies within 3 to 5 minutes. This new method being rapid as well as more effective than other methods in terms of visualization and measurement of zone size could easily be performed for screening a large number of microbial cultures.