Concentration technique for Viable but Non-Culturable organisms
Muhammad Muhsin Fathuddin, Solomon John Obidah
Abstract
This protocol is based on existing protocols, such as those for resuscitating microbes, freeze-drying, and selective isolation. It merges the parts of the three protocols to create a new protocol for isolating microbes via resuscitation, centrifugation, and selective isolation. The first part involves resuscitating the microbes; the second part subjects the medium to centrifugation (concentration of the microbes); the third and final part involves the selective growth in a selective broth and subsequent growth on selective agar.
Before start
Wear necessary safety gear before commencing the work
Steps
Resuscitation of Microorganism
To the resuscitation broth media (e.g., peptone water, tryptic soy broth, Luria-Bertani broth, etc.), add the sample, making a 10% solution, i.e.,10mg in90mL.
Incubate the broth culture at 37oC for 24-48 hours
Centrifugation of the resuscitation media
The grown broth culture is then centrifuged5000rpm,4°C
Then, concentrate the cell suspension and decant the culture supernatant.
Selective medium growth of desired organism
Mix cell pellets with the desired selective medium broth. Suspend cells thoroughly by using a vortex mix100rpm until complete cell resuspension is achieved aseptically.
Then, incubate at the desired temperature24h 0m 0s, which can be extended up to168h 0m 0s.
From the above selective broth, subculture onto a selective agar for the desired organism.
incubate the selective agar plate at the required temperature for 24–48 hours. then observe for the desired growth and colony morphologies
Store the isolate for further tests (Biochemical, Molecular, etc.)
