Jul 03, 2024

Public workspaceConcentration technique for Viable but Non-Culturable organisms

DOI
dx.doi.org/10.17504/protocols.io.261ge53x7g47/v1
  • 1Ahmadu Bello University;
  • 2Modibbo Adama University
Muhammad Muhsin Fathuddin
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DOI: dx.doi.org/10.17504/protocols.io.261ge53x7g47/v1
Protocol Citation: Muhammad Muhsin Fathuddin, Solomon John Obidah 2024. Concentration technique for Viable but Non-Culturable organisms . protocols.io https://dx.doi.org/10.17504/protocols.io.261ge53x7g47/v1
Manuscript citation:
Chauhan, A., & Jindal, T. (2020). Microbiological methods for environment, food and pharmaceutical analysis. Springer Nature.
Fonseca, F., Cenard, S., & Passot, S. (2015). Freeze-drying of lactic acid bacteria. Cryopreservation and freeze-drying protocols, 477–488.
Oxoid (2024) CM0862 Listeria Enrichment Broth. (n.d.). Oxoid. Retrieved May 17, 2024, from http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0862&c=UK&lang=EN

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 16, 2024
Last Modified: July 03, 2024
Protocol Integer ID: 99930
Keywords: Centifugation, Selective Medium, Concentration, VBNC, Resuscitation
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.
Image Attribution
Image Attribution: The flowchart represents the process of bacterial isolation and cultivation from a food sample for laboratory analysis. The steps involved include 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.
Guidelines
Laboratory protocols must be followed, and the environment must be kept as sterile as possible.
Materials
Petri-dish, Centrifuge Machine, Falcon tubes, Wire-loop, bunsen burner, spatula, vortex shaker, incubator
Safety warnings
Aseptic techniques should strictly be followed
Ethics statement
No animals are harmed/used in this experimental setup
Before start
Wear necessary safety gear before commencing the work
Resuscitation of Microorganism
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.,Amount10 mg inAmount90 mL .

Incubate the broth culture at 37oC for 24-48 hours
1d
Incubation
Centrifugation of the resuscitation media
Centrifugation of the resuscitation media
30m
30m
The grown broth culture is then centrifugedCentrifigation5000 rpm, 4°C, 00:30:00
30m
Centrifigation
Then, concentrate the cell suspension and decant the culture supernatant.
5m
Selective medium growth of desired organism
Selective medium growth of desired organism
2d
2d
Mix cell pellets with the desired selective medium broth. Suspend cells thoroughly by using a vortex mixShaker100 rpm, 00:05:00 until complete cell resuspension is achieved aseptically.

Then, incubate at the desired temperatureDuration24:00:00 , which can be extended up toDuration168:00:00 .
1w 1d
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
1d
Store the isolate for further tests (Biochemical, Molecular, etc.)
Protocol references
Chauhan, A., & Jindal, T. (2020). Microbiological methods for environment, food and pharmaceutical analysis. Springer Nature.
Fonseca, F., Cenard, S., & Passot, S. (2015). Freeze-drying of lactic acid bacteria. Cryopreservation and freeze-drying protocols, 477–488.
Oxoid (2024) CM0862 Listeria Enrichment Broth. (n.d.). Oxoid. Retrieved May 17, 2024, from http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0862&c=UK&lang=EN