Feb 10, 2026
Nile red staining protocol to quantify PHB in Saccharomyces cerevisiae cultivations
- Anna Ylinen1,
- Tuula Tenkanen1
- 1VTT Technical Research Centre of Finland Ltd
External link: https://doi.org/10.1371/journal.pcbi.1014087
Protocol Citation: Anna Ylinen, Tuula Tenkanen 2026. Nile red staining protocol to quantify PHB in Saccharomyces cerevisiae cultivations. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8xwxrv2w/v1
Manuscript citation:
Tenkanen T, Ylinen A, Jouhten P, Penttilä M, Castillo S (2026) PHA synthase variant design using a conditional variational autoencoder. PLOS Computational Biology 22(3). doi: 10.1371/journal.pcbi.1014087
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: February 09, 2026
Last Modified: February 10, 2026
Protocol Integer ID: 242904
Keywords: PHB, PHA, Nile red, phb in saccharomyces cerevisiae, nile red staining protocol, nile red staining, other phas as nile, pha granule, surface of pha granule, saccharomyces cerevisiae, other pha, phb, red staining, producing microbe, nile, other intracellular structures such as lipid droplet
Funders Acknowledgements:
Nessling Foundation
Grant ID: 201700259, 201800005
Centre for Young Synbio Scientists (CYSS) funded by Jenny and Antti Wihuri Foundation
Abstract
Nile red staining can be used to screen PHB producing microbes as it binds on the surface of PHA granules. This protocol has been modified from Spiekermann et al. (1999) (DOI 10.1007/s002030050681) and Zuriani et al. (2013) (DOI 10.1007/s12257-012-0607-z). We recommend three or four technical replicates for each sample. As a negative control you will need a strain that does not produce PHB or other PHAs as Nile red do not only bind to the surface of PHA granules, but also to other intracellular structures such as lipid droplets.
Troubleshooting
Before start
Prepare Nile red solution with a concentration of 30 mg/ml in DMSO.
Dilute all cultivation samples to same OD with water for example in a 1.5 ml Eppendorf tube. OD
2-5 is preferred but range 0.5-10 works as well. You will need 100 μl of diluted sample/ measurement. Therefore, 400 μl + a little extra is needed for 4 replicate measurements.
Pipet 100 μl of each cell sample (all samples now at same OD) to a black 96 well plate.
Pipet 20 μl of the Nile red solution (30 mg/ml in DMSO) to each well and mix by pipetting.
Incubate for 10 minutes in RT and measure the fluorescence with a fluorescence plate reader.
Protocol references
Spiekermann, P., Rehm, B.H.A., Kalscheuer, R., Baumeister, D., Steinbüchel, A., 1999. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Archives of Microbiology 171, 73–80. https://doi.org/10.1007/s002030050681
Zuriani, R., Vigneswari, S., Azizan, M.N.M., Majid, M.I.A., Amirul, A.A., 2013. A high throughput Nile red fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates. Biotechnology and Bioprocess Engineering 18, 472–478. https://doi.org/10.1007/s12257-012-0607-z