Dear Sir. Concerning your issue about the preparation of PHB standard for GC analysis. The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2 =0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots. Key words: Flow cytometry; Nile red; Poly-3-hydroxybutyrate; Herbaspirillum seropedicae; Azospirillum brasilense I think the following below links may help you in your analysis:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264536/pdf/1414-431X-bjmbr-1414-431X20165492.pdf

http://aem.asm.org/content/66/8/3415.full

https://pdfs.semanticscholar.org/0ae0/10e187cea17a8a0086d38399987bf794f8a8.pdf

Thanks

Thank you, Sir for your answer ^^

Here is a paper where the methylation conditions were studied. Their conditions are quite different from yours:

https://ac.els-cdn.com/S187661021735138X/1-s2.0-S187661021735138X-main.pdf?_tid=c6d06bb5-d44b-4bbd-8c12-06d4a58547fa&acdnat=1525957422_d2489eac10c36833eb2bd3dc8f39e268

thank you, sir for your answer.

Actually i have found that solvent evaporation during heating is the main reason of my problem.