find attachment it will useful to you. if possible search for any media that is used to detect the PHA production by bacteria, then directly go for chromatography.

Refer to the attachments

Hello Shailesh,

Best of luck for your work.

May be u can grow your microbial strains under rich carbon and limited nitrogen conditions and then stain them with Sudan Black...a simple protocol which detect the presence of lipid granules...if you find this staining positive. May be u can go for the granules extraction protocol...which is well described in all the research articles and then go for GC. But dont forget to prepare methyl esters of extracted PHA before applying to GC.

Best wishes,

Rachana Bhatt.

Thank you very much Chintan Bhagat ,Hema Ramchandran and Dr.Rachana Bhatt

Hi Shailesh Sawant,

Please refer to the following for the protocol of P(3HB) analysis using GC:

The P(3HB) content and composition in the lyophilized cell were determined using the gas chromatography (GC). Approximately 20mg of lyophilized cells were subjected to methanolysis in the presence of methanol and sulfuric acid [85%:15% (v/v)]. The organic layer containing the reaction products was separated, dried over Na2SO4, and analyzed by GC according to the standard method with benzoic acid as an internal standard (Braunegg et al., 1978). GC analysis was done using Shimadzu GC-2014 equipped with flame ionization detector (FID). High purity nitrogen was used as carrier gas at a static flow rate of 1.69 ml min-1. The products were separated in an ID-BP1 capillary column, 30 m x 0.25 mm x 0.25 µm film thickness (SGE). The injection split ratio was 20:1. The injection port and detector temperatures were set at 180 and 200 °C, respectively. Initial oven temperature was set at 110°C and maintained for 1 min, and further increased to a final temperature of 180°C (maintained for 2 min) at a heating rate of 15°C min-1.

Refer also to the actual Brandl et al, 1988 work, attached. My lab has used this, or a version of this PHA detection protocol with great success.

For initial screening/ selection nile red based plating works well (simpler as well). GC or GC-MS could detect % composition.. Brandl et al.,1988 really works..