problem in making a proper smear for nile blue staining of an unknown yeast - (Feb/02/2012 )

i have isolated an organism which looks like an yeast under the microscope and i want to find out if it is capable of producing polyhydroxybutyrate. the problem im facing is that the smear is loaded with weird looking things (not cells as i have performed gram staining and they wont ever stain). due to such interfering background im unable to confirm if the fluorescence is from cells or surroundings.

Yeast-like fungus should stain gram postive. Are you sure it;s a pure
culture?

As Phil Geis mentioned, make sure the culture is pure. Also, depending how you prepare Nile blue A, it might not get dissolved properly (hence the "weird looking things"). We're not sure what you saw exactly.

As for the cells fluorescing, then it's likely a positive for PHB. Then again, Nile blue is lipophilic so you know what this means: GC or HPLC. But by an large, a positive screening with Nile blue usually gives a positive results later on when you're confirming it by chromatography.

What I did with for staining during the time when the microscope wasn't available was I followed the methods of Kitamura and Doi (http://www.springerlink.com/content/e89612600770q703/).