Single chains of conjugated polymers e.g. MEH-PPV (poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene) have become interesting objects for single molecule spectroscopy (SMS) studies. However, most of the experiments so far were performed without full awareness of the isolation status of the polymer chains in host matrices. We used steady-state and time-resolved fluorescence methods and 2D polarization single molecule imaging technique to unravel the isolation/aggregation status of MEH-PPV in spin-coated films prepared at different conditions. It turned out that a sample showing isolated bright spots in fluorescence images could be obtained in a very broad concentration range of MEH-PPV when toluene was used as a solvent and PMMA as a matrix. If the MEH-PPV concentration was not sufficiently low, a substantial fraction of the fluorescence spots should be assigned to individual nano-aggregates rather than truly isolated chains of the polymer. Contrary to single aggregates, truly isolated MEH-PPV chains showed blue-shifted emission spectra, mono-exponential fluorescence decay dynamics with relatively long lifetimes (0.4-1.2 ns), and high polarization anisotropy. We argue that insufficient control of the concentration in the published SMS studies of MEH-PPV resulted in incorrect assigning of some spectroscopic properties of single aggregates to isolated MEH-PPV chains. We believe this to be the main origin of discrepancies among the published data in this field.