ASSOCIATION OF POLYPHOSPHATE WITH PROTEIN IN FREEZE-SUBSTITUTED SCLEROTIA OF SCLEROTINIA-MINOR

In freeze-substituted sclerotia stained with aqueous toluidine blue O, metachromatic material was found throughout the cytoplasm in discrete granules. It was also distributed evenly throughout spherical and elo ngate protein bodies. This material stained at low pH and was extracte d by cold acid, indicating that it was polyphosphate. Retention of met achromatic material was much greater than previously reported in chemi cally fixed, conventionally processed sclerotia. X-ray microanalysis o f dry-cut, unstained sections of freeze-substituted sclerotia confirme d that phosphorus was distributed evenly throughout the protein bodies and was not localised in discrete granules but phosphorus levels in t he cytoplasm were very low. It is concluded that polyphosphate is lost during conventional preparation procedures but retained in dry-cut, u nstained sections of freeze-substituted material. However, when freeze -substituted sections were stained with toluidine blue O, water solubl e polyphosphate was extracted and subsequently precipitated in the cyt oplasm as polyphosphate granules. Therefore it is considered that poly phosphate granules are an artefact, and that protein bodies are the ma jor site for storage of phosphorus in this fungus.