Development and Use of Fluorescent In Situ Hybridization Probes for the Detection and Identification of “ Microthrix parvicella” in Activated Sludge

Abstract

Summary Four 16S rRNA directed oligonucleotide probes (MPA probes) specific for the activated sludge bulking and foaming filamentous bacterium “ Microthrix parvicella ” were designed and evaluated for the in situ detection and identification of this organism. A method for successful permeabilization of “ M. parvicella ” cells employing mutanolysin was developed. Hybridization stringency for the probes was empirically determined with activated sludge samples because “ M. parvicella ” cannot be cultured to give adequate amounts of biomass. A probe complimentary to the 16S rRNA of most bacteria (EUB338) was used to confirm the presence and accessibility of sufficient numbers of ribosomes in “ M. parvicella ”. None of a wide range of pure cultures of bacteria gave positive hybridization signals with any of the MPA probes. Three of the developed probes (MPA60, MPA223 and MPA645) were highly specific for filaments morphologically identified as “ M. parvicella ” in activated sludge samples while one probe (MPA650), required the use of two competitor probes to be highly “ M. parvicella ”-specific. None of the cells morphologically identified as “ M. parvicella ” gave positive hybridization signals with a previously reported probe for high mol%G+C gram positive bacteria. “ M. parvicella ” filaments in activated sludge plants from Australia, France and Germany bound all four MPA probes suggesting that the same genotype is present in each of these countries. A combination of in situ hybridization probing and staining with DAPI showed segments of the “ M. parvicella ” filaments that contained large amounts of polyphosphates were low in ribosomes. From this, we concluded that the storage of polyphosphates could be a survival strategy for “ M. parvicella ”.