Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems

Abstract

Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein-and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems.

Desulfotomaculum genus‐ and subgenus‐specific 16S rRNA hybridization probes for environmental studies

“…Treatment of PFA-®xed cells with cell wall lytic enzymes, such as lysozyme or mutanolysin, also increased the permeability of Gram-positive lactococci, enterococci and streptococci (Beimfohr et al, 1993), Streptomyces scabies hyphae (Hahn et al, 1992), and Microthrix parvicella (Erhart et al, 1997). Furthermore, ®xation with PFA for a short time (1 min) resulted in the successful permeabilization of several mycolic acid-containing actinomycetes (de los Reyes et al, 1997).…”

“…To determine optimal FISH conditions for probes S-G-Dtm-0229-a-A-18 and S-*-Dtm(bcd)-0230-a-A-18, 11 hybridization conditions were evaluated, representing formamide concentrations from 0% to 70% in 5% steps (from 0% to 30%) and in 10% steps (from 30% to 70%) in the hybridization buffer with equivalent formamide concentrations in the wash buffer. Equivalent formamide concentrations in the wash buffer were simulated using various concentrations of NaCl, based on empirical formulae (Stahl and Amann, 1991;de los Reyes et al, 1997). We initially attempted to determine the optimal formamide concentration by quanti®cation of probe-conferred¯uorescence for target and non-target cells for the different formamide concentrations using IPLAB SPECTRUM image analysis software (Signal Analytics).…”

“…Hybridizations were conducted for 2 h in the dark at 468C. The slides were rinsed with wash buffer (0.1% SDS, 20 mM Tris-HCl and Y M NaCl, where Y varied depending on the desired stringency and corresponded to the percentage formamide in the hybridization buffer; Stahl and Amann, 1991;de los Reyes et al, 1997), immersed in 50 ml of wash buffer at 488C for 20 min and stained for 1 min with DAPI solution (6.26 mg ml À1 DAPI in 0.1 M Tris-HCl, pH 7.2, and 0.9 M NaCl). Hybridizations with the Cy3-labelled bacterial probe (S-D-Bact-0338-a-A-18) were performed as described by Amann et al (1992).…”

High levels of nitrifying bacteria in intermittently aerated reactors treating high ammonia wastewater

“…Mixed liquor samples (3 mL) were collected from reactors and fixed with 3 volumes of PFA fixative on ice for 2 h, as previously described [12]. Fluorescent in situ hybridization (FISH) was performed as previously described [27] using Cy-3 labeled probes ( Table 2). Images were visualized with a Nikon Optiphot epifluorescence microscope (Nikon, Japan).…”

“…For slot-blot membrane hybridizations, oligonucleotide probes were 5 0 -end labeled with [c-32 P]ATP (ICN Radiochemicals, Irvine, CA) and T4 polynucleotide kinase (Promega Corp., Madison, WI) and purified with a Quickspin Oligo column (Roche Molecular Biochemicals, Indianapolis, IN). Membranes with immobilized RNA were hybridized as previously described [27] and washed at the appropriate wash temperatures ( Table 2). The results were expressed as percentages of the total rRNA as measured with the universal probe.…”