Development and use of fluorescent in situ hybridization probes for the detection and identification of ''Microthrix parvicella'' in activated sludge
Development and use of fluorescent in situ hybridization probes for the detection and identification of ''Microthrix parvicella'' in activated sludge
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DOI：
10.1016/s0723-2020(97)80078-1
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发表时间：
1997-06-01
影响因子：
3.4
通讯作者：
Blackall, LL
中科院分区：
文献类型：
--
作者：
Erhart, R;Bradford, D;Blackall, LL
针对活性污泥膨胀和泡沫丝状菌“Microthrix parvicella”设计了4种16S rRNA定向寡核苷酸探针（MPA探针），并对其进行了原位检测和鉴定。一种成功渗透“M”的方法。开发了使用变溶素的“小”细胞。探针的杂交严格性是用活性污泥样品凭经验确定的，因为“M.不能培养“小细胞”以产生足够量的生物质。使用与大多数细菌的16S rRNA互补的探针（EUB 338）来确认M中存在足够数量的核糖体并且可接近。小囊菌“。没有一个广泛的纯培养物的细菌与任何MPA探针的阳性杂交信号。三个开发的探针（MPA60，MPA223和MPA645）是高度特异性的长丝形态鉴定为“M”。而一种探针（MPA650）需要使用两种竞争性探针才能高度M。“细小细胞”特异性。没有一个细胞在形态上被鉴定为M。与先前报道的高mol%G+C革兰氏阳性菌的探针杂交，得到阳性杂交信号。'' M。来自澳大利亚、法国和德国的活性污泥工厂中的“细小”丝状体结合了所有四种MPA探针，表明在这些国家中的每一个中存在相同的基因型。原位杂交探针和DAPI染色的结合显示了“M”的片段。含有大量多磷酸盐的细小纤维的核糖体含量低。由此，我们得出结论，多磷酸盐的储存可能是一种生存策略。小囊菌“。
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''.