Floating Filters, a Novel Technique for Isolation and Enumeration of Fastidious, Acidophilic, Iron-Oxidizing, Autotrophic Bacteria

Bruyn, Johanna C. de; Boogerd, Fred C.; Bos, P.; Kuenen, J.G.

doi:10.1128/aem.56.9.2891-2894.1990

Cited by 38 publications

(13 citation statements)

References 9 publications

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“…This observation is supported by the often reported close relationship between nitrite accumulation and total cell number in the batch cultures of ammonia oxidizers [29]. The direct counts of total cell numbers gave no indication of cell viability in cultures, and such information may only be deduced from MPN, macrocolony, or microcolony techniques [20–22]. MPN counting is the traditional tool to enumerate viable ammonia‐oxidizing bacteria, based on their ability to grow in liquid medium [5, 30].…”

Section: Discussionmentioning

confidence: 80%

“…Torrella and Morita [19] used the microcolony technique to study the relationship between cell size and colony formation of heterotrophic bacteria in sea water. In the 1990s similar techniques have been used to isolate and enumerate autotrophic iron‐oxidizing bacteria [20] and to enumerate viable bacteria in the marine environment [21]. Binnerup et al [16] and Winding et al [22] introduced the microcolony technique as a tool for studying the viability of soil microorganisms.…”

Section: Introductionmentioning

confidence: 99%

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Microcolony formation as a viability index for ammonia-oxidizing bacteria: Nitrosomonas europaea and Nitrosospira sp.

Hesselsøe¹,

Sørensen²

1999

FEMS Microbiology Ecology

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A microcolony technique was developed to estimate viability of autotrophic ammonia‐oxidizing bacteria. The technique was based on the ability of cells to divide on membrane filters. The numbers of microcolony‐forming units were followed in liquid batch cultures of two different type strains of Nitrosomonas europaea (ATCC 19718 and NCIMB 11850) and two isolates of Nitrosospira sp. In the Nitrosomonas europaea ATCC 19718 it was observed that a relatively small (<0.5%) fraction of the cells was able to form microcolonies when harvested during exponential phase, while this fraction became larger (10–25%) when the culture entered stationary phase. The same trend was observed for the Nitrosomonas europaea NCIMB 11850 and Nitrosospira isolates. By comparison, a larger and apparently constant fraction of cells showed viability as judged by conventional most probable number counts. The results indicate that a specific fraction of the cell population in batch culture is capable of microcolony formation. This fraction is strongly growth phase‐dependent and may represent a sessile cell type which is capable of surface growth on membrane filters.

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Section: Discussionmentioning

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Microcolony formation as a viability index for ammonia-oxidizing bacteria: Nitrosomonas europaea and Nitrosospira sp.

Hesselsøe¹,

Sørensen²

1999

FEMS Microbiology Ecology

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“…To obtain a pure culture, the enriched culture was repeatedly diluted to extinction until a culture with uniform cell morphology was observed under the light microscope (Eclipse 80i, Nikon) [23]. Thereafter, the most diluted tube indicating cell growth was further diluted and filtered through 0.2 µm polycarbonate filters (Whatman) [24]. The filters were floated on surface LSM medium in Petri dishes and incubated in plastic containers under 20 % methane atmosphere.…”

Section: Isolation and Cultivationmentioning

confidence: 99%

Methylococcus geothermalis sp. nov., a methanotroph isolated from a geothermal field in the Republic of Korea

Awala

Bellosillo

Gwak

et al. 2020

International Journal of Systematic and Evolutionary Microbiology

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A Gram-stain-negative, aerobic, non-motile and coccoid methanotroph, strain IM1T, was isolated from hot spring soil. Cells of strain IM1T were catalase-negative, oxidase-positive and displayed a characteristic intracytoplasmic membrane arrangement of type I methanotrophs. The strain possessed genes encoding both membrane-bound and soluble methane monooxygenases and grew only on methane or methanol. The strain was capable of growth at temperatures between 15 and 48 °C (optimum, 30–45 °C) and pH values between pH 4.8 and 8.2 (optimum, pH 6.2–7.0). Based on phylogenetic analysis of 16S rRNA gene and PmoA sequences, strain IM1T was demonstrated to be affiliated to the genus Methylococcus . The 16S rRNA gene sequence of this strain was most closely related to the sequences of an uncultured bacterium clone FD09 (100 %) and a partially described cultured Methylococcus sp. GDS2.4 (99.78 %). The most closely related taxonomically described strains were Methylococcus capsulatus TexasT (97.92 %), Methylococcus capsulatus Bath (97.86 %) and Methyloterricola oryzae 73aT (94.21 %). Strain IM1T shared average nucleotide identity values of 85.93 and 85.62 % with Methylococcus capsulatus strains TexasT and Bath, respectively. The digital DNA–DNA hybridization value with the closest type strain was 29.90 %. The DNA G+C content of strain IM1T was 63.3 mol% and the major cellular fatty acids were C16 : 0 (39.0 %), C16 : 1 ω7c (24.0 %), C16 : 1 ω6c (13.6 %) and C16 : 1 ω5c (12.0 %). The major ubiquinone was methylene-ubiquinone-8. On the basis of phenotypic, genetic and phylogenetic data, strain IM1T represents a novel species of the genus Methylococcus for which the name Methylococcus geothermalis sp. nov. is proposed, with strain IM1T (=JCM 33941T=KCTC 72677T) as the type strain.

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“…To successfully cultivate a given microorganism, it is important that all its physiological and metabolic needs are met ( Leadbetter, 2003 ). Therefore, several innovative culturing approaches have been developed, including complex microfluidic and laser manipulation systems ( Fröhlich and König, 2000 ; Zhang and Liu, 2008 ; Liu et al, 2009 ; Yamaguchi et al, 2009 ), high-throughput cultivation technologies based on dilution-to-extinction and microencapsulation ( Connon and Giovannoni, 2002 ; Zengler et al, 2005 ; Ben-Dov et al, 2009 ), diffusion devices that allow the exchange of small molecules with the environment ( Kaeberlein et al, 2002 ; Bollmann et al, 2007 ; Nichols et al, 2010 ), filters and membrane systems to simulate the natural environment ( de Bruyn et al, 1990 ; Ferrari et al, 2008 ), co-culture approaches ( Ohno et al, 2000 ; Nichols et al, 2008 ), formulation of new media compositions based on metagenomic information ( Tyson et al, 2005 ) and establishment of growth conditions to mimic the natural environment with low-nutrient media and longer incubation times ( Song et al, 2009 ). Collectively, these approaches have made significant breakthroughs by increasing the diversity and recovery rates of bacteria retrieved in culture and enabling the cultivation of ecologically relevant microorganisms, like the SAR11 clade ( Rappé et al, 2002 ; Bollmann et al, 2007 ).…”

Section: Introductionmentioning

confidence: 99%

Sample Processing Impacts the Viability and Cultivability of the Sponge Microbiome

et al. 2016

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Sponges host complex microbial communities of recognized ecological and biotechnological importance. Extensive cultivation efforts have been made to isolate sponge bacteria, but most still elude cultivation. To identify the bottlenecks of sponge bacterial cultivation, we combined high-throughput 16S rRNA gene sequencing with a variety of cultivation media and incubation conditions. We aimed to determine the extent to which sample processing and cultivation conditions can impact bacterial viability and recovery in culture. We isolated 325 sponge bacteria from six specimens of Cymbastela concentrica and three specimens of Scopalina sp. These isolates were distributed over 37 different genera and 47 operational taxonomic units (defined at 97% 16S rRNA gene sequence identity). The cultivable bacterial community was highly specific to its sponge host and different media compositions yielded distinct microbial isolates. Around 97% of the isolates could be detected in the original sponge and represented a large but highly variable proportion (0.5–92% total abundance, depending on sponge species) of viable bacteria obtained after sample processing, as determined by propidium monoazide selective DNA modification of compromised cells. Our results show that the most abundant viable bacteria are also the most predominant groups found in cultivation, reflecting, to some extent, the relative abundances of the viable bacterial community, rather than the overall community estimated by direct molecular approaches. Cultivation is therefore shaped not only by the growth conditions provided, but also by the different cell viabilities of the bacteria that constitute the cultivation inoculum. These observations highlight the need to perform experiments to assess each method of sample processing for its accurate representation of the actual in situ bacterial community and its yield of viable cells.

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Floating Filters, a Novel Technique for Isolation and Enumeration of Fastidious, Acidophilic, Iron-Oxidizing, Autotrophic Bacteria

Cited by 38 publications

References 9 publications

Microcolony formation as a viability index for ammonia-oxidizing bacteria: Nitrosomonas europaea and Nitrosospira sp.

Microcolony formation as a viability index for ammonia-oxidizing bacteria: Nitrosomonas europaea and Nitrosospira sp.

Methylococcus geothermalis sp. nov., a methanotroph isolated from a geothermal field in the Republic of Korea

Sample Processing Impacts the Viability and Cultivability of the Sponge Microbiome

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