Rapid and multiple in situ identification and analyses of physiological status of specific bacteria based on fluorescent in situ hybridization

Abstract. Benthic foraminifera are an important component of the marine biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically, these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but does not allow discrimination between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a new and useful approach to identify living cells possessing an active metabolism. Our work is the first test of the suitability of the FISH technique, based on fluorescent probes targeting the 18S rRNA, to detect live benthic foraminifera. The protocol was applied on Ammonia group and Miliolids, as well as on agglutinated polythalamous (i.e., Leptohalysis scottii and Eggerella scabra) and soft-shelled monothalamous (i.e., Psammophaga sp. and saccamminid morphotypes) taxa. The results from FISH analyses were compared with those obtained, on the same specimens assayed with FISH, from microscopic analysis of the cytoplasm colour, presence of pigments and pseudopodial activity. Our results indicate that FISH targets only metabolically active foraminifera, and allows discerning from low to high cellular activity, validating the hypothesis that the intensity of the fluorescent signal emitted by the probe is dependent upon the physiological status of cells. These findings support the usefulness of this molecular approach as a key tool for obtaining information on the physiology of living foraminifera, both in field and experimental settings.

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Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent in situ Hybridization (FISH)

Biogeosciences Discussions ◽

10.5194/bgd-7-7475-2010 ◽

2010 ◽

Vol 7 (5) ◽

pp. 7475-7503 ◽

Cited By ~ 1

Author(s):

C. Borrelli ◽

A. Sabbatini ◽

G. M. Luna ◽

C. Morigi ◽

R. Danovaro ◽

...

Keyword(s):

In Situ Hybridization ◽

Rose Bengal ◽

Benthic Foraminifera ◽

Fluorescent In Situ Hybridization ◽

Physiological Status ◽

Hybridization Technique ◽

Fish Analysis ◽

Type I ◽

Active Metabolism

Abstract. Benthic foraminifera are an important component of the marine living biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but this approach does not allow discriminating between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a potentially useful approach identifying living cells with active metabolism cells. In this work, we tested for the first time the suitability of the FISH technique based on fluorescent probes targeting the 18S rRNA, to detect these live benthic protists. The protocol was applied on the genus Ammonia, on the Miliolidae group and an attempt was made also with agglutinated species (i.e., Leptohalysis scottii and Eggerella scabra). In addition microscopic analysis of the cytoplasm colour, presence of pigments and, sometimes, those of pseudopodial activity where conducted. The results of the present study indicate that FISH targeted only live and metabolically active foraminifera. These results allowed to identify as "live", cells improperly classified as "dead" by means of the classical technique (Type I error) and vice versa to identify as dead the foraminifera without rRNA, but stained using Rose Bengal (Type II error). In addition, the comparative FISH analysis of starved and actively growing cells demonstrated that individuals with active metabolism were stained more intensively than starved cells. This finding supports the hypothesis that the physiological status of cells can be directly related with the intensity of the fluorescent signal emitted by the fluorescent probe. We conclude that the use of molecular approaches could represent a key tool for acquiring crucial information on living foraminifera specimens and for investigating their ecological role in marine sediments.

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1796: Analysis of Chromosomal Aneuploidy in Patients with Oligoasthenoteratozoospermia(OAT) Using Fluorescent in-situ Hybridization

The Journal of Urology ◽

10.1016/s0022-5347(18)31984-0 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 596-597

Author(s):

Joseph P. Alukal ◽

Bobby B. Najari ◽

Wilson Chuang ◽

Lata Murthy ◽

Monica Lopez-Perdomo ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Chromosomal Aneuploidy

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Faculty Opinions recommendation of Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.715897919.791352828 ◽

2012 ◽

Author(s):

Magdalena Zernicka-Goetz ◽

Anna Ajduk

Keyword(s):

In Situ Hybridization ◽

Clinical Experience ◽

Fluorescent In Situ Hybridization ◽

Preimplantation Diagnosis

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Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems

BMC Developmental Biology ◽

10.1186/1471-213x-11-43 ◽

2011 ◽

Vol 11 (1) ◽

pp. 43 ◽

Cited By ~ 87

Author(s):

Gilbert Lauter ◽

Iris Söll ◽

Giselbert Hauptmann

Keyword(s):

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Differential Detection ◽

Zebrafish Brain ◽

Detection Systems

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Fluorescent in situ hybridization (FISH): A useful diagnostic tool for childhood conjunctival melanoma

European Journal of Ophthalmology ◽

10.1177/11206721211030775 ◽

2021 ◽

pp. 112067212110307

Author(s):

Raquel María Moral ◽

Carlos Monteagudo ◽

Javier Muriel ◽

Lucía Moreno ◽

Ana María Peiró

Keyword(s):

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Pediatric Population ◽

Diagnostic Technique ◽

Histopathological Diagnosis ◽

Fish Analysis ◽

Conjunctival Melanoma ◽

Adequate Treatment ◽

First Case

Introduction: Conjunctival melanoma is extremely rare in children and has low rates of resolution. Definitive histopathological diagnosis based exclusively on microscopic findings is sometimes difficult. Thus, early diagnosis and adequate treatment are essential to improve clinical outcomes. Clinical case: We present the first case in which the fluorescent in situ hybridization (FISH) diagnostic technique was applied to a 10-year-old boy initially suspected of having amelanotic nevi in his right eye. Based on the 65% of tumor cells with 11q13 (CCND1) copy number gain and 33% with 6p25 (RREB1) gain as measured by the FISH analysis, and on supporting histopathological findings, the diagnosis of conjunctival melanoma could be made. Following a larger re-excision, adjuvant therapy with Mitomycin C (MMC), cryotherapy and an amniotic membrane graft, the patient has remained disease-free during 9 years of long-term follow-up. Case discussion: Every ophthalmologist should remember to consider and not forget the possibility of using FISH analyses during the differential diagnosis of any suspicious conjunctival lesions. Genetic techniques, such as FISH, have led to great advances in the classification of ambiguous lesions. Evidence-based guidelines for diagnosing conjunctival melanoma in the pediatric population are needed to determine the most appropriate strategy for this age group.

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Comparison of culture, polymerase chain reaction, and fluorescent in situ hybridization for detection ofBrachyspira hyodysenteriaeand “Brachyspira hampsonii” in pig feces

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638714563064 ◽

2014 ◽

Vol 27 (1) ◽

pp. 41-46 ◽

Cited By ~ 9

Author(s):

Bailey L. Wilberts ◽

Hallie L. Warneke ◽

Leslie P. Bower ◽

Joann M. Kinyon ◽

Eric R. Burrough

Keyword(s):

Polymerase Chain Reaction ◽

In Situ Hybridization ◽

Fluorescent In Situ Hybridization ◽

Chain Reaction ◽

Polymerase Chain

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Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽

10.3390/foods10071502 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1502

Author(s):

Jorge García-Hernández ◽

Manuel Hernández ◽

Yolanda Moreno

Keyword(s):

In Situ Hybridization ◽

Vibrio Parahaemolyticus ◽

Fluorescent In Situ Hybridization ◽

Potential Method ◽

Viable Count ◽

Hybridization Technique ◽

Fish Technique ◽

Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

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