scholarly journals Novel Approaches in Genetic Toxicology of Pesticides by Applying Fluorescent in Situ Hybridization Technique

10.5772/13426 ◽  
2011 ◽  
Author(s):  
Davor Zeljezic ◽  
Marin Mladinic
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Hybridization Technique ◽  
Genetic Toxicology ◽  
Novel Approaches

scholarly journals 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 ◽  
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.

scholarly journals Technical Note: Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent In Situ Hybridization (FISH)

2011 ◽  
Vol 8 (8) ◽  
pp. 2075-2088 ◽  
Author(s):  
C. Borrelli ◽  
A. Sabbatini ◽  
G. M. Luna ◽  
M. P. Nardelli ◽  
T. Sbaffi ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Benthic Foraminifera ◽  
Fluorescent In Situ Hybridization ◽  
Microscopic Analysis ◽  
Technical Note ◽  
Physiological Status ◽  
Hybridization Technique ◽  
Marine Biota ◽  
Classical Studies

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.

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

2010 ◽  
Vol 7 (5) ◽  
pp. 7475-7503 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document