Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH)

2009 ◽  
Vol 55 (4) ◽  
pp. 465-472 ◽  
Author(s):  
Ryohei Ueno
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Cell Walls ◽  
Fluorescent In Situ Hybridization ◽  
Rapid Identification ◽  
Oligonucleotide Probes ◽  
Logarithmic Growth Phase ◽  
Prototheca Wickerhamii ◽  
Algal Genus

Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

scholarly journals Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures

2000 ◽  
Vol 38 (2) ◽  
pp. 830-838 ◽  
Author(s):  
Volkhard A. J. Kempf ◽  
Karlheinz Trebesius ◽  
Ingo B. Autenrieth
Keyword(s):  
In Situ Hybridization ◽  
Blood Culture ◽  
Fluorescent In Situ Hybridization ◽  
Growth Index ◽  
Rapid Identification ◽  
Blood Cultures ◽  
Oligonucleotide Probes ◽  
Two Samples ◽  
The Family

Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients.

Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization

1998 ◽  
Vol 64 (8) ◽  
pp. 3059-3062 ◽  
Author(s):  
Elena Bidnenko ◽  
Carine Mercier ◽  
Josselyne Tremblay ◽  
Patrick Tailliez ◽  
Saulius Kulakauskas
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Cell Walls ◽  
Fluorescent In Situ Hybridization ◽  
Cell Level ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Identification Of Bacteria

ABSTRACT Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.

scholarly journals Development of Species-specific rDNA Probes for Giardia by Multiple Fluorescent In Situ Hybridization Combined with Immunocytochemical Identification of Cyst Wall Antigens

2005 ◽  
Vol 53 (8) ◽  
pp. 917-927 ◽  
Author(s):  
Stanley L. Erlandsen ◽  
Edward Jarroll ◽  
Peter Wallis ◽  
Harry van Keulen
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Fluorescent Antibody ◽  
Environmental Water ◽  
Small Subunit ◽  
Oligonucleotide Probes ◽  
Variable Regions ◽  
Different Strains ◽  
Species Specific

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17–22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

scholarly journals Differentiation of Methanosaeta concilii andMethanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy

1999 ◽  
Vol 65 (5) ◽  
pp. 2222-2229 ◽  
Author(s):  
Sylvie Rocheleau ◽  
Charles W. Greer ◽  
John R. Lawrence ◽  
Christiane Cantin ◽  
Louise Laramée ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Outer Layer ◽  
Fluorescent In Situ Hybridization ◽  
Methanosarcina Barkeri ◽  
Confocal Scanning Laser Microscopy ◽  
Oligonucleotide Probes ◽  
Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Confocal Scanning Laser

ABSTRACT Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe.Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcinaspecies. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

1998 ◽  
Vol 64 (9) ◽  
pp. 3336-3345 ◽  
Author(s):  
Alison H. Franks ◽  
Hermie J. M. Harmsen ◽  
Gerwin C. Raangs ◽  
Gijsbert J. Jansen ◽  
Frits Schut ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Blot Hybridization ◽  
Dry Weight ◽  
Oligonucleotide Probes ◽  
Fecal Flora ◽  
Dot Blot ◽  
Clostridium Histolyticum

ABSTRACT Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the speciesBacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and theClostridium lituseburense groups. Another probe was designed for the genera Streptococcus andLactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectalegroup. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectalegroup-specific probe detected a mean of 7.2 × 1010cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, andStreptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.

scholarly journals Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization

1999 ◽  
Vol 65 (12) ◽  
pp. 5554-5563 ◽  
Author(s):  
Edward F. DeLong ◽  
Lance Trent Taylor ◽  
Terence L. Marsh ◽  
Christina M. Preston
Keyword(s):  
In Situ Hybridization ◽  
Surface Waters ◽  
Fluorescent In Situ Hybridization ◽  
Oligonucleotide Probes ◽  
Planktonic Cells ◽  
Cell Hybridization ◽  
Group I ◽  
Cell Densities ◽  
Seawater Samples

ABSTRACT Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4′,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50–56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 105/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or “ghosts,” as was suggested in a previous report.

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