As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed [1]. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

<p>Fluorescent sensors provide a powerful analytical tool for the intracellular detection of metal cations. In some cases, these fluorescent metal-chelating sensors have helped elucidate the function of metal cations within complicated cellular systems. However, most measure or sense changes in the bulk concentration of a metal species and do not respond to those involved in a specific cellular event. For instance, misregulated copper and iron are implicated in neurodegenerative disease and cancer because of their ability to catalytically propagate the formation of the hydroxyl radical through reaction with hydrogen peroxide. A fluorescent sensor that is unresponsive to metal binding until activation by intracellular hydrogen peroxide could potentially pinpoint the location of this oxidative reaction and provide an understanding of the relationship between copper/iron and hydrogen peroxide. </p><p> Described here is the development of two fluorescent prochelators that show a selective fluorescence response to iron or copper only in the presence of hydrogen peroxide. A boronic ester masked spirolactam-based prochelator displays a copper-selective turn-on response after oxidation with hydrogen peroxide in organic solvents as determined by absorbance and fluorescence spectroscopy. However, a competing mechanism occurs in aqueous solution due to hydrolytic instability of the masked prochelator and results in a separate copper-dependent turn-on response as verified by liquid chromatography-mass spectroscopy. A second fluorescent prochelator design relies on metal-dependent fluorescence quenching after oxidation of a self-immolative boronic ester in both organic and aqueous solvents. Cellular microscopy studies show that the sensor's fluorescence intensity is unchanged until incubation with exogenous hydrogen peroxide, which resulted in a decreased fluorescent signal that is restored upon competitive chelation. Both of these prochelators provide a template for future applications and designs with improved properties.</p><p> Two additional chapters describe the development of a UV-activated iron prochelator and a new fluorescently tagged metal chelator. The UV-activated prochelator is protected with two nitrophenyl groups that are photolyzed with 350 nm light within 10 minutes to reveal a high affinity iron triazole-base chelator. A chelator of this nature may provide protection from UV-induced iron liberation and oxidative stress. A second triazole-based chelator with an embedded coumarin fluorophore was prepared as a potential metal sensor. However, this design showed off-target fluorescence responses, thus it cannot be utilized in its current form for metal detection.</p> / Dissertation

Detection and treatment of bacterial kidney disease (BKD) was investigated. Experiments were conducted to evaluate the quantitative, fluorescent antibody technique (QFAT) that is used to detect, identify, and quantify both typical and 'bar form' Renibacterium salmoninarum cells. Smears of kidney tissue from naturally and artificially infected salmonids, both with and without chemotherapy, were quantitatively examined throughout the course of R. salmoninarum infections. Detection and quantification by QFAT has been reported to provide assessments of prevalence and severity of R. salmoninarum of individual fish. These assessments and the occurrence of 'bar forms' of R. salmoninarum have been used as an indication of recovery within a population. 'Bar forms' were observed in kidney tissue smears of fish that survived bacterial challenge when treated with erythromycin. The 'bar form' was also detected when rainbow trout were artificially infected with lower doses of live R . salmoninarum and in fish that were injected with irradiation-inactivated R. salmoninarum cells. By examining R. salmoninarum cultures in vitro by QFAT, it was determined that 'bar forms' did not occur on artificial media even when antibiotics were incorporated into the agar. When QFAT was compared to direct fluorescent antibody technique (DFAT) and quantitative enzyme linked immunosorbent assay (ELISA), it was determined that QFAT had similar sensitivity as ELISA but was more sensitive than DFAT. QFAT was also used to predict minimum mortality. Experiments were also conducted to evaluate drug regimes to treat both artificial and natural R. salmoninarum infections. Erythromycin was administered by intraperitoneal injection in different doses and at selected days post infection. Erythromycin decreased percent mortality and increased mean day to death, but did not completely eradicate R. salmoninarum from infected test animals. Sarafloxacin and erythromycin were incorporated into daily ration of artificially infected test animals. Contrary to erythromycin, sarafloxacin did not decrease mortality or increase mean day to death when tested in vivo against R. salmoninarum. A new drug, A-77143, was tested in vitro to determine if it was bactericidal and its minimum inhibitory concentration. When A- 77143 was compared to other antibiotics, it had a relatively low minimum inhibitory concentration and was shown to be bactericidal against the eight strains of R. salmoninarum tested. / Graduation date: 1993

<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.

When cultivated properly in bioreactors, microalgae have been found to produce vast amounts of biomass. In the case of diatom cultivation where the organisms will fall out of suspension quite easily, paddle wheels or pumps are the primary means to maintain the necessary velocity in the raceway. This study will focus on the potentially harmful shear stress these devices may impart onto the organisms. The system used to impart shear stress to a diatom culture was a cone and plate viscometer. Cells were counted using a fluorescein diacetate staining method with a fluorescent and brightfield microscope. Under the white light all cells were visible while only the healthy cells were visible under fluorescent light. The sample was exposed to shear stress with the cone and plate viscometer at 6 Pascals for 10 minutes and compared against a non-sheared sample. For each sample, 5 pairs of white and fluorescent light images were captured, counted, and averaged. A non-sheared sample was paired with a sheared sample to calculate the decrease in cell viability. The slope was calculated from the plot of shear stress and cell viability for 9 strains. In each case shear stress resulted in a significant decrease in cell viability; however, there was no statistical difference between strains. While effective, this method would be impractical for a commercial algae cultivation facility as the viscometer in this study costs approximately $100,000. Therefore, tests were performed to determine if a rotary mixer could be substituted for the viscometer. The hypothesis was that the cell damage was a product of shear stress and exposure time. For the viscometer test, the shear exposure was 3600 Pa s. Two rotational mixer tests were performed, one at 1250 RPM for 7 hours and one at 313 RPM for 28 hours, providing the same 3600 Pa s shear exposure. After staining, cell viability decreased 35.62% and 11.07% in the 1250 RPM and 313 RPM test, respectively. This difference was significant compared to the 6.04% decrease in the viscometer test. The increased cell damage was attributed to turbulence in the mixer tests and the basis for further study.

Els trastorns genòmics són un grup de malalties genètiques causades per reorganitzacions cromosòmiques de segments de DNA de més de 1Kb que resulten de la inestabilitat del genoma en regions que presenten una arquitectura genòmica particular, caracteritzada per la presència de duplicacions segmentals (low-copy repeats – LCR). Els LCR afavoreixen el malaparellament d’aquestes regions durant la meiosi fent-les susceptibles a la recombinació homòloga no al·lèlica (non-allelic homologous recombination – NAHR) que genera diferents tipus de reorganitzacions cromosòmiques en funció de la orientació dels LCR, del número i tipus de cromàtides implicades en la recombinació. Entre les reorganitzacions cromosòmiques generades per NAHR, les delecions i duplicacions són les causants de la majoria de trastorns genòmics. Mentre que el risc de recurrència de trastorns genòmics s’ha establert per consens en ser inferior al 0.5%, recentment s’han descrit haplotips específics que podrien predisposar a aquestes regions a la NAHR i per tant incrementar el risc de transmetre certs trastorns genòmics a la descendència. Aquests haplotips de predisposició a la NAHR poden ser inversions de les regions crítiques o bé variacions del número de còpies dels blocs d’homologia que formen els LCR. La tècnica d'hibridació in situ fluorescent (FISH) en nuclis descondensats d'espermatozoides proporciona una eina útil per estimar la freqüència de NAHR a través dels seus productes. Pel que fa a l’anàlisi d’haplotips de predisposició, la tècnica de FISH sobre fibres estirades cromatina (Fiber-FISH), que permet obtenir una resolució de fins a 1 Kb, presenta un gran potencial per l’anàlisi de l’arquitectura genòmica dels LCR complexes. Així doncs, en aquest treball es va analitzar la freqüència de delecions i duplicacions de les regions 7q11.23, 15q11-q13 i 22q11.2, així com la freqüència d’inversions de la regió 15q11-q13 mitjançant FISH en espermatozoides en una sèrie d’individus control i de tres poblacions problema que consistien en pares amb descendència afecta per tres trastorns genòmics causats per deleció: la síndrome de Prader-Willi (SPW), la síndrome de Williams-Beuren (SWB) i la síndrome de DiGeorge (SDG). A més, es va utilitzar la tècnica de fiber-FISH per mapar LCR complexes. Les dades obtingudes en la població control varen indicar unes freqüències similars de delecions i duplicacions de les regions analitzades i unes freqüències d’inversions de la regió 15q11-q13 que eren un grau de magnitud superiors a les anteriors. Es van observar increments significatius d’anomalies cromosòmiques en espermatozoides d’individus amb descendència afecta per trastorns genòmics: en la població de pares amb descendència afecta per la SPW, 4 dels 16 individus van mostrar increments de delecions i inversions, mentre que 6 dels individus presentaven increments de delecions. Els resultats van suggerir que els increments d’anomalies en espermatozoides són independents de l’origen genètic de la SPW en la descendència. Aquests increments són un reflex de la inestabilitat que presenta la regió 15q11-q13 que la predisposa a diferents tipus de reorganitzacions cromosòmiques. En la població de pares amb descendència afecta per la SWB, 3 dels 15 individus mostraven increments significatius de delecions, mentre que en la població de pares amb descendència afecta per la SDG es van observar increments en 2 dels 10 individus analitzats. Aquests individus s’han de considerar de risc, en el sentit que presenten un risc incrementat de transmetre una anomalia cromosòmica a la descendència. L’anàlisi de les freqüències de delecions i duplicacions de diferents regions en aquests individus van mostrar que els increments d’anomalies cromosòmiques observats en espermatozoides d’individus amb descendència afecta per trastorns genòmics poden tenir el seu origen en la presència d’haplotips de predisposició o en un increment generalitzat de la susceptibilitat a la NAHR. Els increments significatius de delecions i inversions en espermatozoides d’individus amb descendència afecta per trastorns genòmics indiquen un increment de la freqüència del fenomen de NAHR intracromàtide. Es va demostrar que la tècnica de Fiber-FISH permet mapar LCR complexes i avaluar la presència de diferents haplotips. Els estudis de FISH en espermatozoides, dirigits a determinar la incidència de delecions i duplicacions, aporten una informació valuosa en el consell genètic reproductiu en pares amb descendència afecta per trastorns genòmics. Un resultat de FISH en espermatozoides alterat, independentment del valor numèric, s’interpretarà com l’evidència d’anomalies en el procés de recombinació i és indicatiu d’un factor de risc. / Genomic disorders are a group of human diseases caused by chromosomal reorganizations of DNA segments longer than 1 Kb resulting from the genomic instability of regions that show specific architectural features, characterized for the presence of low-copy repeats (LCR). LCR favor the mispairing of these regions during meiosis thus increasing their susceptibility to non-allelic homologous recombination (NAHR) that triggers different types of chromosomal rearrangements according with the LCR orientation, the number and type of chromatids involved in the event. Among the chromosomal rearrangements generated by NAHR, the most frequently involved in genomic disorders are deletions and duplications. NAHR events are thought to be sporadic, thus the risk of recurrence have been established in less than 0.5%. However, there have been recently described some haplotypes that could predispose these regions to NAHR thus increasing the risk of transmission of certain genomic disorders to the offspring. Those predisposing haplotypes are both inversions of the critical regions and copy-number variations of the blocks that build the flanking LCRs. The use of fluorescence in situ hybridization (FISH) on decondensed sperm nuclei provides a useful tool to estimate the frequency of NAHR events throughout its products. Besides, the methodology of FISH on stretched DNA fibers (Fiber-FISH) that allow a resolution up to 1 Kb, has a great potential for mapping complex LCRs. In this work we analyzed the frequency of deletions and duplications of the 7q11.23, 15q11-q13 and 22q11.2 regions, plus the frequency of 15q11q13 inversions using sperm-FISH in a series of control individuals and in three populations consisting on fathers with a deletion-caused genomic disorder affected offspring: Prader-Willi syndrome (PWS), Williams-Beuren syndrome (WBS) and DiGeorge syndrome (DGS). Furthermore, we used the Fiber-FISH methodology to study complex LCR. Data obtained in the control population showed equivalent frequencies of sperm deletions and duplications in the regions analyzed and frequencies of 15q11q13 inversions that were an order of magnitude higher than the previous anomalies. We observed significant increases of sperm anomalies in some individuals with genomic disorders affected offspring: in the populations of PWS fathers, 4 out of 16 subjects showed higher frequencies of deletions and inversions, while 6 subjects showed increases of deletions. Results suggested that the increases of sperm anomalies are independent of the genetic origin of the PWS in the offspring. The significant increases of sperm anomalies are a reflex of the genomic instability of the 15q11-q13 region that makes it prone to different types of chromosomal rearrangements. In the populations of WBS fathers, 3 out of 15 individuals showed significant increased frequencies of anomalies and in the population of DGS fathers we observed 2 out of 10 subjects that showed higher frequencies of anomalies. These subjects should be considered risky individuals, because they show an increased risk of transmission of genomic disorders to the offspring. From the analysis of deletions and duplications of different regions in each individual, our results pointed out that the increased frequencies of sperm anomalies had their origin in the presence of predisposing haplotypes and a generalized susceptibility to NAHR events. The significant increases of sperm deletions and inversions suggested that the fathers with genomic disorders affected offspring have an increased frequency of the intrachromatid NAHR mechanism. There have been demonstrated the potential of the Fiber-FISH methodology to map complex LCRs and for the assessment of variable haplotypes. The methodology of sperm-FISH for analyzing the incidence of deletions and duplications, offers valuable information for the reproductive genetic counseling in fathers with offspring affected by a genomic disorder. An altered sperm-FISH result, independently of the numerical value, will be interpreted as the evidence of anomalies in the process of recombination and, therefore, indicates a risk factor.

<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.

Water-soluble fluorescent metallic clusters at sizes comparable to the Fermi wavelength of an electron (~0.5 nm for gold and silver) were created and their photophysical properties were investigated at the bulk and single molecule levels. We employed biocompatible dendrimer and peptide to prepare a series of strong fluorescent gold and silver clusters with chemical or photo reduction methods. Facilitated by the well-defined dendrimer size, electrospray ionization mass spectrometry indicates that the fluorescent silver nanocluster size ranges from 2 to 8 Ag atoms. The correlation of emission energy with the number of atoms, N, in each gold nanocluster is quantitatively fit for the smallest nanoclusters with no adjustable parameters by the simple scaling relation of EFermi/N1/3, in which EFermi is the Fermi energy of bulk gold. The transition energy scaling inversely with cluster radius indicates that electronic structure can be well described with the spherical jellium model and further demonstrates that these nanomaterials are multi-electron artificial atoms. Fluorescence from these small metal clusters can be considered protoplasmonic, molecular transitions of the free conduction electrons before the onset of collective dipole oscillations occurring when a continuous density of states is reached. In addition, very strong single molecular Stokes and Antistokes Raman enhancement by fluorescent silver clusters was observed. Pushing to larger sizes, we also created ~ 2nm diameter glutathione encapsulated luminescent gold nanoparticles. Distinct from similarly sized but nonluminescent gold nanoparticles, these 2 nm gold nanoparticles show bright, long lifetime emission but no plasmon absorption. The emission might arise from charge transfer between gold atoms and the thiol ligand. Providing the missing link between atomic and nanoparticle behavior in noble metals, these highly fluorescent, water-soluble gold and silver nanoclusters offer complementary transition energy size scalings at smaller dimensions than do semiconductor quantum dots. The unique discrete excitation and emission and strong Stokes and antistokes Raman enhancement coupled with facile creation in aqueous solution open new opportunities for noble metal nanoclusters as biological labels, energy transfer pairs, and other light emitters in nanoscale electronics.

Fluorescent nano-diamond(FND) is an unique fluorescence bio-labeling materials, which exhibit good fluorescence yield, excellent photostability, and non-toxicity. The emission color of FND is determined by the defect centers in the diamond crystal. When the defect center composed of one vacancy and two nearest-neighborhood nitrogen substitutes, it forms a H3 center. H3 center has a zero-phonon line at 496nm , and a broadband green emission around 530 nm,. When the FND contains lots of H3 centers, the emission color is green, hence it¡¦s called green FND(gFND). Since H3 centers composed of two nitrogen substitutes, it is naturally to fabricate the gFNDs by diamonds with high nitrogen substitutes. However, H3 center is not the only products when the diamond contains many nitrogen substitutes, and high density of vacancies. Other type of defect centers (NV-, NV0, ¡K) exhibit lower energy gap, and quench the emission of H3 centers. In this thesis, it aims to study the spectroscopic homogeneity of the gFNDs. Comparing the intensity of the scattering images and the corresponding fluorescence images, it provides the information of the relation between particle size and the density of color centers. Furthermore, images with different color filters are compared to provide the information of the composition of defect structures. Fluorescence lifetime image is performed for the emission dynamics of the nano-particle. The results indicate that the decay lifetime has an relation to the emission intensity. When the nano-particle contains more color centers, it quenches the emission from H3 centers more.

Sponge are primitive multi-cellular organisms. They are important sources of secondary metabolites. In the previous studies indicated that the sponges harbor stable symbiotic microbial consortia. The mechanisms for maintenance and transmission of microbial consortia to the next generations are still not fully understood. The sponge, Cinachyrella australinesis, was chosen to further investigate relationship of the symbiotic bacteria within to the host. Fluorescent in situ hybridization ¡]FISH¡^was employed with non-specific ¡]EUB338¡^and specific oligonucleotide probes for bacteria. The sponge was cryo-sectioned¡]1£gm¡^and hybridized with fluorescent probes. The distribution and ratios of the bacteria in the sponge agreed with those of previous studies indicating that the symbiotic bacteria of C. australiensis are stable and endosymbiotic in nature.