Journal articles on the topic 'Tubes dilution. eng'

Abstract An accurate blast count is pivotal in the diagnosis, classification and prognosis of patients with myelodysplasia. Blast counts in all previous classification schemes are based on morphologic assessment of marrow aspirates with a poor correlation to blast counts determined by flow cytometry. A significant problem in blast enumeration by flow cytometry is the variable hemodilution of the marrow during collection for flow cytometric analysis. Blast counts can vary depending on which aspirate tube is used for flow analysis, e.g., 2.4%, 1st 5ml tube; 0.62%, 2nd 5ml tube; 0.58% 3rd 5ml tube. Morphologists circumvent this problem by selecting a region for assessment close to a spicule with minimal blood dilution. Cell surface antigens can be used to distinguish mature cells found in blood as distinct from immature cells identified in marrow. CD16 intensity on neutrophils reaches a maximum at the band/segmented stage of development with a low coefficient of variation, thereby becoming a marker for mature myeloid forms. A simple method to distinguish immature from mature myeloid cells was developed to assess extent of blood contamination in marrow aspirates using a combination of CD16, CD13, and CD45. The average mature neutrophil content of a marrow was determined from phenotypically normal bone marrow biopsy specimens, assumed to have minimal blood contamination. The proportion of dimCD16 cells gated on the myeloid cells based on CD45 and right angle light scatter in 31 biopsy specimens was 82% (range 69–93, SD=6.2) (Figure 1). A value of 80% (rather than 82%) was used for the subsequent calculations to correct for the excess mature neutrophils found in an aspirate as compared to the biopsies (Corrected Blasts = [80 / % dim CD16 myeloid] x determined blast count). To test this hypothesis bone marrow aspirates were diluted with blood at different ratios to mimic blood marrow hemodilution. Blasts (defined as CD45 dim, low right angle light scatter, HLA-DR positive, CD11b negative) were determined for the various dilutions, then corrected based solely on the proportion of dim CD16 myeloid cells (Figure 2). A marrow from an MDS case was also diluted (1:5 v/v) with blood for comparison. The original marrow contained 80% dim CD16 myeloid cells with a blast count of 9.2%. After dilution, only 12% dim CD16 cells were detected with 1.1% blasts, however upon correction (6.67), the blast count was 7.3%, close to the original determination. This approach may provide for more standardization and consistency in the determination of blast counts in MDS marrow specimens using flow cytometric analysis. Figure 1, CD16 of marrow myeloid cells. Figure 1,. CD16 of marrow myeloid cells. Figure 2, Uncorrected/Corrected Blast Count Figure 2,. Uncorrected/Corrected Blast Count

ABSTRACT A number of hydrothermal vent sites exist on the summit of the Loihi Seamount, a shield volcano that is part of the Hawaiian archipelago. The vents are 1,100 to 1,325 m below the surface and range in temperature from slightly above ambient (10°C) to high temperature (167°C). The vent fluid is characterized by high concentrations of CO2 (up to 17 mM) and Fe(II) (up to 268 μM), but there is a general paucity of H2S. Most of the vents are surrounded by microbial mats that have a gelatinous texture and are heavily encrusted with rust-colored Fe oxides. Visually, the Fe oxides appeared homogeneous. However, light microscopy revealed that the oxides had different morphologies, which fell into three classes: (i) sheaths, (ii) twisted or irregular filaments, and (iii) amorphous oxides. A morphological analysis of eight different samples indicated that the amorphous oxides were overall the most abundant; however, five sites had >50% sheaths and filamentous oxides. These latter morphologies are most likely the direct result of microbial deposition. Direct cell counts revealed that all of the oxides had abundant microbial populations associated with them, from 6.9 × 107 to 5.3 × 108 cells per ml of mat material. At most sites, end point dilution series for lithotrophic Fe oxidizers were successful out to dilutions of 10−6 and 10−7. A pure culture was obtained from a 10−7 dilution tube; this strain, JV-1, was an obligate, microaerophilic Fe oxidizer that grew at 25 to 30°C. A non-cultivation-based molecular approach with terminal-restriction fragment length polymorphism also indicated the common presence of Fe-oxidizing bacteria at Loihi. Together, these results indicate that Fe-oxidizing bacteria are common at the Loihi Seamount and probably play a major role in Fe oxidation. A review of the literature suggests that microbially mediated Fe oxidation at hydrothermal vents may be important globally.

The aim of this study was to examine the preservation of sentul crossbreed chicken semen in ringer lactate egg yolk diluent supplemented with various monosaccharide. Semen was collected from three roosters using massage method. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen with more than 70% motility was divided into four tubes. Each of them diluted with ringer lactate egg yolk glucose (RLEYG), ringer lactate egg yolk fructose (RLEYF), ringer lactate egg yolk xylose (RLEYX) and ringer lactate egg yolk mannose (RLEYM). Semen was stored in refrigerator (5o C) for sixty hours and evaluated every twelve hours for spermatozoa motility and viability. Results showed that no significant difference (P>0.05) among diluents used on spermatozoa quality parameters after dilution and during preservation. Semen quality decrease during storage and at sixty hours of storage, the motility and viability of spermatozoa ranging from 48.33±2.56 to 55.42±2.26% and 58.59±2.87 to 64.83±2.42%, respectively. This research conclude that glucose, fructose, xylose and mannose can be used as energy source for roosters semen during preservation.

Background: In contrast to its role as poison, hydrogen sulfide (H2S) is recently considered as a gaso-transmitter which mediates important physiologic functions in humans. Evidence is accumulating to demonstrate that inhibitors of H2S production or therapeutic H2S donor compounds exert significant effects in various experimental models. Carbonic anhydrases (CA) are a group of zinc-containing metalloenzymes that catalyse the reversible hydration of carbon dioxide. CAs activity in erythrocytes (CAI and CAII) has recently been observed to be associated with various pathological conditions especially in diabetes mellitus, hypertension and lipid disorders. Alteration of this enzyme activity has been reported by the effect of advanced glycation end products methylglyoxal and reduced glutathione. Aims and Objectives: As H2S, being a mediator of many physiological functions and synthesized in vivo, may affect functions of many intracellular proteins like carbonic anhydrase, the objective of this study is to find out if there is any change in the carbonic anhydrase activity under the effect of H2S- donor NaHS in dose dependant manner using RBC model in vitro.Materials and Methods: Blood sample was collected from forty (40) numbers of healthy volunteers of 18-40 years of in heparin containing vials and packed cells were prepared immediately by centrifugation The packed erythrocytes were washed three times with normal saline and diluted (1:10) with the normal saline. One ml each of diluted packed cells was taken in eight test tubes. Serial dilutions of NaHS (1to 250 µMol/L) was added to all the test tubes except for the first test tube where only normal saline was added and incubated at room temperature for one hour. Haemolysates was prepared from the erythrocytes with equal volume of distilled water in each tube and the CA activity was determined in the haemolysates using standardized method.Results: There is significant increase of CA activity in dose dependent manner under the effect of NaHS and also compared to the activity of hemolysate prepared without NaHS. Conclusions:Our study for the first time demonstrated that the Carbonic Anhydrase activity of erythrocytes is significantly increases by the effect of NaHS and this study reveals some important biological role of H2S and carbonic anhydrase.Asian Journal of Medical Sciences Vol. 7(3) 2016 23-27

Ejaculates were collected twice a week from the bulls, via an artificial vagina, during two weeks. The suitable ejaculates obtained for sperm density (≥ 1.4 × 10 9 spermatoz oa / ml) and for motility (≥ 75%) were used for dilution and freezing of semen. A Tris - based extender (Tris 297.58mM, citric acid 96.32mM, fructose 82.66mM, egg yolk 15% (v/v), glycerol 5% (v/v), gentamicin 0.1 ml / 100ml, pH 6.8 - 7.0) was used as the base extender (cryopreservation diluent). Pooled ejaculate was split into 2 equal aliquots and diluted at 32 °C with base extender containing ferulic acid (100 μM) and no antioxidant (control), respectively. Each aliquot was diluted to a final semen concentrati on of approximately 1.2 × 10 8 sperm/ml (single step dilution), in 15 - ml polypropylene centrifuge tubes. After dilution, semen samples were kept at room temperature for 10 minutes then, the diluted semen samples were aspirated into 0.25 ml French straws, seal ed with polyvinyl alcohol powder and equilibrated at 5 °C for 3 h. After equilibration, the straws were frozen in liquid nitrogen vapour (4 cm above the liquid nitrogen, ~ - 100 o C ) for 10 min and then plunged into liquid nitrogen for storage, - 196 o C . In the study, sperm samples containing antioxidant and non - antioxidant were evaluated for spermatozoa motility and membrane integrity after freezing / thawing. In the present study, no statistically significant difference was found between the control and experim ental groups for motility and membrane integrity after freeze - thawing. The application consisted of 4 replications.

ABSTRACT Aim The aim of this study was to evaluate the minimum bactericidal concentrations (MBC) of root-end filling materials ProRoot MTA, MTA Angelus and IRM. Materials and methods Macrodilution broth method was used. Microorganisms used were: Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212) and Streptococcus mutans. Serial two-fold dilutions of root-end filling samples were prepared in macrodilution tubes with concentrations ranging from 1/2 to 1/512. The samples dilutions were incubated for 24 hours. After incubation, 0.1 ml of diluted culture was inoculated onto the surface of supplemented sheep blood agar (Merck, Germany) and all plates were incubated at 37°C in aerobic condition for 24 hours. The MBC was defined as the lowest concentration of root-end filling samples where no growth was recorded. Results MBC of both mineral trioxide aggregate (MTA) products against S. aureus were recorded as 15.62 mg/ml and for IRM 31.25 mg/ml MBC for both MTA groups against E. faecalis were recorded as 31.25 mg/ml and for IRM 62.5 mg/ml. MBC of all root-end filling samples against S. mutans were recorded as 62.5 mg/ml. Conclusion All tested root-end filling materials showed acceptable MBC against S. aureus and E. faecalis. Clinical significance All tested materials can be used safely for filling of a root-end cavity. How to cite this article Koçak MM, Koçak S, Oktay EA, Kiliç A, Yaman SD. In vitro Evaluation of the Minimum Bactericidal Concentrations of Different Root-End Filling Materials. J Contemp Dent Pract 2013;14(3):371-374.

Translocation of a green fluorescent protein (GFP)-tagged Dickeya sp. from stems or from leaves to underground parts of potato plants was studied in greenhouse experiments. Thirty days after stem inoculation, 90% of plants expressed symptoms at the stem base and 95% of plants showed browning of internal stem tissue. The GFP-tagged Dickeya sp. was detected by dilution plating in extracts of the stem interiors (100%), stem bases (90%), roots (80%), stolons (55%), and progeny tubers (24%). In roots, the GFP-tagged Dickeya sp. was found inside and between parenchyma cells whereas, in stems and stolons, the GFP-tagged Dickeya sp. was found in the xylem vessels and protoxylem cells. In progeny tubers, this strain was detected in the stolon end. Thirty days after leaf inoculation, the GFP-tagged Dickeya sp. was detected in extracts of 75% of the leaves, 88% of the petioles, 63% of the axils, and inside 25% of the stems taken 15 cm above the ground level. UV microscopy confirmed the presence of the GFP-tagged Dickeya sp. inside petioles and in the main leaf veins. No blackleg or aerial stem rot and no translocation of the GFP-tagged Dickeya sp. to underground plant parts was observed. The implications for contamination of progeny tubers are discussed.

The following dilutions -710X46, -610X46, -510X46 of Bacillus thuringiensis were used for bioassay against the different larval instar of the potato tuber moth Phthorimaea operculella by the spraying method, the results showed that there was no significant influence in the percentage of egg hatching in comparison with the control. The sensitivity of larval stages was reduced with the increasing the age and exposure period. The study also showed that the larvae infected with B.t. stopped feeding, movement and a general paralysis causing the death of larva after (24-48) hours , and the larva color was changed from the natural waxy colour to brown finally to the black after death.

Abstract Flow injection-inductively coupled plasma mass spectrometry has been evaluated for determining the distribution profile of trace elements along a single strand of hair. Hair was cut into several mm long sections from follicle to the distal end. Each section was solubilized in a capped 1.5-mL polypropylene tube with small volume of nitric acid (typically 50 microL) at room temperature. After dilution an aliquot (50 microL) was introduced into the mass spectrometer by flow injection. The limit of determination was typically 5-50 pg with 5-10% precision (CV), depending on the element examined; this corresponds to sub-microgram/g concentrations of these elements in hair segments. Recent exposure and intake history of individuals to thallium or mercury could be reconstructed by this system.

Abstract A chemical disinfectant against surface-associated bacteria typically uses carriers (e.g., glass disks) that are purposely contaminated with bacteria prior to disinfection. After disinfection, the bacteria are harvested by mechanically separating them from the carrier surface to form a suspension of cells in a dilution tube. Bacterial clumps in the tube are disaggregated using mechanical or chemical techniques, thereby creating a well-mixed suspension of single cells suitable for enumeration. Efficacy is quantified by comparing the viable cell count for a disinfected carrier to the viable cell count for sham-disinfected (control) carrier. A test is said to be biased (invalid) if the observed efficacy measure is systematically higher or lower than the true efficacy. It is shown here for the first time that the bias attributable to the harvesting and disaggregating steps of a disinfectant test can be measured. For some conventional biofilm harvesting and disaggregating techniques, laboratory checks showed either negligible bias or important bias, depending on the disinfectant. Quantitative bias checks on the harvesting and disaggregating steps are prudent for each combination of carrier material, microorganism, and disinfectant. The quantitative results should be augmented by microscopic examination of harvested disinfected and control carriers and of the disaggregated suspensions.

The recent Euro 4 and 5 environmental steps for L-category vehicles (e.g., mopeds, motorcycles) were mainly designed to reduce the emissions of particulate matter and ozone precursors, such as nitrogen oxides and hydrocarbons. However, the corresponding engine, combustion, and aftertreatment improvements will not necessarily reduce the solid particle number (SPN) emissions, suggesting that a SPN regulation may be necessary in the future. At the same time, there are concerns whether the current SPN regulations of passenger cars can be transferred to L-category vehicles. In this study we quantified the errors and uncertainties in emission measurements, focusing on SPN. We summarized the sources of uncertainty related to emission measurements and experimentally quantified the contribution of each uncertainty component to the final results. For this reason, gas analyzers and SPN instruments with lower cut-off sizes of 4 nm, 10 nm, and 23 nm were sampling both from the tailpipe, and from the dilution tunnel having the transfer tube in closed or open configuration (i.e., open at the tailpipe side). The results showed that extracting from the tailpipe 23–28% of the mean total exhaust flow (bleed off) resulted in a 24–31% (for CO2) and 19–73% (for SPN) underestimation of the emissions measured at the dilution tunnel. Erroneous determination of the exhaust flow rate, especially at cold start, resulted in 2% (for CO2) and 69–149% (for SPN) underestimation of the tailpipe emissions. Additionally, for SPN, particle losses in the transfer tube with the closed configuration decreased the SPN concentrations around 30%, mainly due to agglomeration at cold start. The main conclusion of this study is that the open configuration (or mixing tee) without any instruments measuring from the tailpipe is associated with better accuracy for mopeds, especially related to SPN measurements. In addition, we demonstrated that for this moped the particle emissions below 23 nm, the lower size currently prescribed in the passenger cars regulation, were as high as those above 23 nm; thus, a lower cut-off size is more appropriate.

Egg quality has been widely studied, mainly because defects in quality can pose risks to public health, as well as economic losses.Nevertheless, studies about fungiin eggsare scarce. The objective was to compare the fungal microbiota from washed and unwashed eggs in the rainy season and dry season of the year. This exploratory research consisted in the analysis of large size white table eggs acquired from 48 different lots. Two manufacturers were sampled considering the main characteristic of washed or unwashed eggs. From each lot, a 30-egg pack were purchased and six of those eggs were used for mycological analyzes. The eggs were analyzed externally with 0.1% peptone salt solution wash of the eggshells and internally with aliquots being sampled from a pool made from the six eggs content. Samples were inoculated in Potato Dextrose Agar and isolated colonies were passed to test tubes. When sporulated, the isolates were subjected to decimal dilutions using 0.1% Tween 80 to dissociate the conidia. Microcultures were carried out for optical microscopy observation of the reproductive structures of fungi, stained with lactophenol. Aspergillusspp. was the most frequently isolated fungi isolated, with A. nigerand A. flavuspredominant in the dry season, while A. fumigatusand A. terreusin the rainy season. Low numbers of fungi were identified from egg shells, with a higher amount from unwashed eggs. The seasondid not influence the numberof fungi in eggs, despite having influenced the fungal diversity.

In Togo, the abusive use of the root of Cassia sieberiana D.C. in traditional medicine, contributes gradually to the rarefaction of the species. The general objective of this study is to promote the use of vital organs of Cassia sieberiana in traditional medicine in Togo. The identification of secondary metabolites of the extracts (cyclohexane, dichloromethane and methanol) was carried out by GC-MS and by CL-MS / MS. The antibiotic susceptibility test was performed according to the well diffusion method and the MICs and MBCs according to the tube dilution method. Compounds such as sitosterol α-acetate, β-sitosterol, emodin, chaetochromine, luteolin, (±) -catechin, naringenin 5-O-rhamnoside, guibourtinidol- (4 alpha-> 6) -catechin and (-) - epiafzelechin are found in the root and in the stem bark. The identified molecules give the different methanolic extracts, an antibacterial effect on all the germs tested. At the end of this study, it appears that the chemical composition of the stem bark is almost similar to that of the root bark. The leaves would be better placed for the treatment of bacteria tested.

The ‘H2S test’ is being advanced for microbiological water quality testing where conventional coliform based methods are impractical or too expensive. It involves ambient temperature incubation of water samples with nutrient formulated to generate hydrogen sulphide when ‘faecal’ bacteria are present. Recently a WHO review identified several concerns including the limited number of comparative studies, formulation variability, and false positives and negatives. In response we have compared the H2S test's ability to detect and quantify faecal contamination in an aquifer impacted by septic tank leachfields with measurements obtained concurrently using conventional bacterial indicators, coliphages, faecal sterol biomarkers, Cryptosporidium and Giardia. Like these other analytes, H2S testing detected a contamination gradient ranging from high (septic liquid) to moderate (exfiltration zones), to background (e.g. domestic bores), corresponding to indicator removal + dilution by factors >106. Presence/absence tests could not distinguish between heavily and slightly contaminated waters, whereas multi-tube testing (e.g. 10 × 10 mL arrays) did. It was concluded that while the WHO review concerns are justified, the H2S test performance shows promise in sanitary survey work, can be improved by employing an mpn approach and has potential to aid in the protection of source water and identifying contaminated groundwater.

The removal of seminal plasma has a significant effect on semen characteristics. The aim of the study was to evaluate the effect of seminal plasma removal on sperm characteristics following semen dilution with Triladyl® or Bioxcell® extenders during cryopreservation. Semen samples were collected from 6 matured South African indigenous bucks for a period of 8 weeks by means of electro-ejaculation. Semen samples were pooled and then divided into 4 aliquots (Triladyl® -washed and non-washed or Bioxcell® -washed and non-washed) and diluted (1:4 vol/vol). Assessment of sperm motility characteristics was done by computer-aided sperm analysis (CASA) technology. Evaluation of mitochondria membrane integrity was done using JC-1 staining solution. Triladyl® and Bioxcell® washed semen sample groups were centrifuged at 1500 × g for 10 min, and seminal plasma was separated from sperm pellet using 1-mL plastic pipettes. After dilution of all semen sample groups, they were cooled by placing tubes into water (25°C) and then immediately placed in a 5°C fridge for equilibration for 2 h. At the end of equilibration period, all aliquot semen sample (final dilution concentration of 150 × 106 sperm/mL) groups were loaded into straws (0.25 mL) per treatment groups and placed horizontally 5 cm above the liquid nitrogen vapour for 10 min. At the end of freezing process, all semen straws per group were plunged directly into liquid nitrogen (−196°C) and stored until thawed. Frozen-thawed semen samples per treatment were analysed for sperm motility characteristics by CASA. JC-1 staining solution was also used during evaluation of mitochondria membrane integrity of frozen-thawed semen samples per treatment. Significant differences (P < 0.05) among mean values of semen parameters were determined by Tukey’s method. Sperm total motility rate of non-washed semen in Bioxcell® (85.0 ± 3.4) and Triladyl® (73.9 ± 13.8) was significantly reduced (P < 0.05) by cryopreservation compared with fresh (98.9 ± 1.2) semen sample. There was greater (P < 0.05) sperm mitochondrial membrane damage due to cryopreservation in non-washed semen group extended with Bioxcell® (50.2 ± 20.1) compared with semen extended with Triladyl® (31.3 ± 26.8) and fresh (16.6 ± 14.2) semen sample. Sperm total motility rate in Bioxcell® (68.2 ± 13.5) and Triladyl® (63.1 ± 15.1) groups on the non-washed semen were significantly reduced (P < 0.05) following cryopreservation, compared with fresh (98.3 ± 2.7) semen sample. The percentage of sperm with damaged mitochondria membrane in washed semen was significantly reduced (P < 0.05) in Triladyl® (21.6 ± 16.8) group compared with Bioxcell® (34.7 ± 14.9) group and fresh (35.0 ± 20.8) semen sample. In conclusion, seminal plasma removal reduced sperm motility rate in both extenders (Bioxcell® and Triladyl®) following post-thaw. In addition, sperm mitochondria membrane damage was higher in non-washed semen samples extended with Bioxcell® extender.

Buck slaugthering produce waste such as testicles including epididymis which contain fertile sperm. Utilization of cauda epididymis as the sources of sperm for producing goat frozen sperm was not reported yet. The aims of this study were improving the frozen-thawed sperm using stabilization and multistep methods which recovered from the waste of buck slaughtering as the source of sperma. Cauda epididymis spermatozoa which was washed then diluted using extender 1 (Tris-citrate-antibiotics) and extender 2 (extender 1- glycerol-egg yolk). The extender 2 addition was performed by single or multistep methods then freezed. Modification in the pre freezing proces were performed by comparing the conventional equilibration and stabilization methods. The sperm suspension was incubated in 4°C for 2 hours after filling-sealing into straws on the equilibration group whether the stabilization group was cooled in tube 15 mL. All cooled straws from both groups were placed 4 cm horizontally on liquid nitrogen surface for 10 minutes and then plunged into liquid nitrogen for storage. The evaluation of motility parameters such as pattern of the movement and motility percentation were done followed the standard methodology. The student t-test, correlation and one-way ANOVA were used for data analysis with P<0.05. The results showed that multistep dilution method could increase the motility (25.0 ± 1.8 %) compared with single step (18.3 ± 1.7 %). Pre freezing method with stabilization also resulted higher motility (24.2 ± 2.0 %) than equilibration method (17.5 ± 2.8 %). The pattern of the movement were not different between all methods and its combination. The multistep dilution method and stabilization cooling method as well as its combination seems could increase the quality of frozen-thawed cauda epididymis spermatpzoa of local buck.

Diazotrophic bacteria previously isolated from internal tissues of naturally regenerating lodgepole pine ( Pinus contorta var. latifolia (Dougl.) Engelm.) seedlings were tested for their ability to colonize and fix nitrogen (N) in pine germinants in two experiments. Surface sterilized pine seed was sown in glass tubes containing an autoclaved sand – montmorillonite clay mixture that contained a N-limited nutrient solution labeled with 15N as 0.35 mmol·L–1 Ca(15NO3)2 (5% 15N label). Pine seed was inoculated with one of three of the following bacterial strains: Paenibacillus polymyxa P2b-2R, P. polymyxa P18b-2R, or Dyadobacter fermentans P19a-2R, and seedlings grew for either 27 or 35 weeks. At the end of each plant growth period, P. polymyxa strain P2b-2R was detected in the pine rhizosphere but not inside plant tissues. Pine foliar N concentrations were not affected by bacterial inoculation but significant foliar 15N dilution was observed in seedlings treated with strain P2b-2R (30% and 66%, P < 0.05, in the first and second experiments, respectively). This strain also reduced seedling biomass in both experiments but effects were significant only in the second experiment (36%, P < 0.05). Notwithstanding the negative effect of bacterial inoculation on seedling growth, pine seedlings inoculated with strain P2b-2R derived 30% and 66%, respectively, of their foliar N from bacterial N fixation in two seedling growth experiments. These results demonstrate the possibility that some endophytic diazotrophs facilitate pine seedling growth in N-poor soils.

Preliminary studies found that progressive motility of ram sperm declined ~75% when stored at 4°C for 24 h, and continued to decline over time when using extenders supplemented with 5% egg yolk. The current study evaluated the effects of different combinations of extenders, ethylene glycol (EG), egg yolk, and penicillamine, hypotaurine, and epinephrine on ram sperm progressive motility during storage. Semen collected from 3 Katahdin and 2 Suffolk rams by electroejaculation was distributed across treatment combinations consisting of either TRIS citrate or milk extender supplemented with 5 or 20% (v/v) egg yolk, ± 1% ethylene glycol (EG) and ± 20 µM penicillamine, 10 µM hypotaurine and 2 µM epinephrine (PHE). For each semen collection, TRIS citrate extender was prepared from a 4× solution so that the TRIS, citric acid and fructose concentration were constant at 300, 94.7, 27.8 mM, respectively, regardless of semen dilution factor. A 4× milk extender was also used so that the extender contained 10% (w/v) milk powder, regardless of semen dilution factor. Both extenders were supplemented with 50 µg mL−1 of gentamicin. Semen was diluted in extender to a final concentration of 300 million sperm/mL in 1.5-mL tubes, and cooled to 4°C over a 2- to 3-h period. Semen was evaluated initially and daily for 3 days, using computer-assisted sperm analysis. Repeated-measures data were analysed using the mixed model (JMP 12.0 software; SAS Institute Inc., Cary, NC, USA) for main effects of extender, supplements, and their interactions. Nonsignificant interactions were removed from the model before reanalysis. Data are presented as LSMeans ± standard errors. Initially, sperm progressive motility averaged 41 ± 6.2% across treatments. After an initial decline, overall progressive motility did not change (P > 0.05) significantly (mean of 22.3 ± 1.6 and 23.05 ± 1.3% at 48 and 72 h, respectively). Over time and across treatment combinations, mean progressive motility was maintained to a greater extent (P < 0.01) by milk than TRIS-based extender (28.2 ± 1.1 v. 18.9 ± 1.1%, respectively). Across extenders, progressive motility of sperm was similar (P = 0.50) for 5 and 20% egg yolk (22.2 ± 1.4 v. 24.4 ± 1.4). Addition of 1% EG increased (P < 0.01) progressive motility (25.8 ± 1.05 v. 21.3 ± 1.05). Addition of PHE also increased (P < 0.01) progressive motility from 20.9 ± 1.04 to 26.3 ± 1.04%. There was an interaction between EG and % egg yolk, primarily due to an effect on sperm stored in TRIS citrate extender. Addition of 1% EG to extender containing 5% egg yolk improved (P < 0.01) progressive motility from 18.5 ± 1.5 to 26.9 ± 1.5%). Addition of 1% EG to TRIS citrate extender also increased (P < 0.05) progressive motility, from 14.6 ± 1.5 to 23.2 ± 1.5%. Results indicate that milk extender supplemented with 1% EG, PHE, and either 5 or 20% egg yolk is capable of maintaining progressive motility of ram semen at ~60% of its initial value when stored at 4°C for up to 72 h. Additional studies are needed to evaluate pregnancy rate after insemination of ewes with stored semen.

Ralstonia solanacearum (Smith) Yabuuchi, Kosako, Yano, Hotta, and Nishiuchi, the cause of brown rot of potato (Solanum tuberosum), was detected for the first time in Turkey in 1995 in five potato fields in the Nevsehir Province of the central Anatolia Region and was eradicated under measures mandated by the government. Occurrence of the pathogen was not reported in other parts of the country. However, in 2006, brown rot symptoms were observed in potato (cv. Marabel) fields in the Balikesir Province of the Aegean Region. Symptoms and signs included wilting, browning of stem vascular tissues, and ooze exudation from the transversely cut stem. On tubers, brown discoloration of the vascular ring was observed. Creamy bacterial ooze emerged from the vascular ring a few minutes after cutting. In advanced stages, bacterial slime oozed from the tuber heel end (stolon) and “eyes” causing soil particles to adhere. Isolation of bacteria from diseased stem and tuber tissues on mSMSA medium (1) consistently resulted in white, fluid colonies with red coloration in the center. On the basis of biochemical, immunofluorescence (IF), and real-time PCR tests, 10 representative isolates (one per affected field) were identified as Ralstonia solanacearum. They were further identified as biovar 2 according to metabolization of maltose, lactose, and D (+) cellobiose but not mannitol, sorbitol, and dulcitol. In the IF tests, fluorescent cells were observed at antibody dilutions from 200 to 12,800. The expected real-time PCR products were generated using biovar 2-specific primers (2). Pathogenicity tests were performed by injecting a bacterial suspension (106 CFU/ml) into the stem of 2-week-old tomato seedlings (cv. Alta F1). Inoculated plants (five plants per isolate) were incubated for up to 2 weeks at 25°C and 70 to 80% humidity. Wilting symptoms developed within 5 to 10 days. No symptoms were observed on controls inoculated with sterile water. The bacterium was reisolated and identified as R. solanacearum biovar 2 as described above. The incidence of the disease in the affected fields varied between 20 and 40%, and surveys showed that approximately 163 ha were infested. Phytosanitary measures that were taken included a prohibition of production of host plants in the infested areas, tracing and testing programs to identify the source of the bacterium, and measures to prevent any further spread of the bacterium to new areas. To our knowledge, this is the first report of R. solanacearum biovar 2 on potato in the Aegean Region of Turkey. References: (1) J. G. Elphinstone et al. EPPO Bull. 26:663, 1996. (2) M. Ozakman and N. W.Schaad. Can. J. Plant Pathol. 25:232, 2003.

Abstract Postnatal CD34+ cells expressing vascular endothelial growth factor receptor 2 (KDR) generate hematopoietic or endothelial progeny in different in vitro and in vivo assays. Hypothetically, CD34+KDR+ cells may comprise hemangioblasts bipotent for both lineages. This hypothesis is consistent with 2 series of experiments. In the first series, in clonogenic culture permissive for hematopoietic and endothelial cell growth, CD34+KDR+ cells generate large hemato-endothelial (Hem-End) colonies (5% of seeded cells), whereas CD34+KDR− cells do not. Limiting-dilution analysis indicates that Hem-End colonies are clonally generated by single hemangioblasts. Sibling cells generated by a hemangioblast, replated in unicellular culture, produce either hematopoietic or Hem-End colonies, depending on the specific culture conditions. Identification of endothelial cells was based on the expression of VE-cadherin and endothelial markers and with lack of CD45 and hematopoietic molecules, as evaluated by immunofluorescence, immunocytochemistry, and reverse transcription–polymerase chain reaction. Furthermore, endothelial cells were functionally identified using low-density lipoprotein (LDL) uptake and tube-formation assays. In the second series, to evaluate the self-renewal capacity of hemangioblasts, single CD34+KDR+ cells were grown in 3-month extended long-term culture (ELTC) through 3 serial culture rounds—that is, blast cells generated in unicellular ELTC were reseeded for a subsequent round of unicellular ELTC. After 9 months, 10% blasts from tertiary ELTC functioned as hemangioblasts and generated macroscopic Hem-End colonies in clonogenic culture. These studies identified postnatal hemangioblasts in a CD34+KDR+ cell subset, endowed with long-term proliferative potential and bilineage differentiation capacity. Although exceedingly rare, hemangioblasts may represent the lifetime source/reservoir for primitive hematopoietic and endothelial progenitors.

Abstract Introduction/Objective Significant technical issues are associated with methods used for the measurement of estradiol.The objective of this study was to qualify an electrochemiluminescent (ECL) assay for the quantification of 17β-estradiol (E2) in rat serum. Hemolysis has been identified as a factor that interferes with accurate measurement.The impact of hemolysis was also assessed. Methods Approximately 1.0 mL of whole blood was collected from male and female rats into separate red top tubes and processed to serum. The LoQ for E2 was evaluated by analyzing the low calibrator or at least 6 serum samples diluted to produce a value at the low end of the reportable range 8 times in the same run.The mean, standard deviation, and %CV were calculated for each sample.The data set was analyzed by plotting the data and determining the concentration at the intersection of the precision profile curve. Linearity of dilution was performed using commercially available calibration verification material and E2 stripped rat serum.The correlation coefficient, the slope, and the % Nominal were calculated. Intra assay precision was evaluated by analyzing 8 consecutive times in a single run one rat serum sample that was not diluted or spiked. This analysis was performed during the evaluation of the LoQ.The mean, SD and %CV were calculated. The interference of hemolysis with the E2 assay was tested by analyzing at least 5 rat serum samples/pools spiked with hemolyzed rat serum at different hemoglobin concentrations.The %RE was calculated. Results The LoQ assays were acceptable. For all samples tested, the % CV was less than or equal to 25%.The LoQ was verified to be 8.50 pg/mL. The %CV was 15.6%. For samples with estradiol concentrations below the LoQ, a value of 4.25 pg/ml was reported. Linearity of dilution for E2 was acceptable.The correlation coefficients were greater than or equal to 0.9000, the slopes were between 0.7500 and 1.2500, and the % nominals for each level were between 75-125%. The intra-assay precision was considered acceptable with a %CV of 8.6%. There was no hemolysis interference in the assay when samples were spiked with hemoglobin concentrations of up to 70 mg/dL, based on the %RE of less than or equal to 25% of non-hemolyzed samples. Conclusion Qualification of the ECL method, demonstrates the assay is suitable for the determination of E2 in serum samples from rats and absence of hemolysis interference up to 70 mg/dL of hemoglobin concentration.

Abstract Dietary fibers can influence a dog’s overall health, but high concentrations of soluble dietary fibers can cause soft stools. An in vitro model could be useful to predict the rate fibers are fermented once they reach the colon. Pet food companies are constantly searching for new ingredients to differentiate their products from competitors. Miscanthus grass (MG), pea fiber (PF), and sorghum bran (SB) are novel fiber sources that could be alternatives to standards like cellulose (CE) and beet pulp (BP). The objectives of the study were to determine the effects of fiber source on organic matter disappearance (OMD), estimated organic matter disappearance (EOMD), and fermentation end-product concentrations using an in vitro fermentation procedure and dog fecal inoculum. Total dietary fiber (TDF) residues from MG, CE, BP, PF, and SB were fermented in vitro with buffered dog feces. Fecal samples were collected and maintained in anaerobic conditions until the dilution and inoculation. Test tubes containing the fibrous substrates were incubated for 4, 8, and 12 h at 39 °C. Short-chain fatty acids (SCFA), branched-chain fatty acids (BCFA), OMD, and EOMD were determined for each fiber source and time point. Beet pulp had the highest OMD, EOMD, and SCFA production of all tested fiber sources (38.6% OMD, 26.2% EOMD, 2.72 mmol SCFA/g of substrate). Sorghum bran led to greater concentrations of BCFA (59.86 µmol/g of substrate) and intermediate OMD and EOMD compared to the other tested fibers. Cellulose and MG were poorly fermented with the lowest OMD, EOMD, SCFA, and BCFA compared to other fibers. In conclusion, MG could be used as an insoluble minimally fermentable replacement fiber for CE in dog foods.

The aim of this research was to know the effectiveness of guava filtrate supplementation in coconut water- egg yolk dilution on quality of liquid semen stored at 5oC of Bali cattle. Semen collected from a five year old Bali cattle using artificial vagina. Semen of good quality were kept in six tubes based on treatment then stored at 5oC. Treatments of the research were P0 : coconut water 80% + egg yolk 20% without guava filtrate; P1 : coconut water 80% + egg yolk 20% + 0.8% guava filtrate; P2 : coconut water 80% + egg yolk 20% + 0.9% guava filtrate; P3 : coconut water 80% + egg yolk 20% + 1.0 % guava filtrate; P4 : coconut water 80% + egg yolk 20% + 1.1 % guava filtrate and P5 : coconut water 80% + egg yolk 20% + 1.2 % guava filtrate. Each treatment was replicated 8 times making 48 experimental units. Results of the study showed that percentage mean of motility, viability, MPU, and TAU of spermatozoa after three days storage for P0 were : 42.20%, 41.85%, 39.08% and 40.90%; P1 : 50.40%, 53.89%, 52.99% and 54.67%; P2 : 54.67%, 56.97%, 54.51% and 54.36%; P3 : 17.00%, 29.96%, 29.64% and 29.64%; P4 : 23.38%, 24.64%, 21.06% and 24.45%Jurnal Veteriner Maret 2019 Vol. 20 No. 1 : 20 -29 pISSN: 1411-8327; eISSN: 2477-5665 DOI: 10.19087/jveteriner.2019.20.1.20 Terakreditasi Nasional, Dirjen Penguatan Riset dan Pengembangan, online pada http://ojs.unud.ac.id/index.php/jvet Kemenristek Dikti RI S.K. No. 36a/E/KPT/201621PENDAHULUAN Salah satu solusi yang dapat digunakan untuk pengembangan program Inseminasi buatan (IB) secara cepat dan mudah pada sapi bali adalah penggunaan semen cair. Penggunaan semen cair dapat meningkatkan kinerja IB pada sapi bali di Nusa Tenggara Timur (NTT). Keunggulan lain semen cair dapat diproduksi menggunakan bahan pengencer herbal berbasis bahan lokal dan peralatan yang sederhana serta mudah diperoleh dan tidak tergantung dengan persediaan nitrogen cair. Hasil akhir dari metabolisme spermatozoa adalah terbentuknya radikal bebas berupa derivat oksigen di antaranya adalah single1 oksigen (1O2), tripel1 oksigen (3O2), superokside anion (O2-), hidroksil radikal (OH) dan nitrit oxide (NO-) yang semuanya disebut radical oksigen species (ROS). Single1 oksigen dapat merusak ikatan rangkap pada asam lemak sehingga dapat menyebabkan kerusakan Deoxyribo Nuclead Acid (DNA) dan protein. Single1 oksigen bila bereaksi dengan asam amino histidin akan membentuk enzim yang dapat menyebabkan denaturasi protein. Kerusakan spermatozoa pada penyimpanan suhu 5%C akibat radikal bebas dan cold shock inilah merupakan penyebab utama disfungsi semen (Sharma et al., 2000). Oksidasi fosforilasi yang terganggu menyebabkan peningkatan radikal bebas dalam semen. Kadar radikal bebas yang terganggu menyebabkan peningkatan radikal bebas dalam semen. Kadar radikal bebas yang tinggi dalam sel dapat mengoksidasi lipid, protein dan DNA. Lipid membran plasma semen memiliki fosfolipid dengan kadar yang tinggi menyebabkan semen rentan terhadap radikal bebas (Sanoeka dan Kurpisz, 2004). Antioksidan bertindak mengikat asam lemak tak jenuh dan mencegah terjadinyareaksi berantai. Pada proses penyimpanan semen akan terjadi kerusakan membran plasma spermatozoa akibat terbentuknya perioksidasi lipid. Antioksidan-pemutus rantai seperti yang terkandung dalam jambu biji dapat menghambat perioksidasi lipid dalam membran melalui radical peroxyl (RO) dan alkoxyl (ROO) pengurai. Pengunaan jambu biji yang difilter dalam pengencer air kelapa kuning telur dapat menjaga kualitas spermatozoa (motilitas, keutuhan akrosom, viabilitas dan morfologi spermatozoa) semen cair sapi bali selama penyimpanan pada suhu 5%C. Dosis jambu biji yang difilter yang terbaik dalam pengencer air kelapa kuning telur, akan terbaik pula dalam mempertahankan kualitas spermatozoa sampai tujuan IB. Adapun tujuan penelitian ini adalah menguji berbagai level pemberian filtrat jambu biji (FJB) dalam pengencer air kelapa kuning telur terhadap motilitas, viabilitas, membran plasma utuh (MPU) dan tudung akrosom utuh (TAU) spermatozoa sapi bali yang disimpan pada suhu 5%C.METODE PENELITIAN Penelitian ini telah dilakukan di Laboratorium Reproduksi milik Yayasan Wiliams dan Laura yang berlokasi di Tilong, Desa Oelnasi, Kec. Kupang Tengah, Kab. Kupang, Nusa Tenggara Timur, dan berlangsung selama delapan bulan. Materi yang digunakan dalam penelitian ini adalah semen sapi bali yang ditampung dari satu ekor sapi bali jantan berumur lima tahun milik Yayasan Williams dan Laura yang telah dilatih, memiliki performans yang baik, dan organ reproduksi normal. Pakan yang diberikan adalah hijauan berupa rumput dan legum dan pemberian konsentrat secukupnya (dedak padi dan jagung giling).and P5 : 9%, 21.25%, 17.56% and 19.30%. Result of statistical analysis showed that there were a significant effect (P<0.05) between treatment on motility, viability, MPU and TAU of spermatozoa of Bali cattle till the third day of storage. It can be concluded that the supplementation of guava filtrate 0.9% in dilution of coconut water 80% - egg yolk 20% had been able to maintain motility, viability, MPU and TAU of spermatozoa of Bali cattle till the third day of storage at 5oC.

Scales (Hemiptera, Diaspididae) are the dangerous crop and park-ornamental plant pests. They reproduce very quickly and cause great harm to plants, sometimes even lead to their complete destruction. Scales suck juices from plants, cause premature drying, dying and falling off leaves, dry branches, deformation of leaves, fruits and shoots, reducing annual growth of plants. Therefore the fight with these pests is rather topical in the agriculture. In this connection in this work the analysis of bioecological peculiarities of oleander scale on the territory of Azerbaijan, and also the detection of species content of parasites and predators, which regulate their number is conducted. We will mark that oleander scales in the wild there are the entomophages are vermin and predators that reduce their quantity. For realization of biological fight against people we studied the bioenvironmental features of wreckers, and also educed specific composition of vermin and predators that regulate their quantity. In a biological fight against these wreckers, one of basic questions is study of specific composition of these entomophages. The faunistic material on entomophages of this pest was collected from different biocenosis; the researched works were conducted in the laboratory and field conditions in Azerbaijan. The advanced and research studies that we conducted gave an opportunity to educe entomophages oleander scale that inflicts an enormous damage to the agricultural cultures and park-decorative plants. The method of breeding of effective types of entomophages is studied in laboratory terms. Firstly the biology of oleander scale on Apsheron peninsula and in Guba Khachmazskii area was studied. The results of long-term studies showed that oleander scale, having distributed on Apsheron peninsula, on olive trees gives 3 generations. Only adult female animals and maggots of I and II age spend winter. Awakening of the scales on olives takes place in March-April. In II and III decade of April the male animals begin their flight. In Guba Khachmazskii area the biology of this scale, dwelling on oleander bush was studied. On this plant the scale gives 3 generations. Young female animals, and also maggots of I and II age winter. As a result of the works conducted the following entomophages of oleander scale were detected: predator Rhyzobius lophanthae Blaisd, Chilocorus bipustulatus L., Chilocorus renupustulatus L; parasites: Aphytis chilensis Howаrd, Aspidiotiphagus citrinus Graw, Encarsia aurantii (Howard). The habitat of Rhyzobius lophanthae is Australia. At the end of the last century of this predatory beetle left to California, from there left to Italy and in other Mediterranean countries. In 1947 by chance was left to Georgia (Abkhazia). Maybe these useful predators in Azerbaijan were from Georgia. For diluting the entomophages from the local indigenous fauna the potato tubers were used, on which firstly the oleander scales, and then road-beetles Rhyzobius lophanthae, Chilocorus bipustulatus reproduced themselves. In the laboratory conditions the methods of diluting of these Coccinellidae was developed. Thus, firstly the way of diluting parasites of oleander scale was studied and developed – Aphytis chilensis Howard, Aspidiotiphagus citrinus Graw и Encarsia antantii (Howard). It was detected that predatory entomophage-chilocor in the natural conditions is ineffective, as their maggots and chrysalises are affected by other local parasites. In the laboratory conditions this beetle produce itself very well on the potato tubers, infected by oleander scale. However, we should note that among entomophage parasites Aphytis chilensis plays the huge role in destruction of scales. In dependence on the weather conditions this macrophage in the nature can give 3–4 generations. Also it was proved that beetle Rhyzobius lophanthae in the biological fight can be applied against all round scales. This predator is effective entomophage of oleander, olive scale, black pine-leaf scale, white peach scale, European fruit scale and cactus scale. In connection with this, Rhyzobius lophanthae can be applied in the biological fight against oleander scale and presently is irreplaceable and perspective entomophage.

<p>Serum pathogen-free (SPF) embryonated egg for culturing virus has three main steps: a) candling of egg, b) inoculation of egg and c) harvesting of inoculated egg. Furthermore, hemagglutination assay consists of only one step. The method is easy to perform. However, this experiment must be performed in a class 2 biosafety cabinet.</p><p><strong>INTRODUCTION</strong></p><p>More than 200,000 hospitalizations and around 36,000 deaths are associated with influenza infection every year in the United States. As a consequence of consecutive mutations, new influenza viral strains emerge that further complicate the process of drug and vaccine development. The unavailability of cost-effective and efficient methods for the propagation of virus limits the research and hence, further impedes the process of discovering a potent cure. Propagation of influenza virus using serum pathogen-free (SPF) chicken eggs as the source is being recognized as one of the promising, frequently – used, cost effective and efficient method. Therefore, high yield titer of viral stocks obtained from SPF chicken eggs, has resulted in its extensive use in research laborites especially in drug and vaccine production. However, the ability to reproduce in eggs differ with strains of influenza virus. Avian influenza viruses replicated in embryonated chicken eggs. During the incubation phase, the virus duplicates in the cells and make up the chorioallantoic membrane (CAM) — also called the chorialantois. The new viral particles are then produced and released into the allantoic fluid. In this paper we demonstrate how influenza virus is cultured in SPF embryonated chicken eggs and also substantiate the activity of anti-viral drugs by hemagglutination test.</p><p> </p><p><strong>MATERIALS AND EQUIPMENT </strong></p><p><em>Disposables</em></p><p> Glue or varnish</p><p> Needle (size: 22 gauge, 11/2 inch)</p><p> SPF embryonated chicken egg [purchased from (강남농장) Kang Nam Nong Jang, Nam Won City, Korea]</p><p> Sterile plastic pipettes (10 mL)</p><p> Sterilized tubes (15 mL) and rack</p><p> Syringe (1 mL)</p><p> Tips for micropipettors</p><p> Virus [Influenza virus H1N1 (A/ Korea/01/2009); (105.5 EID50/100 µL)].</p><p><em>General equipment and glassware</em></p><p> Automatic pipettor for serological pipettes</p><p> Egg candler</p><p> Egg incubator</p><p> Egg puncher</p><p> Humidifier to maintain 80% humidity environment</p><p> Measuring cylinder (100 mL; sterile one)</p><p> Multichannel pipettor (12 channel and 20-200 μL)</p><p> Single channel pipettor (2-200 µL and 100-1000 µL)</p><p> Sterilized forceps</p><p><em> </em></p><p><em>Major equipment</em></p><p> Desktop centrifuge</p><p> Freeze (-70ºC for storage facility)</p><p> pH meter</p><p> Refrigerator</p><p><em> </em></p><p><em>Solution</em></p><p> Alcohol (70%)</p><p> Ethanol: 30 mL</p><p> Distilled water: 70 mL</p><p> Chicken red blood cells (0.5% v/v)</p><p> Red blood cells: 0.5 mL</p><p> Phosphate buffer saline: 99.5 mL</p><p> Phosphate buffer saline, pH 7.2, (1x):</p><p> NaCl 8 g</p><p> KCl 0.2 g</p><p> Na2HPO 4 1.15 g</p><p> KH2PO 4 0.2 g</p><p> Make up to 1 liter with distilled water and adjust pH to 7.2</p><p> V-form shape 96 well microplate</p><p> </p><p><strong>VIDEO CLIPS</strong></p><p><a href="https://youtube.com/v/DL9UvZsVXjk"><em>In vitro </em>antiviral activity test using SPF embryonated eggs</a>: 7.5 min</p><p><a href="https://youtube.com/v/woSUvVgS4hw">Hemagglutination test</a>: 7.3 min</p><p><strong> </strong></p><p><strong>METHOD 1: PREPARATION OF VIRUS INOCULUM</strong></p><p><em>Candling of SPF egg</em></p><ol><li>Examine the SPF embryonated chicken egg with the help of an egg candler for any cracks or porous shell.</li><li>Discard eggs having cracks or any damage.</li><li>Label each egg with a specific identification number.</li><li>Incubate the eggs for 11 days in an egg incubator pre-adjusted at 35ºC.</li></ol><p><em> </em></p><p><em>Inoculation of egg </em></p><ol><li>Examine the pre-incubated egg again with egg candler before virus inoculation.</li><li>Discard infertile or damaged egg.</li><li>Place the egg with blunt end up into egg tray. Wipe the egg with 70% ethanol.</li><li>Punch a small hole in the shell over the air sac without damaging the egg using an egg puncher.</li><li>Locate the embryo and inoculate 100 µL of virus by inserting the needle into the hole to amniotic cavity.</li><li>Inoculate in two more eggs in same fashion using same syringe and needle. For statistical analyze and better results inoculate three eggs per specimen.</li><li>Discard disposable into a proper safety container.</li><li>Seal the egg hole with glue or varnish and incubate at 35 to 37ºC for 5 days.</li></ol><p><em> </em></p><p><em>Harvesting of inoculated egg </em></p><ol><li>Before harvesting egg is chilled at 4ºC for 4 hours or overnight.</li><li>Label sterilized plastic tube one per egg and arrange tubes in a rack.</li><li>Clean off the top of each egg with 70% ethanol.</li><li>Break the egg shell over the air sac carefully with sterile forceps. Push aside the allantoic membrane with the forceps.</li><li>Aspirate the allantoic fluid with the help of 1 mL or 10 mL pipette and place in a pre-labeled plastic tube (one per egg). Combine the amniotic fluid from the three eggs into one tube.</li><li>To remove any blood or cells, centrifuge harvested fluids at 3,000 rpm for 5 min.</li></ol><p>[The above protocol was followed from WHO manual on Animal Influenza Diagnosis and Surveillance with some slight modification]</p><p> </p><p><em>Check the activity of bacterial strains or drugs</em></p><p>The mixture of bacterial cell-free supernatant or drug and IFV H1N1 (10<sup>5.5</sup> EID<sub>50</sub>/0.1 mL) are injected into allantoic cavity of 11 days old SPF embryonated eggs and inoculated for 5 days at 35ºC on 80% humidity. </p><p><strong> </strong></p><p><strong>METHOD 2: HEMAGGLUTINATION TEST</strong></p><ol><li>Mix chicken RBS with 1x PBS (0.5% v/v).</li><li>Add 100 µL of samples (A, B, C, D), in first row of wells marked as A, B, C, D, respectively.</li><li>From 2<sup>nd</sup> to last row of microplate add 50 µL PBS, and perform 1:2 dilution.</li><li>Add 50 µL of diluted chicken RBS in each well.</li><li>Incubate at room temperature without disturbing the plate for 30 min.</li><li>Observe wells for positive and negative agglutination.</li></ol><p><strong> </strong></p><p>[For hemagglutionation assay 2-fold dilution of treated samples are made in 50 µL with phosphate buffered saline in V-form shape 96 well microplate. Furthermore, serially diluted samples are reacted with equal volume of 0.5% chicken red blood cell at room temperature to allow hemagglutination reaction (Seo et al., 2012, Rather et al., 2015)].</p><p> </p><p><strong>DISCUSSION</strong></p><p>Influenza contributes by a large extent to global burden of disease, therefore, rigorous work continues into understanding the pathogenesis of the disease. In order to accelerate research in this field several methods have been developed for the propagation of influenza virus. Here, we describe a technique to produce influenza virus in chicken eggs. The advantage of this method is that it is highly reproducible and results in large quantities of high titer influenza viral stocks, which is often necessary for <em>in vitro</em> studies. The method is also useful to screen the antiviral activities of different bacterial strains or drugs.</p><p><strong> </strong></p><p><strong>REFERENCES</strong></p><p>Rather IA, Choi KH, BajpaiVK, Park YH. Antiviral mode of action of <em>Lactobacillus plantarum</em> YML009 on Influenza virus H1N1. Bangladesh J Pharmacol. 2015; 10: 475-82.</p><p>Seo BJ, Rather IA, Kumar VJR, Choi UH, Moon MR, Lim JH, Park YH. Evaluation of <em>Leuconostoc mesenteroides</em> YML003 as a probiotic against low-pathogenic avian influenza (H9N2) virus in chickens. J Appl Microbiol. 2012; 113: 163-71.</p><p> </p><div><p><strong>PRECAUTION</strong></p></div><p> Take proper safety measures before handling Influenza viruses.</p><p> This experiment must be performed in a class 2 or higher Bio safety cabinet.</p><p> Only store specimens at -70ºC. Influenza viruses are unstable at -20ºC.</p><p> Avian influenza grows well at 35ºC or 37ºC.</p><p> Avoid any possible contamination.</p><p> </p><p> </p>

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5′ noncoding region (5′ NCR) of HCV. The probe contains a fluorescent reporter at the 5′ end and a fluorescent quencher at the 3′ end. The use of such a probe combined with the 5′-3′ nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5′ NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (103 to 107). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

ABSTRACT An anaerobic, H2-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H2-CO2, formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O2 in the headspace of static, horizontally incubated culture tubes; the concentration of O2 decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O2. In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H2 became significant end products when RD-1 was grown on glucose in the presence of O2. Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H2 were produced. When the concentration of O2 in the headspace exceeded 1% (vol/vol), supplemental H2 was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that ofClostridium glycolicum DSM 1288T, an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288T demonstrated that it had negligible H2- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C.glycolicum DSM 1288T. A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that ofC. glycolicum DSM 1288Tconfirmed that RD-1 was a strain of C.glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O2, (iii) oxic conditions favor the production of ethanol, lactate, and H2by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O2 might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O2.

To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P < 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P < 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P < 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P < 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

Shellfish are frequently contaminated by Campylobacter spp, presumably originating from faeces from gulls feeding in the growing or relaying waters. The possible health effects of eating contaminated shellfish were estimated by quantitative risk assessment. A paucity of data was encountered necessitating many assumptions to complete the risk estimate. The level of Campylobacter spp in shellfish meat was calculated on the basis of a five-tube, single dilution MPN and was strongly season-dependent. The contamination level of mussels (<1/g) appeared to be higher than in oysters. The usual steaming process of mussels was found to completely inactivate Campylobacter spp so that risks are restricted to raw/undercooked shellfish. Consumption data were estimated on the basis of the usual size of a portion of raw shellfish and the weight of meat/individual animal. Using these data, season-dependent dose-distributions could be estimated. The dominant species in Dutch shellfish is C. lari but little is known on its infectivity for man. As a worst case assumption, it was assumed that the infectivity was similar to C. jejuni. A published dose-response model for Campylobacter-infection of volunteers is available but with considerable uncertainty in the low dose region. Using Monte Carlo simulation, risk estimates were constructed. The consumption of a single portion of raw shellfish resulted in a risk of infection of 5–20% for mussels (depending on season; 95% CI 0.01–60%). Repeated (e.g. monthly) exposures throughout a year resulted in an infection risk of 60% (95% CI 7–99%). Risks for oysters were slightly lower than for mussels. It can be concluded that, under the assumptions made, the risk of infection with Campylobacter spp by eating of raw shellfish is substantial. Quantitative risk estimates are highly demanding for the availability and quality of experimental data, and many research needs were identified.

252 Background: Androgen receptor splice variant (AR-V) expression has previously been regarded as a negative predictive biomarker for response to abiraterone and enzalutamide in mCRPC patients. However, recent data questions this association. We designed a whole blood assay to detect AR-V7 and AR-V9, the two most abundantly expressed AR-Vs, and correlated expression with clinical outcomes in patients commencing abiraterone or enzalutamide. Methods: We developed a quantitative real-time polymerase chain reaction assay to detect AR-V7 and AR-V9 from whole blood collected in PAXgene tubes. The assay was applied to samples prospectively collected from 37 mCRPC patients prior to commencing abiraterone or enzalutamide, and at treatment cessation. Patients positive for either AR-V7 or AR-V9 were defined as AR-V-positive, and AR-V-negative if neither variant was detected. AR-V expression was correlated with PSA response rate (chi-square test) and PSA progression-free survival (PSA-PFS) (log-rank test). Assay sensitivity was determined by serially diluting RNA from VCaP prostate cancer cells (known to express AR-V7) to establish a lower limit of detection. Results: The median follow-up was 7.29 months (IQR 4.21-10.55); 9 of 37 patients (24%) were AR-V-positive. We observed similar response rates in AR-V-positive (6/9) and AR-V-negative (18/28) patients (66% vs. 64%, p = 0.896). PSA-PFS did not differ significantly between groups (9.2 months vs. not reached, p = 0.355). Two patients converted from AR-V-negative to AR-V-positive (PSA-PFS 3.35 and 0.60 months respectively), and one patient remained AR-V-positive at baseline and end-of-treatment sampling. The lower limit of detection for AR-V7 was 0.1%, and AR-V7/V9 was not detected in any of the 13 normal male controls. Conclusions: We developed a sensitive and specific whole blood assay for AR-V7 and AR-V9 detection in patients with mCRPC. Neither PSA response rates nor PSA-PFS differed significantly between AR-V-positive and AR-V-negative patients. These data support recent literature questioning the role of AR-V expression as a negative predictive biomarker in mCRPC patients receiving abiraterone or enzalutamide.

Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P < 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P < 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity. This work was supported by AGL-2003-03144.

β-Nerve growth factor (β-NGF) is a neurotrophin with different roles in reproduction that could be regulated by sexual hormone concentrations. The objective of the present study was to characterise β-NGF in the accessory glands and epididymis of male rabbit and to determine whether its expression differentially changed near puberty (week 22) and adulthood (week 37) according to serum testosterone levels and NGF levels in both serum and seminal plasma (SP). Semen and blood were collected every week from 6 males during 3 months and analysed at 3 time points (weeks 22, 30, and 37) by ELISA. The SP was separated from sperm cells by centrifugation (3,000 × g for 15 min at 4°C) and stored at –20°C; blood was collected in EDTA tubes, centrifuged 15 min at 700 × g at 4°C and stored at –20°C too. Reproductive tissues (prostate, bulbouretral gland, and caput and cauda of epididymis) were collected at the beginning (week 22; n = 4) and at the end (week 37; n = 4) of the experiment. For tissue recovery, males were killed and glands and epididymis were dissected, fixed in modified Bouin’s fluid, and mounted in paraffin. The ELISA for β-NGF (abx259154, Rabbit NGF ELISA kit, Abbexa, Cambridge, UK) and testosterone (DE1559, Demeditec Diagnostics, Kiel, Germany) were performed following the kit protocols. For immunohistochemistry (IHC), goat polyclonal anti-NGF antibody was used in a dilution 1:100 (N8773, Sigma Aldrich, St. Louis, MO, USA). The avidin-biotin-peroxidase complex (ABC) method was performed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and then slides were contrasted with hematoxylin. Results of serum testosterone levels revealed a significant decrease (P < 0.05) at week 37 (0.57 ± 0.18 ng mL−1) from week 30 (4.68 ± 0.86 ng mL−1) and week 22 (3.38 ± 0.99 ng mL−1). However, β-NGF levels at week 37 in serum (299.32 ± 60.22 pg mL−1) and in SP (947.29 ± 249.45 pg mL−1) also decreased but there were no significant differences. Also, we found no correlation between β-NGF in serum and SP and testosterone levels. β-Nerve growth factor was immunolocated in prostate of both ages where epithelial cells were highly stained. At 22 weeks old, stain was located mostly in the apical zone of the cytoplasm, whereas at 37 weeks old, the protein was localised in the entire cytoplasm of the cell. Furthermore, the interstitial tissue in adult males had a moderate stain in contrast with younger males, which did not show signal in that tissue. The content in the prostate lumen was stained too. Bulbourethral glands had a low signal in interstitial tissue that seems to be greater at week 37. Caput and cauda of epididymis of both ages were not stained. These results suggest that β-NGF has different immunolocation in the rabbit male accessory glands depending on the age of animal that could be related to changes in serum testosterone levels. However, these differences were not correlated with β-NGF levels neither in blood serum nor in SP. Research funded by AGL2015-65572-C2-2-R Grant and Predoctoral Contract UCM-Santander.

Abstract. Improving the constraints on the atmospheric fate and depletion rates of acidic compounds persistently emitted by non-erupting (quiescent) volcanoes is important for quantitatively predicting the environmental impact of volcanic gas plumes. Here, we present new experimental data coupled with modelling studies to investigate the chemical processing of acidic volcanogenic species during tropospheric dispersion. Diffusive tube samplers were deployed at Mount Etna, a very active open-conduit basaltic volcano in eastern Sicily, and Vulcano Island, a closed-conduit quiescent volcano in the Aeolian Islands (northern Sicily). Sulphur dioxide (SO2), hydrogen sulphide (H2S), hydrogen chloride (HCl) and hydrogen fluoride (HF) concentrations in the volcanic plumes (typically several minutes to a few hours old) were repeatedly determined at distances from the summit vents ranging from 0.1 to ~10 km, and under different environmental conditions. At both volcanoes, acidic gas concentrations were found to decrease exponentially with distance from the summit vents (e.g., SO2 decreases from ~10 000 μg/m3at 0.1 km from Etna's vents down to ~7 μg/m3 at ~10 km distance), reflecting the atmospheric dilution of the plume within the acid gas-free background troposphere. Conversely, SO2/HCl, SO2/HF, and SO2/H2S ratios in the plume showed no systematic changes with plume aging, and fit source compositions within analytical error. Assuming that SO2 losses by reaction are small during short-range atmospheric transport within quiescent (ash-free) volcanic plumes, our observations suggest that, for these short transport distances, atmospheric reactions for H2S and halogens are also negligible. The one-dimensional model MISTRA was used to simulate quantitatively the evolution of halogen and sulphur compounds in the plume of Mt. Etna. Model predictions support the hypothesis of minor HCl chemical processing during plume transport, at least in cloud-free conditions. Larger variations in the modelled SO2/HCl ratios were predicted under cloudy conditions, due to heterogeneous chlorine cycling in the aerosol phase. The modelled evolution of the SO2/H2S ratios is found to be substantially dependent on whether or not the interactions of H2S with halogens are included in the model. In the former case, H2S is assumed to be oxidized in the atmosphere mainly by OH, which results in minor chemical loss for H2S during plume aging and produces a fair match between modelled and measured SO2/H2S ratios. In the latter case, fast oxidation of H2S by Cl leads to H2S chemical lifetimes in the early plume of a few seconds, and thus SO2 to H2S ratios that increase sharply during plume transport. This disagreement between modelled and observed plume compositions suggests that more in-detail kinetic investigations are required for a proper evaluation of H2S chemical processing in volcanic plumes.

Abstract. Improving the constraints on the atmospheric fate and depletion rates of acidic compounds persistently emitted by non-erupting (quiescent) volcanoes is important for quantitatively predicting the environmental impact of volcanic gas plumes. Here, we present new experimental data coupled with modelling studies to investigate the chemical processing of acidic volcanogenic species during tropospheric dispersion. Diffusive tube samplers were deployed at Mount Etna, a very active open-conduit basaltic volcano in eastern Sicily, and Vulcano Island, a closed-conduit quiescent volcano in the Aeolian Islands (northern Sicily). Sulphur dioxide (SO2), hydrogen sulphide (H2S), hydrogen chloride (HCl) and hydrogen fluoride (HF) concentrations in the volcanic plumes (typically several minutes to a few hours old) were repeatedly determined at distances from the summit vents ranging from 0.1 to ~10 km, and under different environmental conditions. At both volcanoes, acidic gas concentrations were found to decrease exponentially with distance from the summit vents (e.g., SO2 decreases from ~10 000 μg/m3 at 0.1 km from Etna's vents down to ~7 μg/m3 at ~10 km distance), reflecting the atmospheric dilution of the plume within the acid gas-free background troposphere. Conversely, SO2/HCl, SO2/HF, and SO2/H2S ratios in the plume showed no systematic changes with plume aging, and fit source compositions within analytical error. Assuming that SO2 losses by reaction are small during short-range atmospheric transport within quiescent (ash-free) volcanic plumes, our observations suggest that, for these short transport distances, atmospheric reactions for H2S and halogens are also negligible. The one-dimensional model MISTRA was used to simulate quantitatively the evolution of halogen and sulphur compounds in the plume of Mt. Etna. Model predictions support the hypothesis of minor HCl chemical processing during plume transport, at least in cloud-free conditions. Larger variations in the modelled SO2/HCl ratios were predicted under cloudy conditions, due to heterogeneous chlorine cycling in the aerosol phase. The modelled evolution of the SO2/H2S ratios is found to be substantially dependent on whether or not the interactions of H2S with halogens are included in the model. In the former case, H2S is assumed to be oxidized in the atmosphere mainly by OH, which results in minor chemical loss for H2S during plume aging and produces a fair match between modelled and measured SO2/H2S ratios. In the latter case, fast oxidation of H2S by Cl leads to H2S chemical lifetimes in the early plume of a few seconds, and thus SO2 to H2S ratios that increase sharply during plume transport. This disagreement between modelled and observed plume compositions suggests that more in-detail kinetic investigations are required for a proper evaluation of H2S chemical processing in volcanic plumes.

It has been proposed that once good-quality sperm are collected, extended, and cryopreserved that the post-thaw quality will remain high regardless of the duration of storage. A previous study has suggested that there is no effect on post-thaw sperm motility of bovine semen stored in liquid nitrogen up to 13 years. However, no studies have reported the effect of extended storage of bovine semen in LN2. In this study, cryopreserved semen from Angus bulls (n = 25) was utilized. Time frames were assigned to each bull based on the year of collection and cryopreservation: time frame 1 (1960–1975), time frame 2 (1976–1991), and time frame 3 (1992–2006). Due to differences in packaging methods across time periods (glass ampules and plastic straws), thawing methods utilized were different and based on the packaging type. Semen packaged in glass ampules (n = 7) were thawed in a 37°C water bath for 80 s, while those in plastic straws (n = 18) were thawed in a 37°C water bath for 30 s. Once thawed all packages were wiped dry and either scribed (glass ampule) or the end cut (plastic straw) and the semen expelled into a 1.5-mL microcentrifuge tube and maintained at 37°C for evaluation. For sperm motility, a 20-µL sample was placed onto a prewarmed microscope slide for analysis. Sperm motility parameters (total motility and progressive motility) were evaluated by 2 experienced technicians and averaged. A hemocytometer was used to determine sperm concentration in a final dilution of 1 : 100. An eosin/nigrosin stain was used to determine sperm morphology. Parameters were analyzed using one-way ANOVA. There was no difference in sperm concentration between time frames 1 and 2 (53 × 106 mL–1 and 59 × 106 mL–1, respectively) or time frames 1 and 3 (53 × 106 mL–1 and 37 × 106 mL–1, respectively). However, time frame 2 exhibited a higher (P < 0.05) sperm concentration compared with time frame 3 (59 × 106 mL–1 v. 37 × 106 mL–1, respectively). There were no differences among time frames 1, 2, or 3 for total (42, 51, and 55%, respectively) and progressive sperm motility (29, 38, and 41%, respectively). Similarly, there were no differences among time frames 1, 2, and 3 for percent normal sperm (80 ± 3.6, 76 ± 5.0, and 71 ± 4.0%, respectively) and abnormal sperm (19 ± 3.6, 23 ± 5.0, and 28 ± 4.0%, respectively). Furthermore, time frames 1, 2, and 3 did not exhibit a difference in primary (9 ± 2.5, 7 ± 2.6, and 6 ± 1.0%, respectively), secondary (2 ± 0.8, 5 ± 1.4, 6 ± 2.0%, respectively), or tertiary abnormalities (7 ± 2.4, 10 ± 2.7, and 16 ± 2.7%, respectively). These data suggest that once good-quality bovine sperm is cryopreserved, the duration of storage (up to 43 years) has no effect on Angus bull post-thaw semen parameters.

Altering the lipid composition of sperm plasma membranes not only affects the ability of sperm capacitation and acrosome reaction, it also affects the way sperm respond to cryopreservation. The objective was to determine if increasing sperm membrane cholesterol levels, by adding cholesterolloaded cyclodextrin (CLC) to boar sperm, alter the cryopreservation sperm to undergo acrosome reaction in vitro. The CLC was prepared as described by Purdy and Graham (2004) with some modification: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents removed using a hot plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Ejaculates (n = 5) from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept for 2 h at 22°C. Afterward, the ejaculates were put at 15°C/ for 60 min. Later, the ejaculates were centrifuged at 15°C at 400g/10 min, the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. These treatments were incubated for 15 min at 15°C. The samples were cooled to 5°C/90 min period and diluted 1:1 with freeze diluent (72.5-mL lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). The sperm were packaged into 0.5-mL straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath 37°C/30 s. A 90% Percoll solution was prepared by diluting 1 mL of 10× PBS with 9 mL of Percoll. A 35% Percoll solution was then prepared by diluting 90% Percoll (0.67 mL) with Medium 199 (1.33 mL). Frozen/thawed spermatozoa (2 mL) were then layered onto 2 mL of 35% Percoll solution in a 15-mL conical tube and centrifuged at 400g/5.5 min. The resulting pellet was suspended with Medium 199 to 100 million cells/mL, and the cells were stained with 5 μL of PI (1 mg mL-1 in water) and 10 μL of FITC-PNA (1 mg mL-1 in 10× PBS). The cells were incubated for 5 min at room temperature to allow PI and FITC-PNA to become incorporated. The acrosomal status of viable cells for each treatment was then determined by epifluorescence microscope at 400× magnification, and the percentage of acrosome reacted cells was calculated as the proportion of FITC-PNA stained and PI negative cells (acrosome reacted, live)/total live cells (PI negative, FITC-PNA positive and negative). Treatment differences for acrosome reaction were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher acrosome reaction (28%) compared with control cells (22%; P < 0.05). Several studies evaluated the ability of bull and stallion sperm treated with CLC to capacity and acrosome react. Adding cholesterol might alter the plasma membrane structure, improving the acrosome reaction in CLC-treated boar spermatozoa. FAPEMIG, Piglandia, CNPq, FACEPE.

Abstract Abstract 2526 Many studies on childhood and adult leukemias have demonstrated that thorough molecular analysis at the time of diagnosis and minimal residual disease (MRD) follow-up are significantly related to the prognosis, and overall- and event-free survival, of patients with acute leukemias. In acute myeloid leukemia (AML), such complex molecular diagnostics and subsequent MRD evaluations are important factors for proper stratification, disease prognosis, assessment of the treatment response, optimal dosage and duration of chemotherapy, and estimation of the optimal timing for hematopoietic stem cell transplantation. Upon diagnosis, the bone marrow samples of AML patients are routinely screened for an array of recurrent chromosomal abnormalities, including chromosomal translocations (e.g. PML/RARa, AML1/ETO, CBFb/MYH11, MLL fusions, BCR/ABL) or leukemia-associated genetic mutations (e.g. mutations in genes NPM1, WT1, FLT3, MLL or CEBPa). Given the fact that laboratories often deal with a limited amount of material sampled for molecular investigations, and the number of possible genomic aberrations and molecular targets is high, it is desirable to implement in routine practice a flexible tool that allows testing for as many genetic abnormalities as possible, while reducing the amount of biological material required for analyses to a minimum. In our laboratory, we have developed a multiplex Real-Time PCR technique allowing us to examine over 75 recurrent chromosomal aberrations in only 10 multiplex PCR reactions (Table 1). The methodology makes use of a set of fluorescently labeled TaqMan hybridization probes, labeled by 5 different fluorophores. Using this methodology we are able to assess the presence of both rare as well as recurrent chromosomal translocations/aberrations in one setting, from a limited amount of starting material. This approach is not only extremely beneficial for the leukemic patient - which is always the primary goal - but also for the overall budgeting of routine molecular screening of diagnostic AML samples.Table 1:List of the AML-associated chromosomal abnormalities included in the 5-Color Multiplex Real-Time PCR system. The multiplexing of individual molecular targets is indicated by the individual PCR tubes (A to J) used in the diagnostic screening of AML in our laboratory. Importantly, a clone-specific chromosomal abnormality found at the time of diagnosis using our 5-Color Multiplex Real-Time PCR system allows us to molecularly follow-up the MRD level with a high sensitivity of 10e-4 to 10e-6, as assessed by serial dilutions of cloned standards harboring the individual aberrant genetic targets. This complex molecular approach considerably helps hematooncologists in clinical decision making and the adjustment and modulation of the treatment of AML patients in respect to their individual needs and their individual disease course. Since 2005 we have molecularly investigated 398 adult AML cases. Using the 5-Color Multiplex Real-Time PCR technique and mutational screening we were able to identify a clone-specific abnormality in 45.2% of cases. The clone-specific genetic markers were then used as specific molecular targets for a clone-specific MRD follow-up. Although in approximately 50% of AML patients we are still not able to identify any clone-specific abnormality to be used for either stratification (recurrent abnormalities) or molecular MRD follow-up (both recurrent and unique/rare abnormalities) of these leukemic patients, our 5-Color Multiplex Real-Time PCR system, being an open platform, enables us to flexibly implement any newly identified chromosomal aberrations to the diagnostic portfolio, thus increase the probability of finding a clone-specific molecular marker with all the positive consequences in respect to the management of patients with acute leukemia. Disclosures: Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

The goal of this research was to investigate the effect of a novel density gradient centrifugation (DGC) treatment on the fertilizing capability of bovine sperm as compared to a standard method. Domestic bull (Bos taurus) semen was used for AI and the production of embryos from in vivo-matured bovine oocytes. In 2004, a preliminary study compared the novel semen treatment using a trypsinized PVP-coated silica particle suspension (Percoll; Sigma, St. Louis, MO, USA) to a standard method (4� dilution with an egg yolk diluent) on the fertilizing capacity in vivo of bovine sperm (de la Rey et al. 2005 J. Reprod. Fertil. 17, 242 abst). Although not statistically significant (P = 0.69), there were more transferable quality embryos recovered from cows inseminated using the treated sperm method v. control (58.9 v. 43%, respectively). In this report we provide the results of two additional trials utilizing the novel semen treatment and substituting Percoll with a silane-coated silica particle medium containing a recombinant trypsin (r-protease). In the second trial (2005), semen samples collected from three bulls were processed by DGC: 2 mL of 40% PureSperm (NidaCon International AB, M�lndal, Sweden) containing recombinant trypsin (TrypLE Select, Gibco/Invitrogen, Carlsbad, CA, USA), which overlaid 2 mL of 80% PureSperm containing 10 µg mL–1 soy-based protease inhibitor (Sigma), was overlaid with the semen sample using a novel centrifuge tube insert (ProInsert, Nidacon) and then centrifuged at 300g for 20 min. The sperm pellets were recovered and washed (500g for 10 min) in 10 mL pre-warmed TL-HEPES medium (Cambrex Corp., East Rutherford, NJ, USA). The washed sperm pellets were then resuspended in the same total volume of pre-warmed Biladyl� A (Minit�b, Tiefenbach, Germany) as the standard method and used to AI a total of 42 (control) and 47 (treatment) superovulated cows three times at 12 h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. In Trial 3 (2006), semen samples collected from three bulls were processed by DGC containing r-protease and a soybean protease inhibitor (BoviPure Pro, NidaCon), media specifically formulated for domestic bull semen. Sperm pellets were washed in 10 mL BoviWash medium (NidaCon). The washed sperm pellet was resuspended in the same total volume of pre-warmed Biladyl A as the standard method and used to AI a total of 23 (control) and 25 (treatment) superovulated cows and embryos evaluated as in Trial 2. The results between control and treated groups were compared using the Mann-Whitney (Wixcoxon rank sum) test. Trial 2 using PureSperm tended to result in higher fertilization rates than for cows inseminated using the standard method (75.2% v. 67%, respectively) but the results were not statistically significant (P = 0.63). Results for Trial 3 indicated that cows inseminated with BoviPure Pro-treated sperm had significantly increased fertilization rates as compared to the standard method (88.4% v. 63.1%, respectively; P = 0.02) and had higher numbers of transferable quality embryos (70.3% v. 51.8%, respectively; P = 0.38). In summary, BoviPure Pro sperm treatment before AI significantly increases fertilization rates and can result in as much as an 18.5% increase in transferable quality embryos as compared to standard methods.

Background: New methodologies have been developed seeking to maximize pregnancy rate in female dogs created in commercial kennels, and also in order to maintain the quality of canine semen after dilution, refrigeration or freezing. One of the main factors that generate damage to sperm is oxidative stress, to minimize sperm damage, selenium and antioxidants like vitamin E are administered, by oral administration, seeking to improve the quality of semen. The objective was to study the effect of vitamin E and selenium, by oral administration, in the quality of fresh, refrigerated and frozen semen in adult dogs French Bulldog breed.Materials, Methods & Results: Semen samples were collected from 5 adult dogs, French Bulldog breed, being 2 semen drawing before the daily oral supplementation with vitamin E and selenium (ESE®) and semen drawing at 20, 40 and 60 days after the beginning of oral supplement. The ejaculated samples were diluted in TRIS - fructose citric acid (3.28 g TRIS-hydroxy-methyl-amino-methane, 1.78 g of citric acid monohydrate and 1.25 g of D - fructose, dissolved in 100 mL of distilled water and added of 20% egg yolk and 6% of glycerol. The characteristics evaluated in fresh semen were: volume (mL), color, appearance, concentration (x106 / mL), sperm motility (%), sperm strength (1 to 5) and morphology (%). For refrigerated and frozen semen were analyzed: sperm motility (%), sperm strength (1-5) and morphology (%). Diluted semen samples were centrifuged at: 1500 g/10 min and “pellets” formed by sperm of each ejaculated, detached from the tube wall were diluted homogeneously in the diluent TRIS type up to the final volume of 1.5 mL. After that, packaged in 0.5 mL French straws, kept under refrigeration at 5ºC/4 h, placed in nitrogen vapor at -120ºC/15 min, and dipped in liquid nitrogen at -196ºC and then stored on identified rachis and stored in liquid nitrogen container until the time of thawing in water bath at 37°C/30 s for semen microscopic analysis. Data from fresh, refrigerated and frozen semen were statistically analyzed by analysis of variance and the average compared by 5% of Tukey test. Fresh semen sperm concentration differed (P < 0.05) between the samples, rising after 40 days after the beginning of oral supplementation with selenium and vitamin E. For the spermatic strength, better score (P < 0.05) was observed at collection 4, in 40 days after the beginning of oral supplementation to dogs. For fresh and refrigerated semen, the total defects, defects of head, acrosome and tail did not differ (P > 0.05) between the samples. Total sperm defects and minor head and tail defects did not differ (P > 0.05) between the samples in post-thawing. Regarding the acrosome defects after thawing, there was a significant reduction (P < 0.05) in samples performed 40 and 60 days after the beginning of oral supplementation with selenium and vitamin E.Discussion: Attention should be paid for what purpose the extenders within the refrigeration or freezing biotech will be used. The managed supplement, by oral administration, containing selenium and vitamin E, influenced beneficially raising the sperm concentration in fresh semen and decreasing the acrosome defects in frozen semen. Oral administration of supplementation with selenium and vitamin E is recommended for improving the quality of fresh and frozen semen in dogs.

Abstract BCL-ABL fusion gene can be found in 100% chronic myelogenous leukemia (CML) and about 25% adult acute lympholid leukemia where its expression level is a crucial parameter for monitoring of minimal residual disease (MRD). Depending on the breakpoint in BCR, exon 2 of ABL (a2) joins with exons 1 (e1), 13 (b2), or 14 (b3), or rarely to exon 19 (e19) of BCR resulting in chimeric proteins of p190, p210 and p230, respectively. The c-ABL gene is one of the best controls for MRD detection by real-time quantitative RT-PCR (RQ-RT-PCR). In most studies published before, PCR probes were labeled by single fluorescence such as FAM, and BCR-ABL and ABL transcripts were detected in separate PCR reactions. Purpose: To design and evaluate a duplex, real-time quantitative RT-PCR (RQ-RT-PCR) system labeled with double fluorescence Taqman probes to simultaneously measure both BCR-ABL and ABL transcripts. Methods: Positive controls are plasmids containing full-length target sequence of BCR-ABLP210 and ABL. 70 cases of CML bone marrow samples collected in Nanfang Hospital from Jane 2005 to July 2008 were examined. Patients were untreated ones and those treated with STI571 or allo-hematological stem cell transplantation. RQ-RT-PCR value=copies of BCR-ABL/copies of ABL. The results are compared with fluorescence chromosomal in situ hybridization (FISH) for BCR-ABL. Probes and primers recommended by Europe Anti-Cancer group in 2003 were used as gold standard control; probe and primers for bcr-ablP210 gene are ENF541, ENP501 and ENR561, respectively; probe and primers for abl gene are ENPr1043, ENF1003 and ENR1063, respectively. Both Taqman probes are FAM labed. For duplex RQ-RT-PCR, HEX labeled probe is used for the ABL gene, along with corresponding primers, ENP541 labeled with FAM fluorescence and ENF501 and ENR561 were also used for BCR-ABLP210. The experiments were carried out in the same tube on a Biorad Opticon2 RQ-PCR unit. The end concentration is 0.3μmoL/L for primers and 0. 2 μmoL/L for probe. The PCR reaction was carried out in 25μL. PCR condition is at 50°C×2min+95°C×10min, then followed 95°C×15s+60°C×1min for 50cycle. Result: Testing using serial dilutions of plasmid positive control suggested that HEX-FAM duplex is readily amplified with the FAM Taqman probes of BCR-ABLP210, and the HEX-ABL results is same with those with FAM-ABL (recommeded by Europe Anti-Cancer group). Coefficiency of variation among different experiments is less than 5%. The 70 CML cases can be divide into 3 groups based on FISH value: ≥10% (n=32), 0.5% 10% (n=27) and negative (n=11). The corresponding RQ-PCR ratio of BCR-ABL/ABL are 0.590±0.264, 0.044±0.041 and (9.46±6.99)×10|4, respectively, P<0.01 between each group. Conclusion: We have established an efficient duplex RQ-RT-PCR method with FAM and HEX Taqman probes. This approach enables acquisition of more information from each test and hence reduces the amount of sample needed for each test. We believe this method will be useful to MRD monitoring in CML and BCL-ABL + B-ALL.

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. These enzymes have great economical potential in industrial, agricultural, chemical, pharmaceutical, and biotechnological applications. Thus, the halophiles isolated from Chott Tinsilt offer an important potential for application in microbial and enzyme biotechnology. In addition, these halo bacterial biosurfactants producers isolated from this Chott will help to develop more valuable eco-friendly products to the pharmacological and food industries and will be usefulness for bioremediation in marine environment and petroleum industry.ACKNOWLEDGMENTSOur thanks to Professor Abdelhamid Zoubir for proofreading the English composition of the present paper.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Akbari, S., N. H. Abdurahman, R. M. Yunus, F. Fayaz and O. R. Alara, 2018. Biosurfactants—a new frontier for social and environmental safety: A mini review. Biotechnology research innovation, 2(1): 81-90.Association, A. P. H., A. W. W. Association, W. P. C. 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AbstractBackgroundGram-negative lipopolysaccharides are potent inducers of inflammation and have been shown to be present in patients with end-stage kidney disease. There are a variety of different manufacturers and assay types to quantify endotoxin levels; however, there is no standard methodology to demonstrate its presence in plasma.MethodsA control group consisting of haemodialysis and non-kidney disease was selected. Five sets of experiments were conducted to try and ascertain the best platform for plasma endotoxin testing. This included: testing of blank tubes; the effects of freezing, thawing and storage on recovery; the effect of different buffers; use of an endpoint assay and comparison of turbidimetric vs. chromogenic kinetic assays.ResultsNo endotoxin was detected in the blood collection tubes. Freezing and thawing per se did not affect spike recovery rates. However, the sequencing of sample dilution relative to freezing had a significant effect on endotoxin recovery. Buffers increased spike recovery at all levels of dilution. No endotoxin was demonstrated with either the turbidimetric or chromogenic kinetic assay at two different dilutions in the haemodialysis controls. The endpoint assay at a 1:5 dilution did not achieve a valid standard curve.ConclusionsThe findings of our study suggest that, when testing plasma samples, either a turbidimetric or chromogenic assay may be used and should be diluted with appropriate buffers to achieve optimal recovery. Studies looking to quantify the presence of plasma endotoxin need to internally validate their assays and specify their validation findings in their results.

Abstract Ignition delay times were measured for methane/O2 mixtures in a high dilution environment of either CO2 or N2 using a shock tube facility. Experiments were performed between 1044 K and 1356 K at pressures near 16 ± 2 atm. Test mixtures had an equivalence ratio of 1.0 with 16.67% CH4, 33.33% O2, and 50% diluent. Ignition delay times were measured using OH* emission and pressure time-histories. Data were compared to the predictions of two literature kinetic mechanisms (ARAMCO MECH 2.0 and GRI Mech 3.0). Most experiments showed inhomogeneous (mild) ignition which was deduced from five time-of-arrival pressure transducers placed along the driven section of the shock tube. Further analysis included determination of blast wave velocities and locations away from the end wall of initial detonations. Blast velocities were 60–80% of CJ-Detonation calculations. A narrow high temperature region within the range was identified as showing homogenous (strong) ignition which showed generally good agreement with model predictions. Model comparisons with mild ignition cases should not be used to further refine kinetic mechanisms, though at these conditions, insight was gained into various ignition behavior. To the best of our knowledge, we present first shock tube data during ignition of high fuel loading CH4/O2 mixtures diluted with CO2 and N2.