Open Access Method Article

Calcofluor white, an Alternative to Propidium Iodide for Plant Tissues Staining in Studies of Root Colonization by Fluorescent-tagged Rhizobia

José David Flores-Félix, Esther Menéndez, Marta Marcos-García, Lorena Celador-Lera, Raúl Rivas

Journal of Advances in Biology & Biotechnology, Page 65-70
DOI: 10.9734/JABB/2015/12444

Aims: To study multiple bacterial colonization In vitro, several limitations are obvious. One of these limitations is the plant autofluorescence generally between green and red fluorescence depending on the plant sections. The most important limitation is the bacterial fluorescence labelling, compromised by different kind of variables. Here we report the use of a secure stain, Calcofluor White, in rhizobial and other kind of beneficial bacteria colonization studies.

Study Design: Root colonization assays were designed to confirm the stability of Calcofluor White stain (Sigmaâ) in root cell walls.

Place and Duration of Study: Every assay developed in this method article was performed using the technical resources at the Department of Microbiology and Genetics in the University of Salamanca (Spain) in 2013.

Methodology: We have labelled rhizobia with two different kinds of fluorescent protein genes (gfp and rfp). We have co-inoculated Lactuca sativa and Daucus carota seedlings with two rhizobia: GFP-tagged Rhizobium sp. PEPV16 and RFP-tagged Mesorhizobium sp. CSLC01. Colonization assays were perfomed in several days post-inoculation, staining inoculated lettuce and carrot roots with Calcofluor White stain (Sigma®). Samples were monitorized for several days, using a fluorescence microscope (NIKON Eclipse 80i).

Results: Bacterial attachment to plant tissues is observed by fluorescence microscopy after their labelling with fluorescent proteins. Our results show how Calcofluor White staining for plant tissues improves bacterial visualization in contrast with tissues stained with propidium iodide, a carcinogenic agent that cannot be used when bacteria are tagged with red fluorescent proteins such as RFP or mCherry.

Conclusion: Calcofluor white is a non-carcinogenic and low toxic compound that has been classically used to stain fungi and plant tissues for different uses. Due to its low wavelength, calcofluor white may be used in combination with several fluorophores. In the present work we showed that this compound is a reliable alternative to propidium iodide for plant tissues staining in multiple rhizobial/bacterial colonization studies.

Open Access Short communication

Production of Protoplasts from Yeast Cells (Saccharomyces cerevisae) Isolated from Palm Wine (Elaeuis guinensis) Using Snail Gut Enzyme from African Giant Snail (Achatina achatina)

Chinenye C. Chijioke-Osuji, Peace N. Ibegbulam-Njoku

Journal of Advances in Biology & Biotechnology, Page 10-15
DOI: 10.9734/JABB/2015/11989

Aims: The aim of this study was to investigate the use of locally sourced snail gut enzyme from African giant snail (Achatina achatina) for yeast protoplasts production.

Place and Duration of Study: Fresh undiluted palm wine was commercially acquired from Umuoke, Obowo in Imo State while African giant snails (A. achatina) were bought from Umuahia main market, Abia State, Nigeria. The study was carried out from June to september, 2013.

Methodology: The snails were starved for 7 days so as to concentrate their gut juice, carefully aspirated using sterile needles and syringes after unshelling the snails. All the extracts from twelve snails were pooled together and centrifuged at 1,500rpm for 20 minutes to remove larger particulates; the supernatant was collected dissolved in 0.1M acetate buffer (pH 5.5). Yeast cells (Saccharomyces cerevisae) were grown in potato dextrose broth and harvested at late exponential phase at 37ºC. 1g of the cell slurry were suspended into 10 ml of 0.1M phosphate buffer (7.2) containing 0.1% Tween 80(osmotic stabilizer). Crude snail gut enzyme extract in 0.1M acetate buffer (pH 5.5) was added to yeast suspension and incubated at 30°C for 120 mins with gentle shaking. The resulting protoplasts were centrifuged at (1000rpm) and suspended in 0.7Mol. NaCl solution as supplement osmotic medium. The effect of lytic incubation time, concentration and osmotic stabilizers were studied. The viability of the cells were assessed by regeneration method.

Results: A wet mount of the harvested yeast cells after staining with lactophenol cotton blue indicate presence of cell walls. However, after incubation of mixture of these cells with snail gut juice at 30°C for 120 mins. The effect of lytic incubation time for the protoplast production shown on Fig. 1, revealed optimal protoplast formation of 2.7 x 107/mL after 80mins of incubation. The enzyme concentration effect on protoplast production shown in Fig. 2 revealed that 100%v/v of gut juice from A. achatina was able to produce 2.0 x 107/mL yeast protoplast. The best supporting osmotic stabilizer during cell wall regeneration was observed with 0.5M sucrose.

Conclusion: This experiment therefore suggests a rapid, inexpensive and efficient procedure for yeast protoplasts production

Open Access Original Research Article

Comparison of In vitro Study of Aspergillus niger and Pseudomonas aeruginosa Biodegradation of Spent Lubricating Oil

E. Stephen, E. O. Emmanuel, L. O. Okwute

Journal of Advances in Biology & Biotechnology, Page 1-9
DOI: 10.9734/JABB/2015/11618

Comparison of biodegradation of spent lubricating oil by Aspergillus niger and Pseudomonas aeruginosa was studied for 16 days. pH, turbidity, nitrate, phosphorus and Gas Chromatographic -Mass Spectrophometry (GC-MS) analyses of the media were carried out. The results showed lower pH and phosphorus levels in A. niger medium compared to Pseudomonas aeruginosa medium and the control while nitrate was higher in Pseudomonas aeruginosa compared to A. niger. The GC-MS revealed that more compounds were degraded by A. niger than Pseudomonas aeruginosa after 16 days. Azulene and benzene were not degraded by both organisms. This study suggests that A. niger grows and metabolize compounds in spent lubricating oil better than Pseudomonas aeruginosa and that neither of the two organisms had the competent enzymes to degrade azulene and benzene in two weeks.

Open Access Original Research Article

Comparison of Two Biopsy Methods in Bovine Embryos

Yasuhiro Ogata, Takemasa Hidaka, Tadami Matzushige, Teruo Maeda

Journal of Advances in Biology & Biotechnology, Page 16-23
DOI: 10.9734/JABB/2015/13028

In this study, the accuracy of sex identification, the viability of biopsied embryos, and the pregnancy rate after biopsied embryo transfer were compared in bovine embryos biopsied by two methods (cutting and pipetting). The cells were more efficiently collected by the pipetting method (2.4±2.0 cells), and the success rate of sex identification was 94.8%. Moreover, the survival, and pregnancy rates of embryo biopsied by pipetting method were 91.7 and 68.0%, respectively, and were numerically higher than those seen in the cutting group (84.4 and 56.5%). From these results, the pipetting method appears to be a simple cell collection method of bovine embryos for sex identification, and assures greater safety of the biopsy process and better embryonic development after biopsy.

Open Access Original Research Article

Crude Flavonoids Isolated from the Stem Bark of Annona senegalensis have Antimicrobial Activity

Mahmoud Suleiman Jada, Wurochekke Abdullahi Usman, Ajayi Opeyemi Olabisi

Journal of Advances in Biology & Biotechnology, Page 24-29
DOI: 10.9734/JABB/2015/11884

This study was aimed at determining the in vitro antimicrobial activity of crude flavonoids isolated from the stem bark of Annona senegalensis. The study was carried out in the laboratory of Biochemistry department, Modibbo Adama University of Technology, Yola, Adamawa State, Nigeria, between July and October, 2013. The antimicrobial activity was carried out using agar well diffusion method. Phytochemical screening of the methanolic stem bark extract of Annona senegalensis revealed the presence of steroids, saponins, anthraquinones, flavonoids, tannins and cardiac glycosides. Crude flavonoid of 1.956g was found to be present in 10g of stem-bark extract of the plant. Streptomycin was used as standard which gave the zones of inhibition of 23mm against Escherichia coli, 26mm against Salmonella typhi and 28mm against Shigella specie while the isolated crude flavonoids from the stem bark of Annona senegalensis gave the zones of inhibition of 18mm against Escherichia coli, 25 mm against Salmonella typhi and 26 mm against Shigella specie. Crude flavonoids exhibited Minimum Inhibitory Concentration (MIC) against Shigella specie and Escherichia coli at 100mg/ml while against Salmonella typhi at 50mg/ml.  In conclusion, the crude flavonoids isolated from the stem bark of Annona senegalensis have antimicrobial activity against the test organisms.

Open Access Original Research Article

N-alkane Solvent-enhanced Biotransformation of Steroid DHEA by Beauveria bassiana as Biocatalyst

Richard Gonzalez, Felipe Nicolau, Tonya L. Peeples

Journal of Advances in Biology & Biotechnology, Page 30-37
DOI: 10.9734/JABB/2015/13541

Aims: The hydroxylation capacity of Beauveria bassiana was enhanced with n-alkane solvents, resulting in a selective and eco-friendly scheme for the synthesis of steroids. A biocatalytic system was engineered to augment the 11α-hydroxylation of dehydroepiandrosterone (DHEA) to valuable intermediates. Exposing and inducing cells into n-alkanes improved the synthesis of 11α-hydroxy derivatives.

Methodology and Results: Reactions were carried out with cells grown with n-dodecane (n-C12) and n-hexadecane (n-C16), resulting in 65%±6.3 conversion of DHEA to androstenediol (40.3%mM) and 3β,11α,17β-trihydroxyandrost-5-ene (22.8%mM), as determined by HPLC and NMR analyses. Experiments without the presence of n-alkanes resulted in 17% conversion of DHEA. Isolated products in this case included: Androstenediol (11.8%mM) and 3β,11α,17β-trihydroxyandrost-5-ene (4.78%mM). Results indicate that only the 3,17-hydroxy derivatives of DHEA undergo the 11α-hydroxylation pathway.

Conclusions: The appearance of the products suggests that the reduction of the C-17 ketone of DHEA is preceded by the 11α-hydroxylation reaction when n-alkanes are present. This differs from reports in the literature, which proposed the activation of an unfunctionalized carbon to 11α-hydroxy-17-oxo derivatives before obtaining a 3β,11α,17β-triol product.

Open Access Original Research Article

Cyclic Intermittent Fasting Influences the Structure of Hepatocyte Nuclear Membrane in Young and Old Rats

M. S. Girych, N. I. Kurguzova, A. I. Bozhkov

Journal of Advances in Biology & Biotechnology, Page 38-50
DOI: 10.9734/JABB/2015/13771

Aims: The present study was undertaken to monitor the changes in structural and dynamic state of hepatocyte nuclear membrane of young (3-months) and old (20-months) rats subjected to intermittent fasting (IF).

Study Design: young (3-month) and old (20-month) rats were individually housed and randomly assigned to one of five groups (with 10 rats per group): (Control)-fed Ad libitum; (1IF)-provided access to a limited amount of food (4g/100g and 2g/100g of food/body weight for young and old rats, respectively) every other day for 10 days; (1R)-refeeded Ad libitum for 20 days after 1IF; (2IF)-provided the same regimen as for 1IF but after successive 1IF and 1R; (2R)-refeeded Ad libitum for 10 days after 2IF.

Methodology: The magnitude of fluidity changes was evaluated through measuring the excimer-to-monomer intensity ratio (E/M) in the pyrene emission spectra. The changes in membrane hydration were assessed using Laurdan generalized polarization (GP).

Results: During two cycles of intermittent fasting/refeeding statistically significant differences in E/M for young animals are observed only after the first refeeding (increase by 11% compared to control). 1IF and 1R in old animals were followed by ~26% and ~18% decrease in E/M value. The second cycle of dietary regimen brought about ~20% and ~36% decrease in E/M of old rats after 2IF and 1R, respectively. The first intermittent fasting resulted in ~74% and ~101% increase in GP value of young and old rats, respectively, and the refeeding period GP parameter underwent ~221% and ~89% increase in comparison with control for young and old rats, respectively. After the second IF following the first refeeding the young and old rats were characterized by ~200% and ~44% increase in GP, respectively. On the contrary, second refeeding leads to ~182% and ~27% increase in GP for young and old rats, respectively.

Conclusion: The main outcome reached is the identification of differences in the effects of re-applicated IF on nuclear membrane fluidity and hydration in animals of different age, suggesting that membrane responses to IF are governed by age-dependent mechanisms.

Open Access Original Research Article

Cytotoxic Effects and Genotoxic Screening of Pharmaceutical Effluents using Onion Bulbs (Allium cepa L.)

Olaitan Olatunde James, Sulola Ebenezer Oluwaleye, Adejumo E. Olufunmilayo, Omoloye Adesina Adebiyi

Journal of Advances in Biology & Biotechnology, Page 51-58
DOI: 10.9734/JABB/2015/12962

Background: Pharmaceutical effluents like other pharmaceutical effluents are toxic waste water generated from drug industries which when discharged directly into the environment without proper handling and treatments may cause deleterious effects on human health and environment.

Aim: This research is aimed at investigating the potential cytotoxic and genotoxic effect of effluents released by three different pharmaceutical industries in Sango Industrial Area of Ogun State in Nigeria using the USEPA recommended Allium cepa test.

Methodology: Each of the effluents were prepared into different concentrations using distilled water (1%, 2%, 3%, 4%, 5%, 10%, 25%, 50% and 100% respectively) and their cytotoxicity on the root length of a series of onion bulbs was observed after three days of exposure. Cytological studies on the root tips of onion bulbs using five concentrations of each effluent were also examined by Aceto-orcein squash technique. Mitotic index which is the ratio of the number of cells dividing to number of cells counted was observed to decrease with increasing concentration of all the effluents compared to the control. Chromosome aberrations induced at different concentrations were observed under microscopes.

Result: Significant results were observed in the Root length Evaluation and Chromosomal Aberration Evaluation tests respectively after three days. Effluent C seems to be the most toxic on the root length of the onion bulbs as it reduced from 1.2cm to as low as 0.30cm, followed by effluent B and A with 0.60cm and 0.70cm respectively. But Effluent A has the most % of aberrant cells with 5.32% and Effluent C with 3.03%. Most of the aberrations induced were vagrants, bridges and sticky chromosomes.

Discussion and Conclusion: The results from this study showed that effluents from these pharmaceutical industries have toxic chemicals and that Allium cepa test in relation to this study has proved to be an effective tool that may be employed by environmental toxicologists in monitoring industrial effluents before they are discharged into the environment.

Open Access Original Research Article

Single Cell PCR Using Y Chromosome Specific Repeat for Sexing

Elham Arbabi Aval, Frouzandeh Mahjoubi, Raheleh Torabi

Journal of Advances in Biology & Biotechnology, Page 59-64
DOI: 10.9734/JABB/2015/13828

Aims: Embryo sexing is one of the important ways for sex selection of offspring. This is a potential method to considerably improve animal breeding and the efficiency of dairy and meat production. A novel repeated sequence specific to male cattle has been identified and named S4. S4 is a 1.5 Kb repeating unit contains various internal repeated sequence. S4 is localized on long arm of the Y chromosome in the region near to ZFY genes. The objective of this study was to establish a simple, sensitive, reliable, reproducible and cost effective PCR based technique for sexing.

Study Design: Case control study.

Place and Duration of Study: Sample: whole blood samples of male and female cattle. Medical Biotechnology Dept. National Institute for Genetic Engineering and Biotechnology, Tehran, 14965/161, Iran, between June 2010 and July 2011.

Methodology: Genomic DNA was extracted from the whole blood samples of male and female cattle. PCR and single cell PCR were performed using specific primers for this region.

Results: By this PCR based methods we could differentiate between female and male genomic DNA.

Conclusion: With this technique we can distinct male from female using as little as 0.1pg DNA. Using this method we could determine the sex of embryo (4 blastomers). This method may optimize for the quantitative detection of Y chromosomes in semen.

Open Access Original Research Article

Ecological Study of Carduus pycnocephalus L. Weed and Associated Species in Hamedan Province, Iran

Mehdi Heidarian, Sayed Mohammad Masoumi, Morteza Atri

Journal of Advances in Biology & Biotechnology, Page 71-78
DOI: 10.9734/JABB/2015/11468

Slender thistle is one of the species of Asteraceae. It is native to: In the Mediterranean region of southern Europe, North Africa, West Asia, East Europe, Caucasus and the Indian subcontinent are scattered. This study carried out for determination associated species on intraspecific variation of Carduus pycnocephalus L. (Italian Thistle) in Hamedan Province (Iran).In this order, vegetation studied to D.S.S method (Determination of Special Station). Based on, 14 special stations for this species were determined. In this investigation, 59 plant species distinguished as associated species that belonging to 53 genera and 19 families. Among the families, Asteraceae and Poaceae have many species. Most of this species are weed plant. The most life form spectrum showed Therophyte that reflects the region's dry climate. Spectrum of plant species is as follow: Hemichryptophyte forms of the plants indicate the possibility of adaptation of Maditerranean and cold temperate affected them. Decreasing of Chamephytes and Hemicryptophytes species and lacking geophytes Indicates a weakening of the vegetation in this area.