FREE ELECTRONIC LIBRARY - Dissertations, online materials

Pages:     | 1 |   ...   | 9 | 10 || 12 | 13 |   ...   | 23 |

«By Yusuf Nur A thesis submitted to The University of Birmingham for the Degree of DOCTOR OF PHILOSOPHY School of Geography, Earth and Environmental ...»

-- [ Page 11 ] --

4.4.7 Concentration of gold with Inductively Coupled Plasma Mass Spectrometry (ICP-MS) In this, project, an Agilent 7500 ICP-MS instrument housed in an air conditioned room was used to measure the concentration of gold in the final solution of the synthesised goldNPs. To determine thes olubility of the AuNPs, samples were ultra-filtered using regenerated cellulose membrane with nominal pore sizes of 1 kDa. 50 ml of each sample was washed three times and ultra-filtered with equal volumes of milli-Q and between the consequent washings 1hour interval was allowed to leave gold nanoparticles to reach a steady state. Replicates of both the unfiltered sample and filtrate were collected and acidified with concentrated nitric acid for further analysis of gold concentration by ICP-MS. The average mean values of each replicate were reported and the gold NP concentrations were calculated by difference.

–  –  –

The surface-charge of all samples was determined using Titrando 809 from Metrohm with 2 dosing 800 units for titrant delivery. The initial pH values of the NPs capped by citrate and the PVP capped NPs which are prepared through hot process were slightly acidic and the actual values lie between 4.6 and 6.2. The high pH of the PVP capped samples reduced by NaOH is obviously due to the large concentration of free hydroxide ions from NaOH in the media. Pre-measured aliquots of nanoparticle solutions were dynamically titrated with volumes of 0.1 mM NaOH solution. The volume addition (10 µL – 200 µL) depended on the rate of pH increase; a stability criterion of 0.5mV/min over 50 seconds was set. Titrations were performed up to pH10 and then back titrations using the same criteria were 0.1 M HCl to pH4. Suitable blanks (without NPs) were run and Gran method was used to find the equivalent point of the acid base titrations as explained below (see section 3.1.9 for theoretical description of the method).

4.4.9 Studying the aggregation of AuNPs in solutions of different ionic strength.

To investigate the effect of ionic strength on the size of the AuNPs, sodium nitrate solutions of different concentrations were prepared. A stock solution of 1M NaNO3 was made by weighing 8.499 g and dissolving in 100 ml of Milli-Q water. From this stock solution another more diluted stock solution of 100 mM of NaNO3 (stock solution 2 hereafter SS2) was prepared. After period of trial and error it was found that citrate capped and PVP capped NPs need to be test in two different ranges of NaNO3 concentrations due to lack of stability of citrate capped NPs in higher ionic strength. Citrate capped NPs were tested in a range of

–  –  –

investigated in higher concentrations of NaNO3 varying from 0 to 100 mM.

4.5 Bacterial growth and media preparation techniques 4.5.1 Media Preparation The bacteria used in this project were pseudomonas fluorescens strain SBW25 provided by Professor Christopher Thomas from school of biomedical science at the University of Birmingham. A detailed description of the pseudomonas fluorescens was given in chapter2 (research background) section 2.3.3. Section 2.4 of the same chapter introduced the current knowledge of the interaction between these bacteria and the NPs in general with emphasis for the need of further research.

Pseudomonas agar base was used to grow bacteria on agar plates. To prepare growth media for the bacteria, 39.72 g of the agar were dissolved into 800 ml of distilled water and then 8 ml of glycerol were added to the boiling agar mixture followed by 15 minutes of sterilisation at 121 oC. After cooling down to 50 oC, the agar was poured into sterile Petri dishes in the clean laminar flow cabinet and stored at 4oC inside fridge. Biweekly, bacteria cells were spread on these fresh agar nutrients to keep them alive. Prior to the investigation of the effect of gold NPs on bacteria their stability in a liquid bacterial growth media need to be tested.

Minimal Davis Media is used for this purpose and ingredients in the media are summarised in Table 4-2 below.

–  –  –

This solution was autoclaved and appropriate dilution was made depending on the concentration of the desired final media. Glucose was dissolved in the media and sterilised by using 0.1µm filter. The total ionic strength of the 5x concentrated stock solution of the minimal Davis media was calculated using Equation 4-2 (Solomon, 2001) and was found to have value of 191 mM.

–  –  –

4.5.2 pH-measurement The pH of the freshly synthesised NPs solutions and bacteria growth media was measured using pH meter from Thermo Electron Corporation. Prior to the actual measurement of the sample pH, The pH meter was calibrated using standard buffers 4, 7 and 10 and between each two buffers the pH sensor was thoroughly washed with milli-Q water. The sensor was

–  –  –

results were recorded.

4.5.3 Sterilisation Techniques Sterilisation is the process through which equipments or solutions are made microorganisms free either by destroying or trapping the microorganisms to separate from solution(Moisan et al., 2002). Among the techniques employed to achieve sterilisation include heating, filtering, radiating and using chemicals depending on the nature of the materials which are to be sterilised(Mendes et al., 2007).

In this project, all glassware and other autoclavable materials were sterilised in a steam autoclave at a temperature of 121oC for 15 minutes and then cooled to room temperature rapidly. During preparation of Minimal Davis Media, all inorganic salts without glucose was dissolved and steam autoclaved while glucose solution was sterilised through membrane filters of nitrocellulose with pore size 0.1µm to remove any bacterial contaminations. When bacterial cells are needed to be centrifuged out of the media, sterile plastic centrifuge tubes were used. Any handling regarding the preparation of the media and sampling of the bacterial culture for the further analysis was carried out in a clean laminar flow cabinet. The floor of the cabinet was treated with 70% ethanol-water. Inoculating metallic loops are sterilised by heating them with the roaring blue Bunsen burner flame until they glow red and all bacterial cells are destroyed.

–  –  – : Chemical Fixation Bacterial samples were grown in MDM media for a period of 12 hrs to reach the exponential growth phase. Similar concentration of Gold NPs of different coating agents and gold ions were exposed to the bacteria for a period of 5 hrs. Bacterial samples were centrifuged (see section 4.4.

1 above) and cells were chemically fixed using 2.5% of gluteraldehyde in phosphate buffer solution for 1 hour. This step was followed by secondary fixation step where the bacterial cells were transferred in 1% tetroxide and kept for another one hour Dehydration Bacterial cells were then dehydrated using a series of gradually increasing alcohol solutions as summarised in Table 4-3 below.

Table 4-3: Concentrations of the alcohol used for dehydration of the bacterial cells.

–  –  – Embedding After treating the cells with the above mentioned series alcohol concentration, propylene oxide /resin (1:1 ratio) was prepared on a rotator in a fume cupboard for 45 minutes. The resin was left on the rotator for another one hour. Samples were then placed just under the

–  –  –

was then polymerised at 60 oC for 16 hours. Ultra Thin Sections To obtain ultra thin sections (50 – 150 µm), diamond knife was used and sections were collected on TEM grid.. Samples were then stained with uranyl acetate before examination with the TEM (see section 3.1.2).

4.5.5 Quantification of Bacterial Growth There are different ways of quantifying bacteria growth. One traditional quantification method is the bacterial colony counting method where the number of colony of bacterial sample on agar plate is counted. Simple comparison of the number of colony in samples treated with chemicals and the number of colony of untreated sample gives information about the bacterial growth inhibition effect of the chemicals under investigation. Another Alternative, faster method for bacteria quantification is the optical density measurement technique explained below.

The growth of the bacteria in the Minimal Davis Media (MDM) was monitored continuously in the whole growth period and phases by measuring the optical density of the bacterial suspension. The more cells are in the media the more cloudy the suspension becomes and the higher the absorbance of the light is. Optical density is measured with UV_Vis (see section 3.1.6 for a detailed description of this technique) and the wave length chosen was 595 nm which is the wave length where most of the light is absorbed by pseudomonas fluorescens (Fabrega et al., 2009).

–  –  –

biomass in the sample. The measurement is based on chemical, photometric and/or physical variables. Of the above mentioned properties, the most implemented one is the sensors that measure the turbidity of the culture such as optical density measurements. It is an easy, fast and non-destructive method (Toennies and Gallant, 1949) for estimation of the bacterial mass in a culture. Here, the amount of light absorbed by the culture is compared to a blank sample and the difference is related to the bacterial population in the culture (Clesceri et al., 1998) Linear trapezoidal method was applied to estimate the area under the growth curve of the bacterial culture. There are three steps in this method: first the area of each trapezoid is measured using Equation 4-3.

–  –  –

Where A is the area of each trapezoid, O1 and O2 are optical density of the culture at time t1 and t2.

Then the area of all trapezoids are added together to give estimation for the area under the curve. Finally, the percentage growth inhibition was calculated using Equation 4-4

–  –  –

The mean and standard deviation of the measurements were calculated using Equation 4-5 and Equation 4-6 below respectively (Miller and Miller, 1993). These equations are build in function of the excel software which automatically calculated and provides the values.

–  –  –

Similarly to carry out the significance tests between sizes measured with different techniques student t-test was calculated using the following equations. First of all pooled estimate of standard deviation was calculated from the two individual standard deviations using Equation 4-7 below.

–  –  –

Where n-2 is the degree of freedom and r is correlation coefficient. Then tdist (t, freedom, tail) function of the excel software was used for this purpose.

–  –  –

5.1 Introduction Synthesised NPs with known properties can give a basic platform as test materials to understand the potential fate, behaviour and biological effects of nanomaterials on organisms and in ecosystems. NPs of the same elements with different coating agents can be synthesised to study how the surface coating affects their fate and behaviour in the environmental relevant conditions. Likewise, the size, shape and the charge of the nanoparticles can be tuned to study the property - response relationship of the environmental organisms. Although it is hard to reach a clear-cut consensus on the number of parameters that should be provided in ecotoxicology studies, scientists suggested that a minimum set of parameters should include the elemental composition of the particles, as well as surface morphology, and imaging by TEM (Bucher et al., 2004). Thus, the first aim of this PhD project is to synthesise a well defined AuNPs of range of sizes and with different coating agents. After synthesis, the particles need to be fully characterised using a number of techniques and their stability will be measured and monitored. Next sections will be devoted to describe the synthesis procedures and the analysis and interpretation of the characterisation results. It will be the task of the next chapter 5 to study the interaction of these synthesised and fully characterised AuNPs with environmental relevant bacteria Pseudomonas fluorescens.

–  –  –

Gold nanoparticles of a range of sizes with different capping agents were prepared using three different synthesis methods as fully described in chapter 4 section 4.2 and its subsections. Special attention was devoted on the quality of the product of the synthesis methods by comparing the properties of the nanoparticles in terms of size distribution, monodispersity, solubility and sphericity of the NPs. For the characterisation of the NPs, a multi-method approach was applied(Domingos et al., 2009a, Baalousha et al., 2012a, Baalousha et al., 2012b) where different but complementing modern analytical and imaging techniques were used to collect relevant and quality physicochemical data for the NPs.

5.2.1 Synthesis and growth of AuNPs In the present work, the synthesis of AuNPs was carried out using two different capping agents: citrate and PVP. Samples with codes G1, G2 and G3 (see Table 4-1 in chapter 4 for the experimental conditions and the concentrations of the reactants used for each sample) used trisodium citrate. When the citrate solution was added into the boiling tetrachloroauric acid solution, an instantaneous colour change was observed. Upon addition of citrate, the initial yellow gold solution turned into a colourless solution which indicates that the gold ions are reduced into neutral Au atoms (Polte et al., 2010). Then the colourless solution became blue followed by a wine-red colour within 5 minutes which remained unchanged. This red colour indicates the formation of stable gold nanoparticles (Faraday, 1857, Turkevich, 1951, Frens, 1973). In like manner, the colour change and the growth of the samples G4, G5 and G6 (see Table 4-1 in chapter 4 for description of the samples) which were reduced by NaOH and stabilised with PVP manifested the same pattern as the above explained citrate capped nanoparticles with a similar time scale. However, the colour change of the samples G7 and

–  –  –

section 4.2.

Pages:     | 1 |   ...   | 9 | 10 || 12 | 13 |   ...   | 23 |

Similar works:

«FEMINIST AWARENESS AND NARRATIVE CHANGE: Suicide and Murder as Transitional Stages toward Autonomy in Women's Protest Writings RACHEL GIORA Suicide and murder are considered two facets of the same coin: they originate in anger. Whereas suicide is the result of turning one's anger against oneself, murder is acting it out against others. Reviewing women's protest writings before and after the feminist revolution in the 1970s, the argument is made that the suicidal heroine in women's protest...»

«1 On PANDAS, the LeRoy Controversy and False Dichotomies By Gunilla Gerland This text can be shared or published in its entirety on Facebook, websites etc. as long as it is not altered, or quoted in parts as long as the author's name is mentioned. An epidemic outbreak of tics disorder among young girls in an area in LeRoy, New York has become international news, and a documentary, The Town That Caught Tourette's, has aired in several countries (1). In the documentary, as well as in reality, the...»

«Public Broadcasters, the Internet, and Democracy Comparing Policy and Exploring Public Service Media Online Hallvard Moe Dissertation for the degree philosophiae doctor (PhD) University of Bergen Public Broadcasters, the Internet, and Democracy Comparing Policy and Exploring Public Service Media Online Bergen, 2008. Copyright: Hallvard Moe (Part I, and Part II, Article 1); Sage Publications (Part II, Article 2, 4, and 5); Intellect Books (Part II, Article 3). Public Broadcasters, the Internet,...»

«Ch. Charan Singh University Meerut Information Brochure 2016-17 for Admission to Regular (CBCS) & Self–Financed Courses of the University Campus 1 Message from the Vice–Chancellor India, the oldest civilisation and the oldest nation of the world, inherits a rich tradition of intellectual exploration. The Rigveda prayer :' आ नो भद्रा क्रतवो यन्तु ववश्वतः' (Let the noble ideas come to us from the entire world.) underlines the openness in...»

«COGNITIVE SCIENCE 8, 1-26 (1984) Discrimination Nets as Psychological Models* LAWRENCE BARSALOU W. Emory University GORDONH. BOWER Stanford University Simulations of human cognitive processes often employ discrimination nets to model the access of permanent memory. We consider two types of dixrimina. ~ tion nets-EPAM and positive-proper&only nets-and argue that they have insufficient psychologi&l validity. ~hei;deficiencies arise from negative properties, insufficient sensitivity to the...»

«VARIATION OF FEEDING REGIMES: EFFECTS ON GIANT PANDA (AILUROPODA MELANOLEUCA) BEHAVIOR A Thesis Presented to The Academic Faculty By Estelle A. Sandhaus In Partial Fulfillment Of the Requirements for the Degree Master of Science in Psychology Georgia Institute of Technology September, 2004 Variation of Feeding Regimes: Effects on Giant Panda (Ailuropoda melanoleuca) Behavior Approved by: Dr. Terry L. Maple, Advisor School of Psychology Georgia Institute of Technology Dr. Mollie A. Bloomsmith...»

«Marathon High School Wendy McPherson, Principal 350 Sombrero Beach Road Marathon, FL 33050 Page 1 Marathon High School School Mission Statement Our mission is to maximize student learning in a safe and orderly environment. School Belief Statements  Students will learn.  Students will focus on learning in a safe and secure environment.  Students will be challenged with a rigorous curriculum.  Students will be provided with a variety of instructional approaches.  Students will be...»

«*** Free EBOOK*** Your Journey of Personal Transformation Begins HERE: An Introduction to Understanding Your Human Design Chart by Evelyn Levenson, MBA Certified Human Design Specialist Your Success By Design Coach Your Journey Begins HERE www.HumanDesignforSuccess.com This Report is for your personal use. You MAY SHARE it, in its entirety with all author and contact information. However, You May NOT Trade, Sell or Resell This Intellectual Property in Any Way. You may not reprint any part of it...»

«THE JOURNAL OF NEUROLOGY AND PSYCHOPATHOLOGY Vol. XVII. APRIL, 1937 No. 68 Oriuuatnal 1apers AN ENQUIRY INTO THE CAUSES OF MESCAL VISIONS BY C. R. MARSHALL, TUNBRIDGE WELLS INTRODUCTION MESCAL hallucinations have recently been investigated in the hope that their elucidation might help to unravel other hallucinatory phenomena. Zucker 1 administered mescaline to patients with hallucinations. From the protocols given many of the effects obtained (coloured lights, tapestry patterns, visions of...»

«Fools Die Mario Puzo Book I Chapter 1 “Listen to me. I will tell you the truth about a man’s life. I will tell you the truth about his love for women. That he never hates them. Already you think I’m on the wrong track. Stay with me. Really—I’m a master of magic. “Do you believe a man can truly love a woman and constantly betray her? Never mind physically, but betray her in his mind, in the very ‘poetry of his soul.’ Well, it’s not easy, but men do it all the time. “Do you...»

«Consulting Psychology Journal: Practice and Research © 2014 American Psychological Association 2014, Vol. 66, No. 1, 1–5 1065-9293/14/$12.00 DOI: 10.1037/cpb0000001 EDITORIAL CPJ4.0: ASPIRATIONS OF A RESEARCH GEEK WITH A THING FOR APPLICATIONS Robert B. Kaiser Kaiser Leadership Solutions, Greensboro, North Carolina It is a cool honor and opportunity to be selected as the fourth editor of Consulting Psychology Journal: Practice and Research (CPJ). This is a unique scholarly publication. Sure,...»

«Organisatorisk segmentering i de danske kommuner: Har Strukturreformen haft en effekt?Abstract: Dette paper behandler spørgsmålet om, hvilken betydning den danske Strukturreform anno 2007 har haft for den organisatoriske segmentering i de nydannede kommuner. Inspireret af henholdsvis historisk og rationel nyinstitutionalistisk teori opstilles to konkurrerende forventninger til Strukturreformens effekter. Først argumenteres der for, at Strukturreformen har givet kommunerne en unik mulighed...»

<<  HOME   |    CONTACTS
2016 www.dissertation.xlibx.info - Dissertations, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.