Microbial Contamination Product Degradation Contamination Invasion Toxic Material Biology Essay

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Microbial contamination causes product degradation and contamination or invasion of toxic material into living cells by the bacteria. Microbial contamination is a serious case especially for those suffer under immunodeficiency syndrome e.g. AIDS patient or those who received chemotherapy. Research also found out that some fungi are potentially dangerous to this group of people due to the biologically active compounds released known as mycotoxins which is lethal to human health. (Cioffi N, 2005) Mycotoxin can be various and an example, aflatoxin produced by species of Aspergilus fungus, it is toxic and potentially carcinogenic. (W.Hudler, 1998) Beside of fungus, pathogenic bacteria can cause different disease and more commonly upset of digestive system.

Surgical sector deeply concern about problems brought by microbial contamination. In 19th century, surgery was at high risk and dangerous because the surgery is not operate under an aseptic conditions. The surgical instrument, surgeon hands were not sterilized before any surgery. (todar, 2008) These caused patient expose to unseen microbes, which caused infection and mortality. The founder for antiseptic surgery is an English surgeon, Joseph Lister who accept the first bacteriology theory of Pasteur's germ theory of disease to surgery at that age, who first used carbolic acid (phenol) as an aseptic agent, periodically spray around the operating room by an aerosol sprayer. (todar, 2008) This method was slowly picked up by other surgeon because it had increase the surviving rate of patients.

Control of microbial growth affected by two basic ways; cidal agent that kill cells, and static agents that inhibit the growth of microorganism. Other than using chemical agents, sterilization also can carry out through some physical technique like using heat by raising temperature to certain degree for sterilization purpose, e.g. incineration, boiling and autoclaving (steam under pressure) which usually operate under 121°C and 15psi for 15minutes and longer if loads is large.

Antibiotic is another type of reagent to resist the growth of the bacteria. It is extensively used in public health and also industrial field. As a result, an increase occurrence of antibiotics resistance genes in many bacterial species is found in human body and animal mainly extensive use of antibiotic in human health, veterinary and agricultural use. The overuse of antibiotics against pathogenic bacteria causes harmful side effects that resulted in the emergence of resistance to antibiotics in bacteria through selection. Antibiotic resistance genes are therefore can be transfer from intestinal bacteria flora to pathogenic bacteria situated at the outside environment. (D.jonkers, 2002)

Hence, extensive research on material that can substitute the function of antibiotic was carrying out. From previous research, some inorganic compounds founded potentially inhibit the growth of microbial chemically, e.g. silver and also copper. Some further research on effect of size of silver and copper on antibacterial activity also reported in journal. However, silver was an expensive element that is less commercial valuable in working as a bactericidal or bacteria static agent. Therefore, further research has to be carrying out on copper nanoparticles testing with different type of bacteria and study the minimal inhibition concentration and minimal bactericidal concentration on those particular bacteria.

copper(II) oxide

The existing copper on earth is about 0.01% relative to all other element exist on earth or it is about 15,000,000 tons compare to abundance of silver where it is only traces and estimated to be 23,000 tons. (Mineral Information Institute, 2010) Copper have been used for disinfection purpose on water purifier, an algaecide, a fungicide, gematocide, molluscicide, antibacterial and antifouling agent for centuries. (I.Perelshtein, 2009) Copper is one of the metallic elements that essential to human health in trace amount. This element will associate along with amino acid and fatty acids for metabolic processes. Low sensitivity of human tissues to copper is another advantage for it to perform the role of disinfectant whereas microorganisms are more susceptible to it. (I.Perelshtein, 2009)

The novel properties of copper oxide were not end here but play an important role in environmental science. It is a very good decomposing agent that used extensively in the wastewater treatment technology. It is a very good oxygen carrier that able to oxidize, detoxifies lots of toxic chemicals such as cyanide, hydrocarbons, halogenated hydrocarbons, and dioxins contained waste with retrievable end product of elemental copper solids in a detoxicification chamber with moderate temperature. (Charlie W.Kenney & Laura A.Uchida, 1986 Apr)

Reactions formula for some example of toxic chemicals,

C6H5OH + 14CuO ƒ  6 CO2O + 3H2 + 14 Cu -------------------------- (oxidation of phenol)

C6Cl5OH +H2O + 9CuO ƒ  6CO2 +5HCl +9Cu ----- (undergoes wet oxidation of Pentachlorophenol, PCP)


Nanotechnology is the newly developed field and is hottest area for research due its fascinating properties offers by its atomic level structure which are cannot achieve by older technology made materials. Nanotechnology started 50 years back but remains dormant because condition 50 years back was not encouraging by the technology available and cost of the process. (National Nanotechnology Initiative, 2001)However, with recent emerge of all kind of technique and cheaper and cost efficient instrument invented nanotechnology once again attracts both industry and also researchers. With the recent finding, a series of novel properties founded through certain metal nanoparticles, which are not achieve by the usual bulky metal cluster e.g. nanotech-enabled sensors may detect cancer cell through smell senses, where the odor profile can be mapped for identification of certain skin cancers. (National Nanotechnology Initiative, 2001) This has interested researchers on unusual properties that nanoparticles readily have.

Nanotechnology is the technology that used the unique phenomena of nanoparticles which ranged from 1 to 100 nm in several applications. There also present of natural nanomaterial through sea spray and erosion; many important functions of organisms are controlled by the nanosized material as well e.g. hemoglobin, the protein molecules which acts as oxygen carrier in bloodstream are 5 nm in diameter. (Natzke, 1998)

Nanotechnologies bring revolution and transformation in multiple industries by changing the old science aspect to something smaller and work more efficient. There are other kinds of benefits and applications in nanoparticles discovered beside the antibacterial activities of copper(II) oxides. Some engineered nanoparticles used commercial products and processes to add strength to composite materials used such as making lightweight tennis rackets, baseball bats and bicycles.

In pharmaceutical industry, some products are reformulated with nanosized particle used to improve their adsorption and make them easier to administer. One of the recent research is by applying nanotechnology to engineer a gel that both stimulate and enhance the growth of nerve cell, which the gel fills the extra cellular matrix (ECM) of the nerve cell to encourage it to growth in much faster rate. (National Nanotechnology Initiative, 2001) If the research is success, it probably can re-grow lost or damaged spinal cord and brain cells.

In textile industry, nanoparticles were used extensively in coating and other fiber enhancing properties, e.g. anti-burning for firefighter cloth, antibacterial cotton wool for pharmaceutical usage, anti-wrinkle properties make cloth more easily to iron and etc. In fact, now researcher is doing research on inventing nanosensor in packaging for detecting food borne pathogens.

Objectives of the Study

Although many researcher discovered presence of antibacterial activities of copper(II) oxide but still lack of knowledge on the efficiency and susceptibility of different bacterial toward the copper(II) oxide. Therefore, the comparison of efficiency is done through by testing copper(II) oxide activity using different gram positive and negative bacteria. It would allow more comprehensive testing on the antibacterial activity of copper(II) oxide had. Sonochemical method is using in the synthesis to prepare very fine nanoparticle of copper(II) oxide. After that, bio-assay was carry out by using both quantitative test through 96 well plate dilution method and qualitative test through disc diffusion method, both bacterial-static concentration and bacterial-cidal concentration can be obtain.



2.1 Synthesis of copper(II) oxide nanoparticles

Copper(II) oxide is one of the oxide metal other than copper(I) oxide that given by copper element. From the light of the antibacterial activity founded with copper itself, many researcher starts to synthesize copper(II) oxide through different method and it give different effectiveness, size and morphology of copper(II) oxide nanoparticles.

Size of nanoparticle always a challenging issue for nanotech researcher. Researcher Ojas Mahapatra and colleague successfully synthesize copper(II) oxide nanoparticles of 80 to 100nm in size by using simple wet base chemical method. In his study, he was using copper carbonate as starting reactants and sodium hydroxide to undergo acid-base reaction to give copper(II) oxide. (Mahapatra, 2009)For investigating the relation of size and the starting concentration, researcher Ojas Mahapatra synthesized the copper nanoparticles with 2 different concentrations, one with 0.57M and other with 1.57M concentration. (Mahapatra, 2009) As a result, the lower concentration gave smaller size of 80-100 nm nanoparticles and 120-160nm nanoparticles were obtained with the higher concentration of copper carbonate. The size of nanoparticles was measured by Agilent Technology's Scanning Probe Microscope. It reports that suspension of copper(II) oxide synthesized with distill water are stable for long time which do not sink or flocculate. However, effect of pH was not report. In another journal, author Guogang Ren and colleague claim that by using thermal plasma technology method allow producing copper(II) oxide nanoparticle with smaller mean size of 60.6nm through continuous gas phase production. (Guogang Ren, 2009) Other than using expensive technology to control the size distribution of nanoparticles synthesized, Dongyun Han research on microemulsion method for narrower size distribution of nanoparticles. He used triton X100 based water in oil reverse micelles system to control the size of nanoparticles. He claims that by using different amount of proportion of water, triton X100 and alcohol would give different size of copper nanoparticles and also the distribution of size having. Dongyun Han used mainly 3 phase in synthesizing copper(II) oxide nanoparticle, (Dongyun Han, 2008)


60% Triton X-100 + 40% cyclohexane

60% 1-pentanol/1-hexanol + 40% cyclohexane

By using different ratio of water to surfactants, series of size and size distribution of nanoparticles produced and the results was recorded. The outcome of his research on molar ratio water to oil (R=4, 7 and 10), Dongyun Han find out that the most narrow distribution of phase system is R=7 which give more than 50% of the total at 25nm, whereas R=4 give 48% at size 15nm and wide size distribution given when molar ratio R=10. (Dongyun Han, 2008)

Besides that, Z.P. Zhang and coworker claims that by manipulates the temperature, we can synthesis other oxidation stage of copper nanoparticles instead of copper(II) oxide. In His study, he tried to synthesis 3 types of copper nanoparticles with manipulating the temperature, CuO, Cu2O and Cu with temperature of 100, 180 and 220 degree Celsius. Copper(II) oxide nanoparticles synthesized from copper(II) acetyacetonatato precursor with oleylamine as medium. He describe that fatty amine products are used as a dispersing agent for better nucleation occur in the solution, it also enhanced the processibility due low friction and abrasion faced by atomic copper. (not complete yet)

Synthesis nanoparticles through sonochemical method are the claim to be the best way to synthesize nanoparticles with both good homogenousity and small size. Fabric surface coating study by Abramov shows that copper(II) oxide nanoparticles synthesize through this way have great homogenousity and size around 10 to 20 nm measured through HR SEM. (O.V. Abramov, 2009) Reactions occur in situ with direct reaction from copper(II) acetate with aqueous ammonia. The medium are adjusted slightly basic with pH of 8-9. The vibration produced by sonicator through sonochemical method, small bulk copper(II) oxide produced collides with surface of cotton wool and bind to it. However, to investigate the chances of leaking ions, Abramov carry some test by immersing the cotton wool in 0.9 sodium chloride water and the results shows only 1% of copper(II) ions detected. (O.V. Abramov, 2009) it indicates that nanoparticles coated on the cotton wool have strongly bind to the fibers when high frequency vibration charged on it, sonic wave distribute the reactants homogenously give narrow size distribution product.

Studying morphology is an interesting topic as well for nanotechnology researcher. When different methods used on synthesis Hongxia Zhang claims that different type of base will give different in surface morphology of nanoparticles. In his study, he success obtain three type of morphology of nano copper(II) oxide particles, cauliflower, feather and nanobelt like morphology by using chemical deposition method. Measurements of size of the nanoparticles operate through SEM and FESEM. The smallest size are nanobelt shape copper(II) nanoparticles follow by feather-like and the largest is cauliflower. Hongxia Zhang shows that by using aqueous ammonia NH3.3H2O with sample A gave cauliflower structure, and reaction with molar ratio 2:1 NaOH to NH3.3H2O give feather like nanocrystalline and reaction with strong base NaOH gave nanobelt structure.

2.2 Antibacterial activity

Some journals also claim that pure copper nanoparticles shows antibacterial activity besides copper(II) oxide nanoparticles.

Antibacterial agent is crucial to have ability that kill the bacteria effectively yet bring harmless to human body. Bacteria  can be divided into variety according to the strain and also gram positive and gram negative. Gram negative bacteria are the bacteria do not retain crystal violet dye in the gram staining protocol. It being claim that the pathogenic capability of gram-negative bacteria is associated with certain components of gram-negative cell walls particularly the lipopolysaccharide layer. Lipoplysaccharide layer contain some non-human cell compound that will trigger innate immune response of cell that will cause inflammation or host toxicity.

There are relatively many way to test the antibacterial activity of the copper nanoparticles but still many relying on the suspending and diffusing principles. Guogang Ren and coworkers used suspension method for doing antibacterial activity. In his study, he claims that concentration of copper need to kill the bacteria is higher than it required for silver nanoparticles. (Guogang Ren, 2009) However, cost of silver nanoparticles in another issues. In his study, 4 different gram positive strain and 3 different gram negative strain were tested. Type of gram positive used include Staphyloccocus aureus, staphylococcus epidermidis SE-51, S.eidermidis SE-4, gram negative are Escherichia coli ETC 9001, proteus spp and Pseudomonas aeruginosa PAOI. (Guogang Ren, 2009) Guogang Ren used time kill assay to run his research. In populations of gram-positive (x4 strains) and gram negative (x3 strains) organisms tested were reduced by 68% and 65%,respectively , in the presence of 1000µg/mL nano CuO within 2 hour. (Mahapatra, 2009) It also report that P.Aeruginosa, S.Aureus, EMRSA-16 and S.epidermidis SE-51 were reduced to zero by 4 h in the presence of 1000µg/mL nano CuO by using mean size of 60.6nm copper nanoparticles with average surface area of 14.6931 m2/g with amount of 5 mg/mL concentration of copper(II) oxide nanoparticles, most of the strains tested which included gram negative and positive are killed. (Guogang Ren, 2009) Guogang Ren claims that CuO nanoparticles were killing a range of bacterial pathogen involved in hospital-acquired infections. He also claims that reduced amount of negatively charged peptidogylcans would make Gram-negative bacteria less susceptible to such positively charged antimicrobials.

Other than copper(II) oxide nanoparticles, Muhammad Raffi tried to investigate the antibacterial activity of elemental copper nanoparticles. He also used suspension method to investigate the antibacterial behavior of copper nanoparticles against Escherichia coli ATCC-15224. The grown axenic culture of E.coli was inoculated into flask containing liquid nutrient growth medium (CM-01) and supplemented with various concentrations of copper nanoparticles (20, 40, 60, 80 and 100 µg/mL) to investigate the minimum bacterical concentration (MBC) of the Escherichia Coli. The samples were taken periodically from the flask to measure optical density at wavelength 625nm using UV-Vis spectrometer to examine the growth of bacterium. (Muhammad Raffi, 2010) The number of bacterial colonies is then observed on solid nutrient agar plates was a function of copper nanoparticle concentration. He reports that CFUs were reduced significantly with increasing copper nanoparticle concentration in the growth medium especially 60 µg/mL and above. (Muhammad Raffi, 2010) He claims that at low concentration of copper nanoparticles, copper acted as a micronutrient for bacteria and causes only a delay in the lag phase, where as higher concentrations bacterial growth ceased. (Muhammad Raffi, 2010) The results shows that at concentration of 20 and 40µg/mL, optical density of the solution raise up sharply at the 5th hour; 60 and 80 µg/mL However, delay the lag phase of the bacteria until 10 hours and the growth rise up sharply after that, whereas 100 µg/mL shows bactericidal effect of copper nanoparticles.

With a different physical principle, Ojas Mahapatra used diffusion method to carry out his bioassay. Instead of working on liquid suspension method, he had done the test through diffusing his particles on the agar plate. In his test, he used 100mg/ml concentration of copper(II) oxide nanoparticle and serial dilution it and put it on socking disc. The incubation process of the test is at 37°C and observes it after 30 h. He claims that the nanoparticles suspension is active against all microorganism tested but reduced after heavy dilution. He reported that copper(II) oxide nanoparticles active against pseudomonas aeruginosa even at low concentration of 1:256 dilution ratio and same happens to klebsiella pneumonia and salmonella paratyphi but shigella was a bit resistant and the copper(II) oxide only effective on it at ratio of 1:128. (Mahapatra, 2009) However, he done a 96-well plate test on HeLa cancer cell line, but it doesn't give an optimistic result. It had been examine under SEM that the HeLa cell wall is less rupture or affected than other type of bacteria. (Mahapatra, 2009) He claims that copper(II) oxide is selectively active against prokaryote cell but not eukaryote cell due to less penetrative of eukaryotic cell. Hence, he claims that copper(II) oxide is potential bactericidal agent since it is less harmful to human cell.

I.Perelshtein and coworkers used a coating method, develops a different prospective of antibacterial activity that can be carry out. In his experiment, he synthesized the copper(II) oxide nanoparticles through sonication method with one step synthesis and coated on a cotton wool. (I.Perelshtein, 2009) He claims that with 1.4 weights percentage of CuO could effectively killed most of the bacteria. In his study, he used a gram positive strain, staphylococcus aureus (ATCC 10407) and a gram negative strain, Escherichia Coli (ATCC 29067) to study the effectiveness of copper(II) oxide nanoparticles on both grams. Concentration of bacteria was prepared according to the standard protocol. To prove that copper nanoparticles could bring antibacterial activity, author also carry out a standard blank by adding a positive sample where the sample was same as the main test sample but adding 0.9% of NaCl instead of adding copper nanoparticles. (I.Perelshtein, 2009) Viable bacteria test was monitored by counting the number of colony forming units from the appropriate dilution on the nutrient agar plate. The antibacterial assay was a time kill assay, where it will periodically examine at time first hour and the second hours. As the result obtained, the time kill assay of E.coli and S.aureus reported 100% killed after one hour time. (I.Perelshtein, 2009) I.Perelshtein claims that copper(II) ions weren't the key component on killing the bacteria. In his reports, he shows that the leaching of Cu2+ ions in the leaching solution only 0.15ppm examine through ICP. (I.Perelshtein, 2009) Besides that, to further confirm the copper ions have no influence on the antibacterial activity, author also carry out suspension method to test on the antibacterial activity off copper(II) ions. It has been reported that no reduction in E.coli after 2 hour where the sample incubate for 24h and 37°C. (I.Perelshtein, 2009)


3.1 Chemicals

3.2 Preparation of CuO Nanoparticles

Sonochemical method was used to prepare the copper(II) oxide nanoparticles. Copper(II) acetate monohydrate was first dissolve in the portion of 1:10 of water to ethanol solution by heating it moderately. Dissolving of copper salt is done by slow adding to a preheated the water/ethanol solution to about 70 °C. It shows a greenish blue colour after the copper salt was dissolved in. The solution was stable without any change of colour more than 24 hours. Suction filtration was carried out to remove any impurity as well as some compound that are not dissolving.

The solution was assured to be stable for about half an hour before carry out the sonication by the probe sonicator. The copper solutions was sonicate fewer than 70% amplitude for 5 minutes to ensure homogenousity is good and 1.5ml of 25% ammonia solution was added to the solution and sonicate it until 1 hour.

The solution will first turn darker blue after the adding of ammonia solution, after sonication, the colour of copper(II) acetate solution become brown in colour. However, there is still some bluish colour shown. Nanoparticles formed are then collected in centrifuge tube by using centrifuge machine at 9000 rpm for 5 mins as shown in figure. The solutions are now separated to 2 layers, dark brown solid nanoparticles are collected at the bottom of the centrifuge tube and the dark blue solution is remaining on top. It indicates that non 100% copper(II) ions was reacted and give copper(II) oxide nanoparticles.

Figure 1: 9000rpm universal 32R

The bluish solution was removed and the nanoparticles are washed by deionised water few times and wash with 95% of ethanol solution. The copper nanoparticles remained are dry at oven at 70°C for 24h. The sample was collected and weighted.

3.3 Characterization of Nanosized CuO

Characterization of copper(II) oxide was carried out by using Fourier transform infrared spectroscopy and x ray diffraction techniques. XRD analysis was conducted by Shimadzu XRD 6000 with Cu Kα radiation (λ= 0.15418nm, scan rate 0.02os-1, range 20-75o). IR spectroscopy analysis was done by using model of Perkin Elmer Spectrum RX1 Fourier Transform infrared spectrometer with KBr pellet.

3.4 Determination of the Percentage Yield

Determination of percentage yield was done by using,

= theoretical mol of copper(II) acetate monohydrate

Copper(II) acetate monohydrate : copper(II) oxide = 1:1


Theoretical weight of copper(II) oxides = mol of copper(II) acetate monohydrate x molecular weight of copper(II) oxides

Percentage yield (%) = x 100%

3.5 Calculation of Particles Size Using Debye -Scherrer Equation

The particles size of synthesized CuO was calculated by using equation below:

Calculation of β


= (FWHM in 2θ x π) / 180o (3.3)

Debye-Scherrer Equation


= k λ / (β cos θ) (3.4)


FWHM = Full width at half maximum

D = Crystal diameter

k = Debye-Scherrer constant, 0.9

λ = X-ray wavelength, 1.5406x10-10 m

β = Width of a diffraction peak

θ = Diffraction angle

3.6 biological assay- antibacterial activities

Biological assays were done by using both the suspension method and also diffusion method. However, both method targeting aspect are different. Suspension method - 96 well plate diffusion method are used to determine the minimum inhibitory concentration and also minimum bactericidal concentration while diffusion method - well diffusion gave the indicatory result where shows the wideness of inhibitory zone through diffusion of nanoparticles; identify how far nanoparticles capable of inhibiting the selected bacteria.

3.6.1 96 well plate dilution test preparation

To ensure that all the apparatus without any contamination, the apparatus are cleaned with deionised water and autoclaved under the steam autoclave unit at 121°C, 15 psi. All the apparatus are taped with sterilization indicator tape for ensuring the apparatus are sterilized. Apparatus autoclaved included different size of micropipette tips head. Nutrient preparation

Mueller Hinton Agar was used as the growing medium for culturing selected bacteria. The culturing process is done one day before the antibacterial testing to ensure the bacteria grown are fresh and healthy for the test.

Agar plate was prepared by pouring an autoclaved Muller Hinton agar solution to the petri dish. The concentration used not complete yet 96 Well plate test method

96 wells plate consists of 12 x 8 wells where it designed to have 4 corners of sterility control, positive and negative controls. Positive control consists of Muller Hinton Broth and the bacteria only and it is to make sure that the bacteria are healthy and shows growing. Where negative grow is consists of copper nanoparticle suspension and the broth, which can make sure without contamination to the particle suspension. 4 corners of sterility controls is to make sure the broth are without bacterial contamination.

Each well was pre-added with 50 µL of MHB and then 50 µL of of copper(II) oxide nanoparticles suspension was added to each first well of selected bacteria and serial dilution was carry out. The prepared bacteria suspensions were added after it with volume of 50 µL.

The bacteria were pre-cultured 24 hrs at 37 °C. One well isolated similar colonies were transferred to 2 to 3 ml of Muller hinton broth with the use of inoculation loop. The concentration of bacteria was tested with 625nm wavelength by using photometer model of photometer with absorption in the range of 0.08 to 0.1 abs. This results in a suspension containing approximately 1 to 2x 108CFU/mL of bacteria. The turbidity of the was adjusted to the range of absorbance according to the standard requirement. The blank used was the pure Muller Hinton Broth. The dilution of 100 factor was done to the inoculums to obtains 1 x 106 CFU/mL within 15 minutes time for preventing the overgrown of bacteria concentration and the inoculums were transfer to each well of 96 well plate after the nanoparticles dilution step was done.

Incubations proceeded in an incubator model of model at 37°C for 24hrs with shaking by using the shaking incubator model with 200 rpm shaking amplitude. Results determination

the results are determine through using chemical indicator P-Iodonitrotetrazolium violet.

3.6.2 Dics diffusion test

Preparation of disc diffusion test is the same as the 96 well plate just instead of dilute the inoculums by 100 factors, it have been direct used to cultured on an agar plate. The inoculum was swipe on to the agar plate by using sterilised cotton wools until the whole surface was covered. sterility control of tetracycline disc with concentration was used and putting it on top of the agar plate after dried. Copper(II) oxide nanoparticles disc of 10 µL were prepared by pipetting the well shake homogenous copper suspension onto it. The discs were placed on four corner with at least 24 mm apart.

Observation was done after incubation of 24 hours at 37°C. Results determination

Results determination was done through measuring the length of the inhibitory zone by using ruler.



4.1.1 Percentage yield of copper(II) oxide

Weight of copper(II) acetate monohydrate (mg)

Theoretical weight of products (mg)

Weight of product (mg)

Percentage yields (%)

















The collection of copper(II) oxide nanoparticles is under centrifuge machine with 9000 rpm rotator speed.

* Collection condition is under centrifuge machine with 7820 rpm, 5 minutes.

4.1.2 Characterization of copper(II) oxide

Characterization of copper(II) oxide nanoparticles was done through both IR spectrometry method and also x ray diffratometry method, XRD.

4.1.3 IR spectroscopy

4.1.4 X ray diffractometry

4.1.5 Results for 96 well plate

Copper(II) Oxide Nanoparticles And Its Antibacterial Activities

Results of 96 well plate:


Minimum inhibitory concentration (MIC), µg/µL

Minimum bacteriacidal concentration (MBC), µg/µL

S.Aureus 6538 (+)











Klebsiella (-)











E.Coli 25922(-)











B.Cereus 11978(+)











E.Coli 35218(-)











Pseudomonas Afruginosa, P.A. (-)











Figure 2 96 well plate test on 5 type of bacteria with starting concentration of 8ug/uL of CuO

Figure 2 B.Cereus Figure 3 S.Aureus 6538

Figure 4 Klebsiella Figure 5 E.Coli 35218

Figure 6 E.Coli 25922

4.1.6 Results for well diffusion


Zone of inhibitory, diameter (cm)

E.coli 35218

40µg/µL (1.7cm), 20 µg/µL (0.8cm)

E.coli 25922

40 µg/µL (1.9cm)


40 µg/µL (1.2cm), 20 µg/µL (1.0cm)


40 µg/µL (1.1cm)

Pseudomonas Afruginosa




Figure 3 zone inhibitory of klebsiella

Figure 4 zone inhibitory of S.Aureus

Figure 5 zone inhibitory of e.coli 25922

Figure 6 zone inhibitory of e.coli 35218

4.2 discussions

Discussion will done in three aspect of characterization of copper(II) oxides nanoparticles, effects of concentration toward the selected strains and also the zone of inhibition of copper(II) oxide nanoparticles toward the selected strains.

4.2.1 Characterization of copper(II) oxide nanoparticles

Synthesis of copper(II) oxide are done in situ generation, which are subsequently deposited when the nanoparticles are formed through irradiation of sonicator. The formation of copper oxide takes place through the ammonium complex, [Cu(NH3)4]2+. Copper ions react with ammonia to form deep blue solution containing [Cu(NH3)4]2+ complex ions. The complex is hydrolyzed and crystalline CuO nanoparticles are obtained. (I.Perelshtein, 2009)

Cu2+(aq) + 4NH3.H2O (aq) ƒ  [Cu(NH3)4]2+(aq) + 4H­2O

[Cu(NH3)4]2+(aq) + 2OH-(aq) ƒ  Cu(OH)2(s) + 4NH3.H2O

Cu(OH)2(s) ƒ  CuO(s) +H2O

(I.Perelshtein, 2009)

The sonochemical irradiation of a liquid causes two primary effects namely, cavitations (bubble formation, growth, collapse) and heating. When the microscopic cavitations bubbles collapse near the surface of the solid substrate, they generate powerful shock waves and microjets that cause effective stirring/mixing of the adjusted later of liquid. Ultrasonic waves cause fast migration of the newly formed copper oxide nanoparticles promote more chaotic condition in the solution. It increase the chances of copper(II) ions to reacts and forms solid copper oxide nanoparticles yet did not give high agglomeration rate, so that it can produce narrow disperse of size of the nanoparticles.

CuO nanoparticles were obtained using sonochemical method with the percentage yield averagely 47-55% . copper(II) oxide nanoparticles. are dark brown in colour and were characterized using XRD and FT-IR. The figure shows XRD patterns of the CuO. It shown that the intense peak of the sample at 2θ is 48.7704°, 38.6738°, 35.4755°, 34.68° which are closely matched with the JCPDS card no.00-048-1548 of 48.7158°, 38.7081°, 35.5430° and 35.4170°, that indicated that it is monoclinic system of copper(II) oxide (ternorite). The cells size of the structure, a = 4.6883Å, b = 3.4229Å and c = 5.1319Å.

The particles size of the CuO was calculated using Debye-Sherrer equation as shown below,


= kλ / β cos θ (4.1)


D = Crystal diameter

k = Debye-Scherrer constant, 0.9

λ = X-ray wavelength, 1.5406x10-10 m

β = Width of a diffraction peak

θ = Diffraction angle, 18.5290o

The width of diffraction peak in degree is 0.6419 degree, or 0.0112 radian.

By substitute the value into the Debye-Scherrer equation, the size of the crystal diameter is 1.3637 x 10-10meter or 13.637 nm.

4.2.2 Effects of concentration toward the selected strains.

There are 2 positive strains selected, which include S.Aureus ATTC 6538 and B.Cerues ATTC 11978; 4 negative strains included klebsiella, E.Coli ATTC 25922, E.Coli ATTC 35218, Pseudomonas Afruginosa. antimicrobial testing on 96 well plate are done duplicate each plate and repeats 5 times.

According to the results test, the effect of concentration toward different selected strain gives different rate of inhibition. According to the 96 well plate test, the most susceptible bacteria to inhibitory effects of copper(II) oxide nanoparticles is E.Coli ATTC 25922 and E.Coli ATTC 35218 and the rest inhibit at concentration of 4 µg/µL concentration. Copper(II) oxide shown no inhibitory effect to Pseudomonas Afruginosa. 200rpm have been selected to ensured that the homogenousity of the nanoparticles in the solution.

E.coli species is the most susceptible species because they are simple bacteria with

There are several inhibiting mechanism of copper(II) oxide toward bacterial. According to the journal reported by Mary Grace, M.Sc., the copper(II) oxide particles black coloured was faded away and releases copper(II) ions. (Mary Grace, 2009) The biocidal action of copper nanoparticles was done by released of the Cu(II) ions on contact with moisture. These copper ions bind with the thio (-SH-) group and (-COOH) carboxyl group of protein molecules of bacterial cell wall. (Mary Grace, 2009) Some adhesion on the surface of membrane also cause deadly to the bacteria through structural change. The structure of the outer cell membrane responsible for the cell permeability was substantially changed for the E.Coli. (Vojislav Stani, 2010) Cu(II) ions cause the cell walls were seriously damaged and lot of contents in the cells leaked. (Vojislav Stani, 2010) (Guogang Ren, 2009) However, this antibacterial property only active toward prokaryotes which is single membrane cell, whereas eukaryotes is double membrane cells e.g. human cell is not vulnerable to the attack. (Mahapatra, 2009) This are probably due to the penetration of the copper(II) ions which proved the importance of copper ions released and binding of copper ions into the internal structure. (Mahapatra, 2009)

4.2.3 Zone of inhibition of copper(II) oxide nanoparticles toward the selected strains

Zone of inihition

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