This investigation was conducted to determine the effect of heavy-metal ions on growth of Escherichia coli and its threshold concentration. Copper(II) solutions with concentrations ranging from 0 to 10 mM were mixed with Escherichia coli culture and nutrient agar in Petri dishes by using aseptic techniques. After 24 hours incubation, number of colonies formed on each agar plates, which represented bacterial growth, were counted and recorded. Experimental results showed that bacterial growth increases as concentration of copper(II) solution decreases. Threshold concentration of copper(II) solution was 2.0 mM, since bacterial growth was inhibited at concentration higher than 1.0 mM. Statistical analysis using Pearson product-moment correlation coefficient with 5 % significance level confirmed the significant negative correlation between concentration of copper(II) solution and number of Escherichia coli colonies formed.
Research and rationale
Heavy-metals are often referred to elements exhibiting metallic properties with atomic number of 21 or above and complex electron configurations. They include transition metals, lanthanides, actinides, and metalloids of arsenic and antimony.  These non-biodegradable contaminants are frequently associated with pollution and eco-toxicity in environment.  However, not all heavy-metals are considered biologically important, either due to their low solubility or availability in natural ecosystems. 
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Heavy-metals, which exist naturally as mineral ores, are released to environment by volcanic eruption and rock weathering.  Its anthropogenic sources can be divided into five categories, including atmospheric deposition, industry, agriculture, waste disposal, metalliferous mining and smelting. Ores extraction by mining and refining processes will cause environmental problems when tailings left in fully or partially opened mining sites are transported by water or wind.  Zinc, copper, lead and traces of other heavy-metals are released by vehicles, which is one of the major pollution sources.  Leaching and runoff during rainy seasons will cause heavy-metals to enter groundwater pathway and accumulate in aquifer, resulting in water and soil pollution.5
According to "Disinfection, sterilization and preservation" by Seymour Stanton Block, 43 heavy-metals with potential to interact with microorganisms had been identified. They include vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, zirconium, niobium, molybdenum, silver, cadmium, indium, tin, antimony, lanthanum, hafnium, tantalum, tungsten, platinum, gold, mercury, thallium, lead, bismuth, cerium, praseodymium, samarium, europium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, thorium, uranium, plutonium, americium and californium.  However, metal ions have to enter and accumulate in cell, either by passive or active transport, to produce any physiological effect. 
Depending on concentration and uptake route, some heavy-metals are vital in metabolism of most organisms as they can form ions with incompletely filled d-orbital and complex compounds.3 For instance, copper, calcium, iron, manganese, nickel, zinc and chromium are essential micronutrients for living organisms.1 They are important since they can act as cofactor and redox catalysts in iron-sulphur proteins, blue-copper proteins and cytochromes. Metals can create concentration and charge gradient across cell membrane to regulate osmotic pressure, assist transportation, quorum sensing and intracellular compartmentation. Furthermore, cellular structures and bio-molecules, such as organelles, membranes, cell walls, proteins and DNA, can be stabilised by electrostatic interactions. However, almost all metals, either essential or nonessential, will form unspecific complex compounds in cells once threshold concentrations are exceeded.7 Therefore, their intracellular concentrations have to be tightly regulated to prevent cellular damage.3
Highly toxic metals with extremely low threshold concentrations are oligodynamic and can be lethal to microorganisms in minute amount.1,  Divalent cations and oxyanions, such as copper(II), manganese(II), chromate and arsenate can interact with ligands and displace essential ions with similar structure and charge from their cellular binding sites.3 Additionally, they can block functional groups of polynucleotide, enzymes, transport and storage system of nutrients.  Cations at high concentration can damage proteins, DNA, organelles and cell membranes, causing mutagenesis and metabolic interference.  For example, mercury, cadmium(II) and silver ions tend to inactivate enzymes by binding irreversibly to sulfhydryl groups.7 Metabolism, morphology and growth of microorganisms will be affected and inhibited. 
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Fig.1: Escherichia coli treated with copper shows cavities in cell walls 
Gram-negative bacillus, Escherichia coli is used in this experiment to represent pathogens, since it can cause gastroenteritis, diarrhoea and urinary-tract-infections. Through this experiment, other uses of heavy-metals, which are normally expelled as pollutants, can be explored. Effect of heavy-metal ions on bacteria and its possibility to be used as antimicrobial can be investigated. Six common heavy-metals, including nickel, lead, copper, zinc, iron and aluminium are tested to identify the ions with strongest inhibitory effect and its threshold concentration in bacteria. Consequently, discarded metals can be retrieved and reused as antimicrobial, to reduce heavy-metal pollution and bacterial infection.
Experimental hypothesis, H1 :
When concentration of copper(II) solution decreases, number of Escherichia coli colonies formed increases.
Null hypothesis, H0 :
There is no correlation between concentration of copper(II) solution and number of Escherichia coli colonies formed.
Manipulated variable: Concentration of copper(II) solution
Responding variable: Number of bacterial colonies formed
Constant variables: Types and volume of bacterial culture used, volume of copper(II) solution, incubation temperature
Weighing bottle, electronic balance, spatula, 1000 cm3 volumetric flask, 10 cm3 measuring cylinder, beaker, sterile micropipette, sterile Petri dishes with lid, marker, Bunsen burner, 250 cm3 conical flask and incubator
Antiseptic soap, 70% ethanol spray, copper(II) sulphate, CuSO4âˆ™5H2O salt (relative formulae mass=244.6), sterilised distilled water, Escherichia coli culture and sterilised molten nutrient agar
0.1 M salt solutions containing nickel(II), lead, copper(II), zinc, iron(III) and aluminium ions respectively were tested by using disk diffusion method to determine the most effective antimicrobial. Two Petri dishes containing nutrient agar and Escherichia coli culture are used. Sterilised paper discs were impregnated with each salt solution respectively and placed on bacterial lawn. Inhibition strength of ions is represent by diameter of clear zones produced, which is measured and recorded in Table 1 after 24 hours of incubation. Four discs soaked in sterile distilled water are used in each Petri dish as control.
Types of salt solution
Mean diameter of inhibition zone (mm)
Results indicate that the strongest antimicrobial is copper(II) solution, followed closely by zinc and nickel(II) solutions. Therefore, copper(II) solution is chosen to be used in main experiment.
Bacterial colony counting and disk diffusion method were tested to determine the most suitable method for this experiment. Two Escherichia coli lawns containing 200 Âµl of 10 mM copper(II) solution and distilled water respectively are prepared, while two discs impregnated in the solution and water respectively are placed on another bacterial lawn. After incubated for 24 hours, number of colonies and diameter of inhibition zones formed on 3 lawns are recorded in Table 2.
Bacterial colony counting
22 colonies formed
247 colonies formed
Clear zone with 8 mm diameter
No clear zone
Bacterial colony counting method is chosen because bacterial growth during absence and presence of metal ions can be counted and compared. Disk diffusion method is rejected because diameter of clear zone formed is very small when using salt solution with extremely low concentration.
Trial was performed to determine the concentration range of metal ions to be tested. 2.446 g copper(II) sulphate is dissolved in 100 cm3 sterilised distilled water to form 100 mM solution, while sterilised distilled water represent 0 mM solution. 75, 50 and 25 mM solutions are prepared by mixing distilled water and salt solutions in proportion according to Table 3. 200 Âµl Escherichia coli culture and molten nutrient agar are mixed thoroughly with 200 Âµl salt solutions of different concentration in 6 Petri dishes respectively. After 24 hours incubation, number of colonies produced are counted and recorded in Table 4.
Solution concentration (mM)
100 mM solution
50 mM solution
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Number of colonies (colonies)
Concentrations ranging between 0 and 10 mM are selected to determine the minimum effective concentration of copper(II) ions in inhibiting Escherichia coli growth. Other concentration ranges are not chosen since copper(II) ions at higher concentrations inhibit bacterial growth completely.
2.446 g copper(II) sulphate salt is weighed by using weighing bottle and electronic balance, and transferred into a clean 1000 cm3 volumetric flask.
Weighing bottle is washed by using sterilised distilled water and poured into the flask. 10 mM copper(II) solution is thus prepared by adding distilled water gradually into the flask until reaching the graduation mark. The flask is swirled carefully between additions of water to ensure complete dissolution of salt.
0 mM solution is represented by distilled water. Solutions with different concentration are prepared by diluting 10 mM solution according to Table 5.
10 mM salt solution
200 Âµl of 10 mM salt solution and Escherichia coli culture is transferred by sterile micropipette to a labelled sterile Petri dish. 20 cm3 molten nutrient agar is poured into the dish, which is swirled gently in a pattern of "8" to mix its content thoroughly and allow agar to solidify.
The above step is repeated to prepare 3 identical agar plates.
Step 4-5 are repeated for 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 mM solutions respectively.
Petri dishes are inverted and incubated for 24 hours.
Number of colonies formed in Petri dishes is counted and recorded in Table 6, and its average is calculated. A graph of number of Escherichia coli colonies formed against concentration of copper(II) solution is plotted.
Aseptic techniques must be used. All equipment and materials needed in experiment should be cleaned and sterilised to kill any bacteria present.
Before and after the experiment, hands should be washed with antiseptic soap and working area is sprayed thoroughly with 70% ethanol spray and wiped clean after 10 minutes, to prevent infection and contamination.
The neck of conical flask containing molten agar should be flamed by using a lit Bunsen burner before and after pouring to prevent bacterial contamination.
Molten agar at 60oC should be used to reduce water condensation in Petri dishes.
When bacterial culture, salt solution and nutrient agar are transferred to Petri dishes, lids should be lifted as little as possible and covered immediately since Escherichia coli is a potential biological hazard which can cause food poisoning.
Micropipette should be used carefully to avoid trapping air bubbles in its tip, which can affect volume of solution measured.
Used micropipette tips should be disposed in biohazard container with disinfectant to prevent bacteria from spreading.
Spilling should be avoided when mixing content of Petri dishes by swirling.
Petri dishes must not be sealed completely to allow aerobic respiration and prevent anaerobic bacterial growth.
Petri dishes must be inverted during incubation to prevent water vapour produced by growing bacteria to condense on colonies and affect their growth.
Incubation temperature should be around 27oC to avoid growth of pathogens.
Incubated Petri dishes must not be opened and should be autoclaved before disposal to prevent exposure and spread of Escherichia coli.
Number of bacterial colonies formed, N (colonies)
Hypothesis test is conducted to examine validity and degree of correlation between concentration of copper(II) solution and number of Escherichia coli colonies formed in this experiment. Pearson product-moment correlation coefficient (PMCC) is used to determine the strength, direction and type of correlation between variables. Ranging from âˆ’1.0 to +1.0, correlation coefficient, r indicates direction and strength of relationship by its sign and value. Correlation strength increases as its value approaches 1.0, while 0 shows no linear correlation between variables. Positive sign indicates positive correlation, and vice versa. 
When sample size, n=11 and significance level, Î±=0.05; critical value, rcri=Ââˆ’0.5214
âˆ´H0 is reject if râ‰¤âˆ’0.5214.
r =âˆ’0.8598<âˆ’0.5214, âˆ´At Î±=0.05, there is an evidence of significant negative correlation between concentration of copper(II) solution and number of Escherichia coli colonies formed.
H0 can be rejected.
Growth of Escherichia coli is represent by number of colonies formed, which can be observed as white circular spots on solidified nutrient agar after incubation. Although the graph fluctuated with anomalous results at 4 and 7 mM concentration instead of a perfect negative trend, the overall negative trend in bacterial growth shown is supported by statistical analysis. Generally, number of bacterial colonies formed decreases as concentration of copper(II) ions increases. This confirms that pre-incubation with minimum 2 mM copper(II) solution can inhibits bacterial growth.
Initially, bactericidal activity is not observed since copper(II) ions are absent, resulting in average formation of 251 Escherichia coli colonies. Colony count is the highest in 1 mM solution, indicating that copper(II) ions in low concentration will promote bacterial growth slightly by 4.8 %. This is because traces of copper are essential for normal growth of Escherichia coli since it acts as cofactor for P-type ATPase, cytochrome oxidase and amine oxidase.3,13
Furthermore, Escherichia coli are able to cope with copper(II) ions at this concentration through reaction of glutathione (GSH) antioxidant. GSHs can prevent cellular damage by deactivating reactive intermediates generated by copper(II) ions and forming glutathione disulfide (GSSG). Normal GSSG-to-GSH ratio, which is used to estimate cellular toxicity, is maintained when GSHs are regenerated from GSSG by glutathione reductase.3
Additionally, bacterial growth in low concentration of copper(II) ions may increase due to lack of competition. Escherichia coli population size decreases as some are killed by copper(II) ions. Therefore, more nutrients are available for surviving bacteria, resulting in increased colony counts. 
However, bacterial growth is inhibited as copper(II) ion concentration increase further and exceed its threshold concentration. Copper(II) solution with higher concentration has stronger inhibitory effect. At 10 mM, colony count has reduced by 90.4 %. Aside from its ability in blocking protein binding sites, disrupting metabolism, transport system and respiratory chain, copper toxicity is believed to be the result of altered balance in pro-oxidant and antioxidant systems of Escherichia coli.10 Metal ions may catalyse formation of GSSG and hydrogen peroxide (HP), from GSH in gram-negative bacteria. Even though GSSG can be reduced by glutathione reductase, the persistent ions will immediately bind to the rejuvenated GSHs. Balance between formation and detoxification of oxidants in cells will be disrupted. GSSG-to-GSH ratio within cells will increase, indicating significant oxidative stress.3
Furthermore, membrane-bound copper can increase bactericidal activity of HP towards Escherichia coli by hundredfold through formation of hydroxyl radicals, which is the strongest oxidant known.7,  Excess HP and radicals can attack sulfhydryl groups and double bonds in bio-molecules, resulting in massive cellular damage and cell death.7
Variations and anomalous results at 4 and 7 mM copper(II) solutions may be caused by experimental errors. Firstly, concentration of solutions may not be accurate due to spilling and mixing error during preparation. Concentration of copper(II) ions presence in solutions may varies when volume of solution measured by measuring cylinder during dilution is not accurate. Slight variations in concentration will affect experimental results, since Escherichia coli are extremely sensitive to presence of copper(II) ions in culture media.12 Validity of results can be improved by using volumetric flask and pipette to measure and dilute solutions more accurately.
Results may be unreliable due to difficulties in counting bacterial colonies. Overlapping of colonies formed may occur. Therefore, spread plate method should be used to ensure that colonies only grow on agar surface, instead of within the agar. Confusion during counting due to vast amount of colonies present may contribute to inaccuracy. Repeated counting was overcome by marking counted colonies with marker on plate. Besides, colonies formed vary in sizes. Some are extremely small and cannot be observed clearly by unaided eyes. Therefore, only spot with minimum 1 mm diameter are considered as 1 colony. Moreover, automatic colony counter can be used for faster and more accurate colony counts.
Additionally, results may be biased due to presence of random errors, including human error and slight variation of surrounding condition which can affect bacterial growth. Their effect on results was reduced by repeating the experiment for at least thrice to observe the trend of inhibition strength and obtain its average results.
Limitations in experiment include genetic differences between individual Escherichia coli. Some bacteria may acquire additional copper-resistance genes through selective mutation, plasmid mediation or conjugation.15 They are well adapted and can survive in copper-rich environments, which will normally overwhelm their copper homeostatic systems.  Colony counts may be higher than usual as more resistant cells survive, despite increasing copper concentration.
Viable Escherichia coli in bacterial culture used and exact amount of copper(II) ions present in each agar plate may vary slightly. Small amount of salt solution and bacterial culture may remain in measuring equipment during measurement and transfer of materials. However,
their effects on results accuracy are very small and can be ignored by assuming their amount is constant for all agar plate.
This experiment can be modified by using other types of bacteria, such as Staphylococcus aureus, to test the inhibitory effect of heavy-metal ions. Since different bacteria may response differently to presence of metal ions with differing minimum effective concentration, experiment on single bacterial species can provide wrong inference on effect and mechanisms of metal toxicity. Further investigation can be carried out to determine the precise threshold concentration of copper(II) ions in Escherichia coli by incubating bacteria with copper(II) solution in concentration range between 1.0 and 2.0 mM.
Experimental results obtained and its statistical analysis confirmed that the lower the concentration of copper(II) solution, the greater the growth of Escherichia coli, which is represents by number of bacterial colonies formed. Since Escherichia coli growth is inhibited at concentrations higher than 1.0 mM, at least 2.0 mM copper(II) solution must be used as antimicrobial to ensure that bacterial growth is inhibited.
Source 1 is up-to-date, since "EoE" provides facts about Earth and environments, which are contributed and constantly reviewed by experts from related fields, even as recently as in March, 2011. Details listed in Source 2 are dependable because this IUPAC technical report was prepared for publication by Commission on Toxicology. Source 3, 5, 8, 10, 12, 13, 15, 16 and 17 are reliable scientific articles written by researchers and published in journals for peer-review. TutorVista and NOVA in Source 4 and 14 provide online tutoring and distance learning programs. Hence, lessons about correlation and heavy-metal water pollution provided in their websites should be dependable. Technical information in Source 6 is factual because it is provided by government agency which is concern about water pollution. As the latest editions of published books, data from Source 7 and 9 are trustable. Information in Source 11 is accurate, since this ecological study was supervised by scholars from several universities.
In conclusion, all references used are reliable since information provided is supported and coincided with each others.