, Research Paper
The inhibitory effects of a mercury compound, silver nitrate, and copper sulfate on Bacillus cereus, Escherichia coli, Micrococcus luteus, and Pseudomonas putida
Certain heavy metals can have harmful or inhibitory effects on microorganisms, a process known as oligodynamic action; for example, some heavy metal ions accumulate in cellular proteins leading to protein denaturation and ultimately cell death (Biology 108 lab manual 2001). A consequence of this is that heavy metals can affect organisms higher up in the food chain, a process called biological magnification. One particular heavy metal, mercury, has an unusual ability to be concentrated in living tissues (Brock and Madigan 1991). In addition to the dubious effects of heavy metals, certain bacteria can have harmful effects on organisms. In May of this year, E. coli O157:H7 was blamed for the deaths of 7 inhabitants of Walkerton, Ontario (Mossman 2001). Therefore, in addition to studying heavy metal effects, it is also important to study bacteria in an attempt to learn how they may be controlled. This experiment attempts to accomplish this.
Recent research in the area of oligodynamic action has focused on the injury by heavy metals in Escherichia coli (Cenci et al. 1985) and the resistance to heavy metals by Pseudomonas aeruginosa (De Vicente et al. 1990). In this study, the inhibitory effects of a mercury compound, silver nitrate, and copper sulfate on Bacillus cereus, Escherichia coli, Micrococcus luteus, and Pseudomonas putida were examined. In particular, the zone of inhibition for each heavy metal in relation to each bacterium was examined. A zone of inhibition can be defined as the diameter of the area where no bacterial growth occurs around a paper disk impregnated with the heavy metal solution. The results of this experiment are expected to show that heavy metal treatments affect the growth of bacterial cultures in a negative way; more specifically, it is felt that the mercury solution will create the largest zone of inhibition. This is based on “general knowledge” that mercury is a toxic heavy metal. Additionally, it is expected that certain bacterial species will not be as sensitive to heavy metal compounds. Once again, based on “general knowledge”, it is felt that Escherichia coli will be the most resistant of the bacterial species.
Methods and Materials
The methods and materials for this lab are outlined on Pages 28 – 30 and 49 – 52 of the Biology 108 Lab Manual (2001). No changes were made to the experimental method.
Table 1 outlines the summarized results for the experiment.
Table 1: The average diameters of the zones of inhibition created by the three different heavy metals on the four different species of bacteria.
Organism Average Diameter of the Zone of Inhibition (mm)
Mercury Compound Silver Nitrate Copper Sulfate Control
Bacillus cereus 34.4 15.5 slight 0.0
Escherichia coli 24.5 13.2 11.2 0.0
Micrococcus luteus 24.2 12.5 0.0 0.0
Pseudomonas putida 27.5 15.0 slight 0.0
As shown by Table 1, the mercury compound affected Bacillus cereus the most followed by Pseudomonas putida. Escherichia coli and Micrococcus luteus were affected by approximately the same amount but to a lesser degree than the other two bacteria. Also indicated by Table 1 is the trend that the silver nitrate solution was less effective at inhibiting bacterial growth as compared to the mercury compound. For this heavy metal, Bacillus cereus and Pseudomonas putida were relatively affected the same amount. Also, Escherichia coli and Micrococcus luteus were affected the same amount but to a lesser degree than the other two. The overall trend of metal effectiveness in preventing bacterial growth is outlined in Table 2.
Table 2: The ability of each heavy metal to prevent bacterial growth.
Heavy metal compound Effectiveness of the heavy metal compound in preventing growth in all 4 of the bacterial species (mm)
Mercury Compound 27.7
Silver Nitrate 14.1
Copper Sulfate 2.8
As indicated by the data in Table 2, the mercury compound had the largest effect on all 4 bacterial species. The silver nitrate solution had an intermediate effect, and the copper sulfate solution had a slight effect. The control data show that the control cultures had no effect on bacterial growth.
Table 1 also shows that some bacteria such as Escherichia coli and Micrococcus luteus are more resistant to heavy metals than other bacteria. This trend is indicated by the smaller zones of inhibition. This trend of increased resistance of certain bacteria is outlined in Table 3.
Table 3: The sensitivity of each of the 4 bacterial species to the 3 heavy metal solutions.
Bacterial Species Sensitivity of each of the bacterial species (mm)
Bacillus cereus 25.0
Escherichia coli 18.8
Micrococcus luteus 18.4
Pseudomonas putida 21.2
As outlined by Table 3, Escherichia coli and Micrococcus luteus are the most resistant (or most insensitive) of the all bacterial species. The data indicate they are approximately equally resistant. Pseudomonas putida is the 3rd most resistant, and Bacillus cereus is the least resistant. To provide further data for analysis, Table 4 outlines another set of data as presented to the class by the T.A..
Table 4: The average values of the zone of inhibitions created by the three different heavy metals on the four different species of bacteria as provided by the T.A.
Organism Average Diameter of the Zone of Inhibition (mm)
Mercury Compound Silver Nitrate Copper Sulfate Control Average Resistance
Bacillus cereus 23.0 18.0 slight 0.0 20.5
Escherichia coli 20.0 13.0 0.0 0.0 16.5
Micrococcus luteus 28.0 20.0 0.0 0.0 24.0
Pseudomonas putida 35.0 25.0 slight 0.0 30.0
Average Effectiveness 26.5 19.0 0.0 0.0
The additional data outlined in Table 4 show that the mercury compound had the largest effect on all 4 bacterial species. The silver nitrate solution had an intermediate effect, and the copper sulfate solution had a slight effect. The control data show that the control cultures had no effect on bacterial growth. In terms of resistance, Escherichia coli was the most resistant of all bacterial species to all three heavy metal solutions. Following behind Escherichia coli in terms of resistance were Bacillus cereus, Micrococcus luteus, and Pseudomonas putida respectively.
As indicated by Tables 2 and 4, the mercury compound had the largest oligodynamic action. The average effect of this heavy metal based on the experimental data was 27.7 mm. Furthermore, Tables 2 and 4 also indicate that the silver nitrate solution had the second largest inhibitory effect. In contrast to this, the copper sulfate solution had only a slight effect. The above statements verify the original hypothesis that the mercury solution would create the largest zone of inhibition. The hypothesis also correctly predicted that the silver nitrate solution would create a zone of inhibition. In contrast to this, the hypothesis incorrectly predicted that the copper sulfate would create a zone of inhibition.
The above stated trend that Hg is better than both Ag and Cu in preventing bacteria growth is verified by two different studies: 1. Cenci et al. ( 1985) noted that the 50 percent inhibition of TDH-activity (a measure of energetic metabolic systems) in Escherichia coli was 7.7 and 50 mM respectively for Hg and Cu. 2. De Vincente et al. (1990) found that the minimum inhibitory concentrations of Ag and Hg in Pseudomonas aeruginosa were 8-16 and 2-4 (mg metallic salt/ml) respectively. At this time, the reason for the decreased ability of Ag and Cu to create a smaller zone of inhibition is not known; however, in general, the reason for the oligodynamic action of heavy metals on bacteria is that certain cellular proteins in the microorganisms have an affinity for metal ions. This results in an accumulation of the metal ions in the cell eventually leading to protein denaturation and cell death (Biology 108 lab manual 2001). The long-term environmental consequences of continued industrial use of toxic compounds (heavy metals) is that certain toxins can become more concentrated in successive trophic levels of a food web, a process called biological magnification (Campbell et al. 1999). As stated in the introduction, mercury has an unusual ability to be concentrated in living tissues (Brock and Madigan 1991).
When comparing Tables 1 and 4, three differences can be noticed. Firstly, the zones of inhibition of the mercury compound on the Micrococcus luteus and Pseudomonas putida are larger in Table 4. Secondly, Table 1 shows that Pseudomonas putida better resists the mercury compound than Bacillus cereus. This trend is reversed in Table 4. Lastly, based on the experimental data, the average zone of inhibition was 15.0 mm for the silver nitrate solution; however for the data shown in Table 4 the average zone of inhibition for the silver nitrate solution was 19.0 mm. At this time, the reasons for these differences are not known.
In relation to the resistance of bacterial organisms, the experimental data showed that Escherichia coli and Micrococcus luteus had approximately equal resistance to the effects of heavy metals. Following behind these bacteria in terms of resistance are Pseudomonas Putida and Bacillus Cereus respectively. This is shown in Tables 1 and 3. This trend contrasts that shown in Table 4 where Escherichia Coli is definitely shown to be the most resistant of the bacterial species. Table 4 also shows that following Escherichia Coli are Bacillus Cereus, Micrococcus luteus, and Pseudomonas Putida in terms of resistance. This ordering contrasts that shown by Tables 1 and 3, and the reasons for this are not clear. It is suggested that in the future more than two zone of inhibition measurements be taken for each metal-bacteria solution to obtain more data points.
Based on the experimental data, the fact that Escherichia coli would be resistant to the heavy metals was predicted by the hypothesis. The hypothesis did not, however, predict the resistance of Micrococcus luteus. The resistance of the Escherichia coli to heavy-metal effects could possibly be related to the fact that Escherichia coli is a gram-negative bacterium. As a result, the heavy-metal ions may have a hard time permeating the outer lipopolysaccharide layer present in gram-negative bacteria. The consequence is increased resistance to heavy metals. The reason for the resistance of the gram-positive Micrococcus luteus is unknown. It might, however, be related to the fact that this type of bacteria may have heavy-metal-resistant plasmids.
The data in Table 1 show one interesting trend: The copper sulfate solution had quite a pronounced effect on the Escherichia coli as compared to the other bacteria. Based on the ability of Escherichia coli to resist the effects of heavy metals and based on the inability of the copper sulfate solution to affect any other bacterial organisms, it is felt that a mistake was made in the experimental procedure (Footnote 2). In particular, it is thought that instead of a Escherichia coli and copper sulfate solution, this solution was probably Escherichia coli and a silver nitrate solution. The reason for this is that the average diameter of the zone of inhibition of the Escherichia coli and silver nitrate solution is very similar to the average diameter of the zone of inhibition of the Escherichia coli and copper sulfate solution.
Table 1 also shows that there weren’t any zones of inhibition around the paper disks of the control. This indicates that the paper disks did not affect the growth of the different bacteria.
Based on the experimental data, the original hypothesis that Escherichia coli would be the most resistant bacteria was verified. The ability of Micrococcus luteus to have approximately the same resistance as Escherichia Coli was not, however, predicted by the hypothesis. In relation to the effects of the heavy metals on the bacteria, the original hypothesis correctly predicted that Hg would have the largest effect. Furthermore, the hypothesis also predicted the ability of Ag to have an effect on the bacteria. The hypothesis did not, however, correctly, predict the very slight effect of the Cu solution on the bacterial cultures.
Biology 108. 2001. Biology 108 laboratory manual 2000-2001 University of Alberta, Edmonton, Alberta, Canada.
Brock, T. D. and M. T. Madigan. 1991. Biology of microorganisms. Prentice Hall, Englewood Cliffs, New Jersey.
Campbell, N. A., L. G. Mitchell, and J. B. Reece. 1999. Biology. Benjamin/Cummings, Menlo Park, CA.
Cenci G., and G. Caldini. 1985. Injury by Heavy Metals in Escherichia coli. Bulletin of Environmental Contamination and Toxicology 34:188-195.
De Vincente, A., M. Aviles, J.C. Codina, and P. Romero. 1990. Resistance to antibiotics and heavy metals of Pseudomonas aeruginosa isolated from natural waters. Journal of Applied Bacteriology 68: 625-632.
Mossman, M. “Months after E. coli outbreak, Walkerton residents still weary of water supply.” http://www.nandotimes.com/noframes/story/0,2107,500291929-500463058-503086695-0,00.html (Feb. 11, 2001).