Water Quality Lab Report MICR 3050 (2)

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Clemson University *

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3050

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Biology

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Dec 6, 2023

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Water Quality Lab Report MICR 3060 Sect 13 15 November 2023 Krisha Jha 1 Water Quality Lab Report (Bacteriological Examination of Water-Presumptive and Confirmed Tests) MICR 3060 Sect. 13 15 November, 2023 Krisha Jha

2 INTRODUCTION Water is a necessity for all human beings to survive, and within a community, most of the population will share the same water source. Due to the inevitable demand, water represents an excellent mode of transportation of infectious diseases. Prior to the requirement of water treatment, diseases such as Cholera and Typhoid Fever would spread quickly through communities. During the 1840’s, a physician by the name of John Snow, isolated the source of the Cholera outbreak in London back to the water supply being contaminated (Brown, 2012). Since the discovery that Snow had made, water safety and sanitation have transformed. Plants now exist to purify water, killing all harmful bacteria using a variety of different methods, but the most popular amongst the others is chlorination. Wastewater is also now cleaned before being released back into the natural environment, such as lakes and rivers. Coliforms constitute one of the key indicators of contaminated drinking water. Coliforms originate in soil and the intestinal tract of humans and other warm-blooded animals. Several characteristics of coliforms are that they are gram-negative, facultative anaerobic, non-endospore forming rods that ferment lactose to produce gas (Brown, 2012). These characteristics make coliforms relatively easy to test for, as they prove useful to water treatment companies, as testing for specific pathogens can become very costly and time consuming. Although coliforms are not necessarily always pathogenic, they serve as a suitable indicator of potential water contamination (Treyens, 2009). Two methods of testing for water contamination are the presumptive and the confirmed test. A presumptive test uses the coliform's ability to ferment lactose and produce gas as an indicator that these organisms are present. Fifteen tubes of lactose broth, five double-strength lactose broth tubes, and ten single-strength lactose broth tubes are inoculated with different

3 amounts of water. After 24 hours of incubation at 37°C, the tubes are observed for their presence of gas. The presence of gas would lead to the “presumption” that coliforms are present in the water. The number of coliforms present per 100 mL of water, or the MPN (most probable number) of coliforms can also be determined using this method (Brown, 2012). Although there is gas present in the tube, and there is a strong possibility of coliforms being present, the gas can be explained by other lactose fermenting bacteria. Therefore, to further confirm that coliforms are the source of the gas, the confirmed test is then performed. For the confirmed test, an EMB and an Endo agar plate are inoculated using a positive tube of lactose broth. Both the EMB and Endo agar are selective for gram-negative bacteria and therefore inhibit the growth of gram-positive bacteria (Brown, 2012). “The dyes Eosin & Methylene Blue inhibit the growth of Gram-positive bacteria. It is different because lactose fermenters form colonies that are dark in color and sometimes have a green metallic sheen (typical for Escherichia coli ); the colonies of lactose non-fermenters appear pale pink” (4). The overall purpose of this experiment was to determine if the water sample is safe for consumption, and if coliforms are present. RESULTS Table 1: Observations for the Presumptive Test. Lactose broth tubes inoculated with assigned water sample results. MPN value per 100 mL of sample and 95% confidence limit included.

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4 Figure 1: Quadrant streak of water sample three onto EMB agar plate, incubated at 37°C for 24 hours Figure 2: Quadrant streak of water sample three onto Endo agar plate, incubated at 37°C for 24 hours

5 Table 1 depicts the results for the presumptive test for water sample three. One out of the five double-strength lactose broth tubes, inoculated with 10 mL of water showed gas production, while only one of the single-strength lactose broth tubes inoculated with 1.0 mL of water showed gas production. However, two of the single-strength lactose broth tubes inoculated with 0.1 mL of water showed gas production. The MPN (most probable number) was found to be 8.1, and the low and high 95% confidence limits were found to be 3.4 and 22 respectively. Figure 1 represents the one of the results to the confirmed test, the EMB agar streak with the positive sample. The streak revealed colonies with black coloring, and dark color, with a metallic green sheen, and small size. Figure 2 represents the results of the confirmed test on the Endo agar streak plate. The colonies on the Endo plate appeared to be hot pink with an area of hot pink tint to the agar in the position of the original quadrant streak. This showed a positive result for the presence of coliforms due to the hot pink colony growth. DISCUSSION The EMB plate revealed that the water did have the presence of lactose coliforms and that they were Escherichia coli , due to their dark color with a green metallic sheen, and their small size. “This metallic green sheen is an indicator of vigorous lactose and/or sucrose fermentation ability of fecal coliforms. A smaller amount of acid production, which is a result of slow fermentation (by slow lactose-fermenting organisms), gives a brown-pink coloration of growth. Colonies of non lactose fermenters appear as translucent or pink” (Genung and Thompson, 1926). The Endo agar also showed a positive result for the presence of coliforms, due to hot pink colony growth. The results for the presumptive test found in Table 1 determine that per 100 mL of the water in sample three, there were approximately 8 coliforms with a 95% probability of there being between 3.4 and 22 coliforms. When the water was placed in the

6 DSLB (10 mL), one of the five tubes showed a positive result, and when the water was placed in the SSLB (1.0 mL), one of the five tubes showed a positive result. However, when the water was placed in the SSLB (0.1 mL), two of the five tubes presented with a positive result. It would be expected that there would be a positive result in each of the three concentrations collected, due to the strong evidence that the coliforms exist in the water from the first five tubes. The more positive results in the least amounts of water could be explained by the concentration of/or placement of water in each SSLB (0.1 mL) tube. There could have been a large possibility that there were no coliforms in the DSLB (10 mL) tubes, and it ended up being in more of the SSLB (0.1 mL) tubes. The suspicions of coliforms in the water were then further investigated through the confirmed test, which solidified the suspicions. The water from a positive lactose fermenting tube was streaked on both an EMB agar plate and an Endo agar plate. Both of these plates are selective for gram-negative bacteria. EMB agar contains peptone, lactose, sucrose, as well as methylene blue and eosin Y dyes. The sugars available in the agar further its selectivity, because only bacteria that can ferment lactose and sucrose can grow on this medium. This is especially a trait of fecal and non-fecal coliforms. While the methylene blue dye inhibits the growth of the gram-positive bacteria, eosin Y dye responds to changes in pH and turns black as the conditions become acidic. Non-lactose fermenting gram-negative bacteria will produce clear or pink colonies on the agar because no acid will be produced. A small amount of acid production indicates the slow fermentation of lactose and results in brown-pink colored colonies (Lal and Cheeptham, 2013) Gram-negative bacteria that quickly ferment lactose produce large amounts of acid and turn the agar black, sometimes with a green metallic sheen (Lal and Cheeptham, 2013).

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7 In Figure 1, the colonies grown on the EMB agar from this experiment were small and black, which led to the conclusion that E. coli were the coliforms present in the water. Endo agar is another source of lactose fermenting gram-negative bacteria. However, it does not allow for clear differentiation between the types of bacteria. This medium contains lactose and decolorized basic fuchsin. When coliforms grow on this agar, a reaction takes place that restores the color to the decolorized basic fuchsin. Those bacteria that ferment lactose with the production of acid and gas will restore the color, bacteria that produce only acid as a byproduct of lactose fermentation will restore only a faint pink color, and bacteria that do not ferment lactose at all will remain colorless (Genung and Thompson, 1926). The bacteria grown on the Endo agar in this experiment seen in Figure 2 restored the color of the decolorized basic fuchsin and are therefore lactose fermenters. The results for both of the agar plates were expected because they were streaked from a positive lactose broth tube. The results of this experiment depict conclusive evidence that coliforms are present in respectable levels in water sample number three. According to the regulations of drinking water in South Carolina, the specific number of coliforms in water does not matter, but their mere presence in five or more percent of samples for a system that collects at least forty samples a month, is not in compliance with the maximum contaminant levels for microbiological contaminants (SCDHEC, 2009). Due to the coliforms present in the water, it is unfit for human consumption, and cannot be distributed to the public.

8 REFERENCES Brown. (2012). Benson's Microbiological Applications: Laboratory Manual in General Microbiology, Short Version, 12 th ed. Mc-Graw-Hill, New York, NY. Genung, Elizabeth F., and Lucy E. Thompson. "Color Diffusion in Endo Agar." Journal of Bacteriology 14.2 (1927): 139-55. American Society for Microbiology. Web. 19 Nov. 2013. <http://jb.asm.org/content/14/2/139.full.pdf+html>. Lal, Archana, and Naowarat Cheeptham. "Eosin-Methylene Blue Agar Plates Protocol." ASM MicrobeLibrary . American Society for Microbiology, 1 Apr. 2013. Web. 19 Nov. 2012. <http://www.microbelibrary.org/component/resource/laboratory-test/2869-eosin methylene-blue-agar-plates-protocol>. Treyens, Cliff. "Bacteria and Private Wells." National Environmental Services Center. National Ground Water Association, 2009. Web. 19 Nov. 2013. <http://www.nesc.wvu.edu/pdf/dw/publications/ontap/magazine/OTWI09_featur s/BacteriaAndPrivateWells.pdf>. United States of America. South Carolina Department of Health and Environmental Control. Bureau of Water. N.p.: n.p., n.d. State Primary Drinking Water Regulations . Department of Health and Environmental Control, 2009. Web. 19 Nov. 2013. <www.scdhec.net/water>

Water Quality Lab Report MICR 3050 (2)

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