The Sanitary Quality of Pond Water and Canal Water of UPR in Cayey P.R. CampusDocument Transcript
4352925828675The Sanitary Quality of Pond Water and Canal Water of UPR in Cayey P.R. Campus JESYKA MELÉNDEZ¹, WILMER FELICIANO¹ & BELINDA ROMÁN² PhD, RISE program UPR Cayey P.R. Grant number 5 R25 GM059429 ¹Undergraduate research students at University of P.R. in Cayey, Department of Biology. ² Mentor in charge of investigation. PhD professor at University of P.R. in Cayey Biology Department. ______________________________________________________________________________ ABSTRACT The aim of this investigation is to study and report about the presence of E. coli in pond water and canal water of UPR in Cayey campus. This experiment was to be conducted by testing for the presence of total coliforms and E. coli with incubation techniques using colilert as the identifier, after which, PCR techniques were used to corroborate this presence. Results acquired with the use of both techniques demonstrated that indeed E. coli was present in all the water samples collected. SPECIFIC AIMS To provide a general overview about the water quality from selected areas of the UPR in Cayey campus by; determining the presence of coliforms in the selected water bodies, (if the presence of coliforms is confirmed) to determine the presence of Escherichia coli in the water and to provide important data about the water quality in the UPR Cayey campus that might avoid potential health risks to the college community. HYPOTHESIS Because of the presence of animals in the campus, we believe there will be a highly significant amount of coliforms present in the water bodies. This is due to the fact that they serve as water supply for these animals. If there is any other source of coliform production it is yet to be seen. We expect the contamination to be higher in exposed areas of the water bodies and diminished in the least exposed. BACKGROUND AND SIGNIFICANCE Fig. 1.1 Diagram presenting the relationship between some coliform types. http://images.google.com.pr/images?hl =es&um=1&sa=1&q=total+coliforms+&aq=f&oq= 38169852203450The Cayey UPR College Campus, located at 205 Antonio R. Barceló Avenue at Cayey Puerto Rico, is home to a wide variety of plant species, two water bodies and wondering animals such as canines and horses. These water bodies have small or none recreational value at the present time but still compose a visibly exposed part of the campus. The purpose of this investigation is to establish the sanitary quality of water of these mentioned bodies. Ten samples from different points in these locations will be collected and tested for the presence of coliforms. “The presence of coliforms in water is designed to indicate the possible presence of fecal contamination and therefore the presence of pathogens” (Anderson and Davidson). Water quality studies are an extremely important part of our community because of the fact that these bacteria “do not change the appearance or taste of water, there is no way to tell if these microorganisms are present” (Well Stewardship Information Series 2002). These types of bacteria may cause side effects such as; diarrhea, cramps, headaches and other symptoms (Skipton et. al, 2008). The possible contamination of these water bodies could be an indicator of contamination in other nearby water bodies. “The presence of fecal coliforms usually indicates recent contamination of groundwater by human sewage or animal droppings, which could contain other bacteria, viruses, or disease causing microorganisms” (Well Stewardship Information Series 2002). Coliforms are generally described as the bacteria present in the intestines of warm blooded animals, and are found in feces and other bodily wastes as well as naturally in soils. Coliforms are classified into a general group of total coliforms as presented in fig. 1.1. E. coli is extremely reliable as a fecal contamination indicator in water. For this reason experiments will first be conducted to identify the presence of coliforms, using the Quanty-Tray Sealer plates for incubation, and later specifically for E. coli. Previous experiments conducted using molecular methods for E. coli identification have proven to be very efficient. PCR and other assays for detecting E. coli have been proven to be reliable enough as to be used for the examination of foods and other samples (Deng et. al, 1996). The use of the PCR- based detection of E. coli is a hundred percent specific for E. coli not amplifying the DNA of any other enteropathogenic bacteria tested (Gunzburg et. al, 1995). This is very important due to the fact that many types of bacteria are abundant in both soil and water but only E. coli is of interest in this investigation. Because the presence of E. coli in the water indicates the possible contamination of the water with pathogenic organisms such as Giardia lamblia and Cryptosporidium parvum, it is important to conduct further experiments in the case that the water samples are contaminated. Cryptosporidium spp., which are carried as cysts in the feces of contaminated hosts, have been recognized as water borne pathogens and can be tested for successfully using a combined immunoflourescence antibody procedures (LeChevallier et. al, 1991). RESEARCH DESIGN AND METHODS Water samples are to be recovered from ten different locations along the water pond and canal, see fig 1.2. Fig.1.3 presents a map of the UPR in Cayey Campus where the water canal and pond are indicated. The samples are then taken to the lab where an aliquot is taken using a 100ml test tube. Each aliquot is transferred to a clean and enumerated 250 ml bottle. The agent colilert is added to each sample and shaken well. The content of the bottle is then transferred into an enumerated test tray. Each one of the test trays is sealed in the Quanty- Tray Sealer. Once sealed, they are placed in an incubator at 35-37C° for 24 hours. The test trays are then removed from the incubator and the yellow cells, which would indicate the presence of total coliforms, are counted. Afterwards, the test trays are exposed to a UV lamp and the florescent cells, which indicate the E. coli presence, are noted. A numerical probability table is used to determine total coliforms. Fig. 1.2., The table below presents the data corresponding each of the water samples such as pH, temperature and location of collection. The fig. references next to the sample number indicate the photo corresponding to each sample location. Number of SampleLocation of Water SamplepH of SampleTemperature of Sample1 (fig. 1.2a)Shore of the pond6.2325°C2 (fig. 1.2b)Middle of the pond6.2525°C3 (fig. 1.2c)Opposite shore of the pond6.2925°C4 (fig. 1.2d) Stream section at water bomb near soccer court7.5425°C5 (fig. 1.2e)Stream section after bridge near water bomb8.0625°C6 (fig. 1.2f)2nd stream sample after bridge8.5125°C7 (fig. 1.2g)Stream section before second bridge8.1425°C8 (fig. 1.2h)Stream section after bridge7.8025°C9 (fig. 1.2i)Stream section at R. Barceló Ave. bridge7.5425°C10 (fig. 1.2j) Stream section after R. Barceló Ave. bridge7.4225°C fig. 1.2a The photo below is that of the shore of the pond. The pond is covered with leaves making water area in the photo unrecognizable -857252747645-8509076200fig. 1.2b The photo below is that of the middle of the pond. The pond is covered with leaves making water area in the photo unrecognizable -1022352802255fig. 1.2c The photo below is that of the opposite shore of the pond. The pond is covered with leaves making water area in the photo unrecognizable -85090512445fig. 1.2d The photo below is that of the stream section at water bomb near soccer court. fig. 1.2e The photo below is that of the stream section after bridge near water bomb. -844552842260fig. 1.2f The photo below is that of the-1022357620 second stream sample after first bridge section. fig. 1.2g The photo below is that of the stream section before second bridge. -1885952834005fig. 1.2h The photo below is that of the -20574021590stream section after section after second bridge. -2057402853690 fig. 1.2i The photo below is that of the stream section at R. Barceló Ave. bridge. -15875362585fig. 12j The photo below is that of the stream section after R. Barceló Ave. bridge. To ensure the presence of E. coli in the water samples even further, a PCR is conducted. The first step in preparation for the PCR is to isolate the bacteria from the water sample. 1.7ml of the water from each of the ten samples is placed in a 2ml screw cap tube. They are microcentrifuged at high speed for 15min. The supernatant is then removed and placed in a clean tube leaving the bacterial pellet in the tube. 175μl of the supernatant are added to the bacterial pellet to re-suspend by vortexing. In order to isolate the bacterial DNA for the PCR, 50μl of lysis solution must be added to the tube containing the supernatant plus the pellet. The solution is mixed several times by inverting or vortexing. The samples are then heated at 56°C for 30 minutes in a water bath. They are removed from water bath and placed at 95°C for ten minutes. The sample is then allowed to cool at room temperature for two minutes and right afterwards the tube is spun at maximum speed in microcentrifuge for five minutes. The 50μl of the supernatant are removed and placed in a clean tube. In preparation for the PCR, tubes are labeled with Pcr rxn pellet. The PCR rxn pellet must be at the bottom. 20μl of the primer solution and 5μl of the bacterial DNA must be added. The tubes containing the dissolved pellet are then mixed gently and spin briefly to collect, then sample at the bottom. 0.2ml of the sample is then transferred to the PCR tube. The samples are then ready for PCR cycling. The initial denaturation period is of five minutes at 95°C followed by thirty cycles beginning at 94°C for forty-five seconds then 50°C for forty-five seconds and finally 72°C for ninety seconds. The final extension will be of 72°C for a period of seven minutes and, upon ending, it will remain at 4°C for as long as needed. Once the PCR cycling is completed, 5μl of 10x loading solution are added to each tube. The gel to be used should have a concentration .5g of agarose. For the running buffer, 25ml of 300ml 50x Buffer dH₂O will be used. The gel will be run at 80V for 20 minutes. Before loading the samples, 5μl loading dye are added to each DNA sample and to the 25μl of the molecular ladder. After electrophoresis, the gel is stained with INSTASTAIN® EtBr Cards for ten to fifteen minutes. PRELIMINARY RESULTS After the Quanty- Tray Sealer incubation at 35-37 C° for 24 hours has culminated. The samples were corroborated for the presence of total coliforms by examining each of the 48 tray cells for yellow cells. The trays were then examined with UV light for fluorescing E. coli containing cells. Fig. 1.4 presents a chart displaying the amount of coliform containing cells and the amount of E. coli containing cells. Only eight of ten samples were incubated due to the fact that there were 76200560705Fig.1.3. The image below presents an aerial photo, retrieved using Google earth, of the UPR in Cayey campus. The two yellow tags indicate the location of the water stream and pond. not enough trays to accommodate all the samples. As can be seen if fig. 1.4, 48 out of 48 cells in all eight water samples contained total coliforms. All but three samples (samples one, two and three) contained E. coli in all 48 cells. As can be seen in the numerical probability table, fig. 1.5, the three samples containing the least amount of E. coli were taken from slightly acidic water areas. This establishes an evident relationship between acidity and bacterial growth. Sample three was expected to have less E. coli due to the fact that it was collected from a shallower part of the pond, where animals are less likely to roam (refer to fig 1.3 for location photo). In general, all three samples collected from the pond had less E. coli presence than those from the stream. This is probably due to the fact that access to the pond by wondering animals is more restricted than to the more accessible, exposed stream. To further guarantee the presence of E. coli in the samples, a PCR was conducted with the extracted bacterial DNA. The first electrophoresis conducted was a failure due to poor bacterial DNA concentration in the samples. For the second PCR, instead of adding 175μl to re-suspend the bacterial pellet, only 75μl were used. This concentrated the sample further and allowed for proper staining with EtBr and visualizing of the gel. An image of the photo taken of the second electrophoresis gel can be seen in Fig. 1.6. It is evident by the thickness and clarity of all the bands that E. coli is present in all ten samples of water taken from the UPR in Cayey Campus. The PCR confirms the previously acquired information by incubation of the test trays containing the colilert. Fig. 1.4., The table below presents the amount of coliform and E. coli containing cells in the test trays. The fluorescing and yellow cells marked with a one (¹) represent the larger cells on the tray while the ones marked with a two (²) represent the smaller cells on the same tray. Sample number¹Fluorescing cell due to E. coli ¹Yellow cells due to coliform presence²Fluorescing cell due to E. coli²Yellow cells due to coliform presence14848184824648204832248348448484848548484848648484848748484848848484848 Fig. 1.5., The figure below presents a numerical probability table indicating the amount of total coliforms and E. coli present in all ten samples in relation to temperature and pH. Fecal ColiformsE. coliTemperaturepHSample #11011.2248.925˚C6.23Sample #21011.2203.525˚C6.25Sample#31011.232.325˚C6.29Sample #41011.21011.225˚C7.54Sample #51011.21011.225˚C8.06Sample #61011.21011.225˚C8.51Sample #71011.21011.225˚C8.14Sample #81011.21011.225˚C7.80Sample #91011.21011.225˚C7.54Sample #101011.21011.225˚C7.42 It is important to clarify the fact that just because E. coli are highly present in the samples, it does not indicate the presence of pathogenic bacteria. Although it is a very reliable indicator for other coliforms (possibly pathogenic) and feces related organisms, further experiments would need to be conducted in order to corroborate their presence. Upon further investigation, if the water samples do indeed contain pathogenic bacteria or organisms, such as Giardia and/or Cryptosporidium, this may indicate contamination of nearby water bodies. In which case, further experimenting will be needed to establish the actual source of the contaminants. Fig. 1.6 The image below is that of the successful gel staining with EtBr. Confirmed presence of E. coli can be seen clearly in all bands (one through ten). The small tail observed in the bands of the positive control and band number one is due to an air bubble defect when the comb was removed. 207034134285 LITERATURE CITED Anderson, K. and Davidson, M. Drinking Water & Recreational Water Quality: Microbiological Criteria University of Idaho, Cooperative Extension System of Agriculture Experiment Station Deng M., Fratamico P., Jensen M., Tice G. 1996. DNA Amplification using Tableted PCR Reagents for Identification of Escherichia coli 0157:H7 Isolated from Foods. Journal of Rapid Methods and Automation in Microbiology 5(1):61-74 Gunzburg S., Tornieporth N., Riley L. 1995. Identification of Enteropathogenic Escherichia coli by PCR Based Detection of the Bundle-Forming Pilus Gene. American Society of Microbiology 33(5):1375-1377 LeChevallier M., Norton W., Lee R., 1991. Giardia and Cryptosporidium spp. in Filtered Drinking Water Supplies. American Society for Microbiology 57(9):2617-2621 Skipton, S., Dvorak, B., Woldt, W., Wirth, S. 2008 Drinking Water Bacteria DHHS Nebraska Well Stewardship Information Series 2002 Total and Faecal Coliform Bacteria in Groundwater REFERENCES EDVO-Kit # 948. The Biotechnology Education Company® ACKNOWLEDGEMENTS Belinda Roman PhD We wish to extend a special thanks to the RISE program and its entire staff for their always enthusiastic support and guidance.