E. Coli : An Indicator Of Water Quality

The first result of importance was the result of the Gram stain. The observations of the unknown bacteria from the slant culture after Gram staining showed that the unknown bacteria were Gram negative bacilli (Image 1). After determining the unknown bacteria was Gram negative, an oxidase test was conducted on a sample from the slant culture. The cotton swap with the sample of bacteria did not change color when the oxidase reagent was applied, thus providing a negative result. With a negative oxidase test, further tests were conducted to determine various characteristics of the unknown bacteria. A MR-VP broth was inoculated with a sample from a slant culture of unknown bacteria. After incubation, the methyl red reagent was added to the broth, and the broth turned red, providing a positive result (Image 2). An EMB agar streak plate was inoculated with a sample from a slant culture of the unknown bacteria, and after incubation, growth was found on the plate, providing a positive result (Image 3). A Citrate agar slant was inoculated, and after incubation, growth was found on the media, providing a positive result (Image 4). A Urea agar slant was inoculated, and after incubation, the agar had changed from a peach color to a bright pink color, providing a positive result (Image 5). Using the flowchart (Figure 1) developed from the Table of Expected Results, the lab partners started at the oxidase test. Given the negative result of the oxidase test, the flowchart is

This lab experiment was done for the purpose of learning how to determine a gram negative bacterium based on multiple tests learned throughout the semester. My gram negative unknown bacterium given to me was Salmonella typhimurium based off of the following tests; Triple Sugar Iron Agar (TSIA), Sulfate Indole Motility (SIM), Methyl Red (MR), Voges-Proskaur (VP), Citrate, Urea Hydrolysis, and Gelatin Hydrolysis. Each test performed gives results such as motility, acid production, fermentation, carbon requirements, or detection of certain coenzymes. With a process of elimination, I determined which bacteria it was not and which bacterium I had, S. typhimurium. The expectation was to master the techniques for each test and utilize the results to determine the unknown bacterium I was given within a two-week period.

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

There were two tubes used in this process: the tube that contained the primary culture and the tube that contained the nutrient agar where the unknown bacteria would grow. First, the inoculating loop was flamed. After removing the caps of both the test tubes, they were flamed to prevent contamination of the unknown bacteria. The inoculating loop was cooled for a few seconds and was then placed into the test tube containing the bacteria. The inoculating loop with the bacteria was placed into the nutrient agar test tube for cultivation. Before the test tubes were capped, they were flamed once again. Also, isolation of the unknown bacteria had to completed. Nutrient agar was placed in the petri dish, and was left to gel for a few minutes. After the agar gelled, the inoculating loop was used to acquire bacteria and streak the unknown onto the plate for

An unknown mixed culture was distributed by the Microbiology instructor labeled with a “3”, which was the assigned group number. The group was informed that there were two unknown microorganisms within the tube. Procedures were then completed in a specified order based on methods taught in the laboratory using the assistance of the assigned

After collecting all eight samples, they were then placed in the Petri dishes. The Petri dishes were both placed in an incubator. The temperature at that time was 80 degrees Fahrenheit. The samples remained in the incubator for over 24 hrs, by which time, quite a lot of growth was formed.

Incubation of both closed tubes took place in an ice bath, insuring the tubes were making contact with the ice, for ten minutes. While the ten minutes was passing five different plates were collected from the TA table: 1 LB plate, 2 LB/Ampicillin plates, 1 LB/Arabinose plate, and 1 LB/Ampicillin/Arabinose plate. Both the +pGLO and the –pGLO were transferred into the 42°C water bath using the foam rack as a holder. The tubes needed to make contact wityh the warm water for 50 seconds. After these fifty seconds were completed the tubes were placed back on ice immediately for another two minutes. The transfer from ice, to hot water, back to ice creates the best bacterial transformation results. Removal of the rack containing the tubes from the ice took place, putting the tubes on the bench top. Using a new sterile pipette, 250 µL of LB nutrient broth were added to each tube, making sure that for the second tube a new pipette was used. A 20-minute incubation at room temperature occurred. Then, using a new sterile pipette for each tube, approximately 100 µL were placed onto the appropriate

The E. coli bacteria are a native flora of humans and animals intestinal track, and it is

A) Based on the data we got some groups had coliforms in their FR water sample but others did not. The same thing occurred with the ML water sample. This was determined by letting the bacteria grow colonies on an EMB plate and these colonial growths indicated that there were coliforms present in the water.

RESULTS: Our main goal was to identify which lakes and ponds tested positive for Coliform or Escherichia coli and to come up with some reasoning as to why this could have happened. Water sampled from 6 different locations: Sebago Lake, Hinckley Reserve, Clarks Pond, a small pond near the turf field at the University of Southern Maine, in Gorham, Maine, and two runoff snowbanks both located on the USM campus in Gotham were all surveyed. These samples were taken into the lab, mixed with a COLISURE packet and pipetted into a well plate. They were then incubated for at least 24 hours at 37˚C to see if they would yield positive or negative results. The COLISURE kit perviously mentioned tests for two things: first for Coliform in the sample

In comparison to the States Standards in table 10, Fecal Coliform for sites 7, 8, 9, 12, 17, and 18, all in the urbanize Buffalo Creek watershed have mean values of 1613.95, 1189.52, 2447.38, 3167.71, 1410.86, 1178.30 CFU/100ml respectively which are within the states watch zone for the 1999 to 2002 parameters. Subsequently, above normal levels were recorded for Buffalo Creek Watershed sites 9, 12, 15, 18 for 2003 to 2008, sites 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 for 2009 to 2010, and sites 8, 9, 13, 14, 15, 16, 17, 18 for 2011 to 2013with fecal coliform concentrations of 1459.56, 1078.11,1340.28,1113.34; 1890.46, 1517.04, 4115.75, 1863.17, 1475.29, 2078.82, 2165.17, 1314.42, 1389.96, 4427.21, 4684.38, 2026.25; and 1172.76, 1928.00, 2507.14,

Data was analyzed at the Little Spokane River at the Verbrugge Environmental Center in Scotia Valley. In terms of the accuracy and methods employed, they were all appropriate in the area of study. Recording data at every 0.5 meter may not have been absolutely accurate since the meter tape may not have been tight enough. The biotic index may not have been accurate because only a small sample was taken from the stream to study. Through the two-sample t-test, the mean gave us a good idea of what the average concentration of E. coli (Escherichia coli) or coliform was in Reach 2 and Reach 3. Over the years, it was comforting to observe that the high concentration level of E. coli (Escherichia coli) in 2013 decreased in 2015. However,

Preparing serial dilutions of the alfalfa water will ensure an accurate count. The plates are incubated until you see visible colonies, usually 18-24 hours. The colonies you see growing on the plate are considered to have started from one viable bacterial unit but because bacteria are usually not found as individuals, the colony you see may have started from a single cell or a group of cells. The results are reported as colony forming units (CFU’s).

- A pure single E.faecalis colony was isolated from the cultured plates and gram stained to confirm the colony growth under microscope.(Fig.12)

A sterile pipette was used to add 0.1ml of E. coli culture to the pH 3.0 tube. This was then repeated for the tubes at pH 7.0 and pH 9.The tubes were then incubated at 37oC for 48 hours. This was then repeated for saline culture of Saccharomyces cerevisiae but incubated for 72 hours at 25oC.