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Microbiology Lab.docx

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McMaster University
Lovaye Kajiura

Microbiology Michelle Selvaratnam & Varda Khan 1212996, 1209502 Sara Halawa May, 5 , 2013 ABSTRACT This experiment was carried out to successfully decontaminate a water sample that was infected with Escherichia coli. The purpose was to decontaminate the Escherichia coli from the water sample using an antibacterial agent as well as to test for the presence of the Lac Z gene in Escherichia coli strains. The testing for the presence of Escherichia coli strains was done using a variety of microbiological techniques including aseptic techniques, serial dilutions and plating techniques. In the first part of the experiment, the contaminated water sample was treated with salt water. The salt water acted as a decontaminating agent, and this allowed the ability to assess its productivity in killing the Escherichia coli bacteria. There were two components used: a control plate (which did not include the salt water sample) and a treatment plate (which included 100 µ of salt water). The control plate and the treatment plate were prepared through a series of 10-fold serial dilutions. Then, the number of bacterial colonies, along with the change in bacteria concentration and the total number of bacteria were calculated. Furthermore, in the second part of the experiment, two strains of Escherichia coli (specifically, NC 4468 and GC 4468) along with a contaminated water sample were plated on an X-gal plate, and this was done to determine the presence of the Lac Z gene which codes for β-galactosidase. As the results displayed, the NC 4468 Escherichia coli strain produced blue colonies- which indicated that there was a presence of the Lac Z gene in the strain. INTRODUCTION The bacterium, Escherichia coli was the central focus of this experiment. Escherichia coli are most commonly found in the lower intestine of warm-blooded organisms. Escherichia coli tend to be harmless, except for their ability to become infectious, causing food poisoning in humans and further even leading to life-threatening conditions (Nordqvist, 2012). The severity of such infections caused by Escherichia coli calls for a need to examine treatment methods that can assist it’s decontamination in water. Salt water treatment method was used to decrease Escherichia coli contamination in the water sample. According to The National Institute of Standards and Technology, salt water has proven to be an effective decontaminating agent due to its pH levels, especially in comparison to regular water (CR-MMRS Rapid Access Mass Decontamination Protocol, 2003). In addition, Escherichia coli were further studied through the examination of the activity between different strains of Escherichia coli and the chemical indicator X-gal. X-gal, is an artificial galactosidase, which produces a blue by-product when digested by β-galactosidase (Carr, 2008). It is important to note that β-galactosidase is a product of the Lac Z gene since the Lac Z gene codes for the enzyme β-galactosidase (Carr, 2008). Furthermore, X-gal is effective as a chemical indicator of Lac Z activity (Bio 1A03 Lab Manual, 2013). In the first part of the experiment, the goal was to find and test a method which would effectively reduce the microbial contamination in water. Therefore, salt water was used as an effective decontaminating agent, and the ability of salt water to act as a decontaminating agent was tested through its effectiveness in decontaminating a sample of water which was contaminated by Escherichia coli. Furthermore, the treatment and control samples were prepared and compared to test the hypothesis that salt water can act as a successful agent in decontaminating Escherichia coli from water. These results were interpreted through calculations of the number of colonies that existed when the sample was treated with salt water. The result predicted was that a smaller number of bacterial colonies would exist in the treated water sample as opposed to the control (untreated) water sample. In Part B of the experiment, the goal was to test for the presence of the Lac Z gene using the chemical indicator X-gal, in two different strains of Escherichia coli (NC 4468, and GC 4468), as well as a sample of untreated water. The hypothesis in this part of the experiment was that the X-gal plate with the Escherichia coli strains and untreated water would portray a change in colour, which would indicate that X- gal was metabolized, further ensuring Lac Z activity (Bio 1A03 Lab Manual, 2013). METHODS: Part A1: Design an experiment 1. The sample was treated with salt water for 20 minutes. Part A2: Preparing the Serial Dilutions 1. The bench was prepared for aseptic procedure. 8 sterile, 2 ml micro centrifuge tubes were -1 -2 -3 -4 obtained and were labelled: C10 , C10 , C10 , and C10 for the control serial dilutions. T10 , T10 , T10 , and T10 for the treatment serial dilutions. Referred to the Biology 1AO3 Spring 2013 lab manual for aseptic procedures. 2. Aseptically added 900 μl (0.9 ml), using the P1000 and sterile blue tips, of sterile saline to each of the 8 tubes. -1 3. Aseptically removed 100 μl from the untreated water sample and added it to the C10 tube. Referred to the Biology 1AO3 Spring 2013 lab manual for the serial dilution process. 4. Step 3 was repeated for the Treatment dilution series. Part A3: Plating the Serial Dilutions 1. A petri plate of sterile nutrient agar was obtained and turned it upside down and divided it into 4 quadrants, each quadrant was labelled: C10 , C10 , C10 , and C10 -3 -4 2. The loop was sterilized and then dipped it onto the tube C10 . The liquid was spread on -4 the surface of the agar in the quadrant labelled C10 . The cap was replaced and turned one quarter and prepared to do the same with the next dilution. -3 -3 3. A loopful of liquid was taken from the C10 tube and was spread on quadrant C10 . Similarly, a loopful was spread from C10 and C10 on the designated quadrants. 4. Steps 1-3 was repeated for the Treatment dilution series. The bundle was incubated at 30˚C overnight. Part A4: Analyzing your results 1. After the plates had been incubated for 24 hours. The number of colonies in the quadrants were totalled and recorded on the Data Analysis sheet and lab notebook. 2. Once the number of colonies was determined, the concentration of bacteria was calculated using the formula given in the Biology 1AO3 Spring 2013 Lab Manual. 3. Plates were discarded in the yellow biohazard waste bin and all other safety precautions were followed. Part B1: Chemical Indicators 1. 1 quadrant was labelled NC 4468, GC 4468, and untreated water. A test tube containing NC 4468 and GC 4468 strain of E. Coli was obtained. 2. Aseptic techniques was used to spread a loopful of NC 4468 on the designated quadrant. This step was repeated for GC 4468 and untreated water. 3. An agar plate that ha been treated with X-gel was obtained and steps 1-2 was repeated. Part B2: Analyzing the results 1. After the plates had been incubated for 24 hours. The number of colonies in the quadrants were totalled and recorded on the Data Analysis s
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