BIOL368: Genetics and Cell Biology Laboratory

BIOL368 Genetics and Cell Biology Laboratory Project Number: 2 Project title: Sterilization and aseptic technique Name: Alan Sanousian Student ID: 27680104 Lab section: Thursday Afternoon Group: 10 Lab partner: Mathieu Harb Date submitted: 04/10/2018 Introduction: The aim of this project is to understand the use of aseptic techniques in research laboratories and utilize them carefully to avoid contamination (via pathogens from different sources e.g dirty hands, nails, hair etc. encountered while handling bacterial culture). A second aim of this project was also the introduction to different sterilization methods, that are a crucial part of proper laboratory functioning e.g. disinfection and autoclaving. Subsequently, an introduction to different inoculation techniques using both a loop or stick (each one used separately) to isolate pure single colonies from different environments and plating them on different media, followed by their quantification was also done. For quantification, either viable cell counting or optical density measurements of varying dilutions of bacterial cell culture was used. (Concordia Biology department 2018). Aseptic techniques are procedures adopted to ensure proper handling of bacterial cultures in laboratory settings. Flame from a Bunsen Burner is used to heat sterilize glassware (high temperatures kill surface pathogens adhered to glassware) in addition to creating a sterile air corridor that pathogens from contaminating the apparatus used, as they usually fall vertically on the sterile work place. Therefore, all sterile glassware should be held next to the flames when in use.(Concordia Biology department 2018). The first part of the project was the use of aseptic techniques for sterilization of apparatus that would be required for the later part of the experiment. Different sterilization techniques e.g. radiation, autoclaving or filter sterilization can be adopted to sterilize glassware and other apparatus (Concordia Biology department 2018). In the present project, autoclaving of the Erlenmeyer flask and all glassware apparatus was done before preparing different media for the plates. Autoclaving involves application of high temperature and pressure in an enclosed pressure chamber for a definite time period to disinfect objects from all sources of living organisms1. For the succeeding task, streaking was done to isolate uncontaminated colonies of E. coli. This was performed using an inoculation loop, or a stick, which was sterilized and then dipped in the E. coli culture inoculum. It was then streaked on the 25% to 30% of the plate in a zigzag motion. The inoculation loop is reheated and streaked on the adjacent quadrant of the first streak. The second streak begun at the end of the first one. The process was then repeated for the third and fourth quadrants of plate. When streaking using a clean stick, the stick is not heated and discarded after use. A new stick is used to streak for every quadrant of the plate. The next part of the project entailed Next objective was to counting single colonies by plating different samples taken at a dilution of 10-7or 10-6 of E. coli culture) on a selective media (LB). The spread plate technique allows counting of bacterial  colony on a nutrient medium by naked. The alternative of quantifying cells that was used was by measuring optical density (OD) of the samples at 600nm. Measuring OD600 reflects as how much light is being scattered by the cell particles in the sample which is referred to as cell turbidity which is proportional to thecell density in the particular dilution. E.coli cells have an OD of 1 at 600nm and contains approximaely5×108 of cells. Based on this standard, the recorded OD was used to calculate the number of cells per milliliter(ml) of the sample. (Concordia Biology department 2018). Finally, the last objective of this project was to assess the ubiquity of microorganisms in different environments. Tryptic Soy Agar(TSA) plates were used to reveal whether the environment can procure bacteria for suitable media for its growth. TSA services the bacteria with essential amino acids and nitrogenous bases (as it contains extracts of casein and soybean). This provides enough nutrition for the growth of diverse bacterial organisms. Another alternative to test for the ubiquity of microorganisms was using Malt Extract Agar(MEA) which is standardized for fungal. Fungal growth demands a high carbon to nitrogen ration under high pH (acidic conditions) and MEA is optimized for such growths. (Concordia Biology department 2018). MATHERIAL AND METHODS All materials, methods and procedures were derived from the following manual: (Concordia Biology department 2018). No modifications were made. Table 1: Contamination experiment description: Sample description Incubation Temperature Plate 1 Hair 37oC Skin Clean fingers Dirty Fingers Plate 2 Swab pencil 37oC Swab phone Swab Labmanuel Swab Table (Bench) Table 2: Viable count for different dilutions of bacteria on solid media 10-5 10-6 10-7 Colony count 1000 317 41 Viable Count (cfu/ml) 3.17× 109 4.10× 109 Sample calculation: cfu= number of colonies/dilution factor/volume in ml cfu=31710-60.1=3.17×109cfu/ml Table2: Viable count result of the class: Sample Calculations: cfu= number of colonies/dilution factor/volume in ml Wednesday Section Group 1 10-6 dilution: 155/10-6/0.1= 1.55 ×109cfu/ml     Tuesday Selection Wednesday Selection Thursday Selection (AM) Thursday Selection (PM)   Dilution  Colony Count Viable Count (cfu/ml)  Colony Count Viable Count (cfu/ml)  Colony Count Viable Count (cfu/ml)  Colony Count Viable Count (cfu/ml)  group 1     10-5  1 1075 1216 1312 10-6  1 1.0 x105 155 1.55 x109 334 3.34 x109 549 5.49 x109 10-7  1 9 45 47  group 2     10-5  32 1708 1656 1720 10-6  1 1.0 x105 350 3.50 x109 872 8.72 x109 453 4.53 x109 10-7  1 56 104 39  group 3     10-5  26 816 964 2432 10-6  1 1.0 x105 498 4.98x109 38 3.80 x109 397 3.97 x109 10-7  1 131 16 26  group 4     10-5  16 811 1669 2248 10-6  5 5.0 x105 214 2.14 x109 760 7.60 x109 420 4.20 x109 10-7  1 14 75 38  group 5     10-5  44 860 1576 1940 10-6  4 4.0 x105 232 2.32x109 256 2.56 x109 403 4.03 x109 10-7  1 37 1 63  group 6     10-5  22 801 1160 1000 10-6  1 1.0 x105 55 5.50 x109 158 1.58 x109 92 9.20 x109 10-7  1 8 - 15  group 7     10-5  23 330 960 1504 10-6  1 1.0 x105 238 2.38x109 18 1.80 x109 524 5.24 x109 10-7  1 39 14 45  group 8     10-5  11 918 1560 1344 10-6  1 293 2.93x109 337 3.37 x109 379 3.79 x109 10-7  1 30 42 48  group 9     10-5  23 1340 784   NA 10-6  6 6.0 x105 294 2.94x109 133 1.33 x109   NA 10-7  1 36 24   NA  group 10     10-5  18 1346 1000 1180 10-6  4 4.0 x105 158 1.58 x109 317 3.17 x109 289 2.89 x109 10-7  1 11 41 41 Table3: Average value and standard deviation of the viable count data Average viable count (cfu/ml) Standard deviation Tuesday Section 2.98x105 0.661 x105 Wednesday Section 2.98 x109 1.34 x109 Thursday Section Afternoon 3.72 x109 2.49 x109 Thursday Section (PM) 4.81 x109 1.82 x109 Class 4.10 x109 2.33 x109 Sample calculation: For calculation average, standard deviation of the Thursday section: average=∑xn=3.34×109+8.72×109+3.80×109+7.60×109+2.56×109+1.58×109+1.80×109+3.37×109+1.33×109+3.17×10910=3.72×109 Standard deviation: SD=Σx-x2n-1 ((3.34×109)-(3.72×109))2 + ((8.72×109)-(3.72×109)2+ ((3.80×109)-(3.72×109))2+ ((7.60×109)-(3.72×109))2+ ((2.56×109)-(3.72×109))2+ ((1.58×109)-(3.72×109))2+ ((1.80×109)-(3.72×109))2+ ((3.37×109)-(3.72×109))2+ ((1.33×109)-(3.72×109))2+ ((3.17×109)-(3.72×109))2 Standard Deviation = (7.674*10-3)/9 = 2.49 x109 Figure 1 : Bar graph showing average values of viable cell count for different sections and class The average viable cell count value for Tuesday section is suspec...