SPRING 2017 ELECTRONIC LABORATORY NOTEBOOK MCB3023 L SECTION 078G CAROLYNA GUILLEN MEHRSA RAZAVI Table of Contents Exploring the Microbiome 2 The Art of Microscopy 4 Life in A Drop of Water 5 Streaking for Isolation 8 Micro-pipetting and Graphing 10 Re Streak for Isolation 13 The Gram Stain 14 Bacterial Growth Curve 16 Saliva Enumeration 18 Gram Stain of Unknown: Identification Begins 21 Inoculation of Selective/Differential Media 1 23 Inoculation of Selective/Differential Media 2 26 Inoculation of Selective/Differential Media 3 28 Inoculation of Selective/Differential Media 4 29 Indole Test 30 Oxidase Test 31 Catalase Test 32 DNA Isolation 33 Loading and Running Gel Electrophoresis 36 __________________________________________________________________________ STD Infection Simulation Using ELISA 37 Test for MRSA Carrier 39 pGLO Transformation 40 Antibiogram Actvity 43 Real Life Dilutions 46 Clean Water Lab 48 Fecal Float 50 1 Exploring the Microbiome January 5, 2017 Purpose: The purpose of this activity is to observe the microbiome that exists in three different areas: the surrounding environment, the air and the human body. Procedure: 1. Label three separate TSA plates with the different sources from which microbial samples will be acquired (i.e air, environment, body). 2. The air sample will be acquired by leaving the labeled petri dish open on the benchtop until the end of the lab period. 3. To sample from the environment, take a sterile cotton swab and dip it in saline solution. Then, take the swab and rub it on an object from the environment. Take the swab and lightly streak the TSA media in the respective petri dish. Close the petri dish and set it aside. 4. Repeat Step 3 for the body sample. 5. Tape all petri dishes closed. 6. Incubate at 37 degrees Celsius for 5 days. Observe colonies after incubation period. Results Figure 1. The picture on the left depicts colonies from microbes in the air. As seen, the culture is mixed. There are colonies that are very small while others are larger. Nearly every colony is flat except for one small yellow one that is convex. The majority of colonies are pale yellow or gold. The margins around most of them are entire, with a few undulate or lobate. The black colony is filamentous and the larger ones are irregular. 2 Figure 2. The image on the left depicts colonies from a sample taken of the microbes on my inner left forearm. As shown, the majority of colonies are pale yellow or gold, a few orange ones, and they are circular. All of the colonies are flat with entire margins. Figure 3. The plate on the left is depicting a colony of microbes isolated from the track pad of my laptop. The colony is round, flat and pale yellow with entire margins. The colony measured .6 cm. This colony will be streaked for isolation and analyzed for the unknown project. 3 The Art of Microscopy January 5, 2017 Purpose: To become familiar with the anatomy of the microscope and acquire certain microscopy skills such as focusing and using oil immersion. Procedure: 1. Acquire a microscope from the cabinet, as well as a prepared microscope slide. 2. Load the slide onto the microscope and secure it. 3. Observe the specimen using the 4X objective lens first. To focus, use the coarse focus knob. Once focus is attained, move up to the 10X lens. Again, focus the image using th coarse focus knob. Once focus is attained, move up to th 40X lens. Only use the fine focus knob now. 4. Once focus is attained on the 40X lens, ask an Undergraduate TA to assist in adding the oil for the oil immersion step. 5. After the immersion oil is added, slowly turn the rotating piece to the 100X objective lens. Use the fine focus knob to acquire a clear and detailed picture. 4 Life in a Drop of Water January 5, 2017 Purpose: To practice creating wet mounts and, to observe and characterize various eukaryotic microbes in water. Procedure: 1. A wet mount was created using a water sample from Lake Alice. 2. The wet mount was observed under the microscope and organisms were identified. 3. Another wet mount was created using a water sample from the sewage system. 4. This wet mount was also observed under the microscope. Several organisms were identified. Results: Pictures of organisms found in the Lake Alice wet mount. Figure 4. Bryozoa 5 Figure 5. Cyanobacteria Pictures of organisms observed in the sewage water wet mount. Figure 6. Hydra Figure 7. Rotifer 6 Figure 8. Gastrotrich 7 Streaking for Isolation January 10, 2017 Purpose: To isolate a pure culture in order to: (1) polish laboratory techniques and (2) have a pure culture for the Unknown Project. Procedure: 1. Decide which colony from which isolate is going to be cultured. The colonies are coming from the environmental samples plated in the lab on January 5, 2017. 2. Label the bottom of a fresh TSA plate with pertinent information (initials, date, isolate sample). 3. Draw a T on the bottom of the TSA plate (as shown below). This will make the quadrants where the bacteria will be streaked. 4. Light a Bunsen burner, establishing a steady, contained flame. Be sure to work close to the Bunsen burner in order to decrease the likelihood of contamination of the plate. 5. Grab the inoculation loop and hold it in the flame until the loop burns bright orange. This is to disinfect the loop before you begin streaking. 6. After disinfecting the loop, cool the loop on the EDGE of the agar in the new TSA plate. This way the bacteria will not be fried by the heat. 7. Take the inoculation loop and LIGHTLY scoop a very small amount of the desired colony. 8. (The first streak pattern will be made in the top quadrant of the TSA plate). LIGHTLY drag the inoculation loop over the agar of the new plate creating a zig-zag (depicted below). 9. Disinfect the inoculation loop in the flame. Cool it on the edge of the agar. Now, starting in the bottom left quadrant, streak the agar and create a zig-zag pattern. Make sure that two of the streaks pass into the first quadrant that was streaked. 10. Disinfect the inoculation loop in the flame. Cool it on the edge of the agar. Now starting in the bottom right quadrant, streak the agar and create a zig-zag pattern. Make sure that two of the streaks pass into the bottom left quadrant previously streaked in Step 9. 11. The streak plate is now ready for incubation. Incubate the plate UPSIDE DOWN at 37 degrees Celsius for the next two days. Figure 9. The image on the left depicts the pattern of streaking that is achieved when following the procedures above. 8 Results: Figure 10. The image on the left depicts a streak plate of an unknown microbe, sampled from the track pad of my laptop. Isolated colonies were obtained. The colonies are also the same yellow color, shape and of similar size. It is likely that this is a pure culture. 9 Micro-pipetting and Graphing January 10, 2017 Purpose: To perform a serial dilution in order to establish precise and proper micro-pipetting skills. Procedure: 1. Acquire an ELISA strip and label the end without a notch with your seat number. 2. Using the 200 microliter pipette, fill each of the wells with 90 microliters of water. To DRAW UP liquid, push the plunger down to the FIRST stop and enter the tip into the water. Slowly release the plunger to draw up the liquid. DISPENSE the liquid into the wells making sure to go down to the SECOND stop. This will ensure that every drop of liquid is expelled. Note: NEVER use the pipette without a tip! 3. Using the 10 microliter pipette, fill the FIRST well with 10 microliters of crystal violet dye. Aspirate the well by slowly pressing the plunger up/down. 4. Dispose of the tip. Draw up 10 microliters from the FIRST well and dispense it into the THIRD well. Dispose of the pipette tip. 5. Using a new pipette tip, aspirate the THIRD well and transfer 10 microliters of solution into the FIFTH well. Dispose of the tip. Aspirate. Leave well SEVEN blank. This will serve as a control. 6. Now, to create replicates of the dilutions performed, transfer 10 microliters of crystal violet into the SECOND well. Aspirate and dispose of the tip. Transfer 10 microliters of the solution to well FOUR. 7. Dispose of the tip. Aspirate. Transfer 10 microliters of the solution to well SIX. Aspirate and dispose of the tip. Leave well EIGHT blank. This will serve as another control. The ELISA strip should appear as depicted on the following page. Figure 11. This photo was added for extra clarity of the procedure 10 Results: Figure 12. This figure depicts the completed ELISA strip with all dilutions. Table 2. Optical density values for each dilution measured at 600nm using a microplate reader. Dilution Optical Density at 600nm 0.01 0.899 0.01 1.138 0.001 0.275 0.001 0.275 Control 0.038 Control 0.038 11 Average Class OD600 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 Optical Densityat Optical Densityat 600nm (O600) 0.2 0 0.01 0.01 0.001 0.001 control control Dilution Series1 Series2 Figure 13. Line series 1 represents average optical density measured by ELISA plate reader of students’ serial dilutions of crystal violet. Error bars reflect standard deviation of all optical density values measured at specified dilution.