Summary: Apsetic Technique

On day 3, I was able to perform the P disc sensitivity test. I asked for the correct media and I was able to inoculate on CNA using aseptic technique. I performed a lawn technique getting bacteria from the T-soy plate inoculated the first day. I used a swab dipped in sterile water and smeared the whole plate forming a lawn. To get the P disc from the container a used forceps to place the disc in the middle of the plate. The plate was place inverted in a candle jar for incubation. I disposed of the used materials using the proper disposal

glycine agar test by sterilizing the loop and tubes by heat, taking the bacteria and streaking it. The plate

1 Q-tip was then swirled around in the water in a test tube for each different type of water for 1 minute and the Q-tip was observed for any changes in color. The Q-tip was then rinsed by tap water from the sink as instructed by the kit’s instructions and properly disposed of. This was done for all the different types of water in both environments. Then for the tap water in both environments, bacteria was tested using the test kits. Using a dropper pipette provided by the kit, water was collected, and placed into a vial that contained a solution known as ‘purple reagent’ at the bottom. The vial was gently swirled and then placed on a table for 5 minutes and was undisturbed. The vial was then swirled again and a test strip that was also provided by the kit was placed into the test vial with the arrows on the test strip point down. The vial was undisturbed for 10 minutes, and the test strip was then observed for reddish lines within 1 minute after the 10 minutes. The test strip, vial, and dropper pipette were not rinsed, and were properly disposed of after

Still holding the vial at a 45 degree angle, the swab was inserted into the vial to soak up the S. marcescens for 10 seconds. Partly lifting the lid of one petri dish, shielding it over top like an umbrella, we inoculated S. marcescens onto the agar growth medium in the petri dish. We repeated these steps 2 more times to inoculate all 3 petri dishes. Once all three plates were inoculated with the S. marcescens we removed our forceps from the alcohol. We air dried them, and then passed them through the flam to sterilize them. Using the sterile forceps we placed the c disc in the center of a petri dish and press down lightly to ensure that it stayed. It is then labeled it C, for the control group, 5 for the replicate number, along with the date and time of the class. Again we sterilize the forceps and place the T disc into the next petri plate. It is labeled the same way but with a T for tetracycline. The T disc contained 30ug of the antibiotic. We repeat the sterilizing of the forceps one more time and place the Chl disc on the remaining petri plate and labeled it Chl for chloramphenicol. The Chl disc contained 30ug of the antibiotic. Once all 3 petri dishes are labeled they were placed upside down to prevent water droplets from falling onto the nutrient agar substrate. The last step to the experiment was to clean up. We placed our swabs in a waste beaker, cleaned up our work stations, and then washed our hands with soap and

The materials that will be used for this experiment are. Overnight to 24-Hour culture of E.coli in TSB. T4 bacteriophage. We will have 8 screw cap tubes that contain 4.5ml of sterile agar that are melted and cooled in 55C water bath. 8 TSA plates were used. 1 tube containing 3ml of TSB was used. We had 2 screw cap tubes, they each contained 9.9ml of TSB. We also had 1ml pipettes with pipette pumps. We also had 1 15ml sterile centrifuge tube. A Bunsen burner. We also had a incubator that was set at 37C. Some micropipettes were used as well as a table top centrifuge.

Before the streak technique was performed, the sample was “vortex” to mix solution, then a loop was sterilized with a flame. The loop was then used to aseptically inoculate a sample from the vial and a lawn was created on the plates, the loop was again sterilized. From the lawn, about 6-8 directional streaks were made using the edge of the loop; then the loop was flamed. The plate was turned slightly to make

In order to observe the different effects of antibiotics and antimicrobials on different bacterial cultures, we utilized the Kirby-Bauer method. With four different bacterial species and two plates for each, one to test antibiotics and the other to test antimicrobials, we created lawns on each plate with the respective bacteria. Before creating the lawns, one must label each plate to indicate the sample, the method, and the types of antibiotics or antimicrobials used. To create a lawn, one sterilizes an inoculating loop with an open flame, allows it to cool, dips it into the sample, and then proceeds to create a dense zig-zag pattern that covers the area of the plate in one direction. Then the plate is rotated 45 degrees and the process is repeated. Finally the plate is rotated 45 degrees once more and the process is repeated again. The creation of a

The first lab preformed was a harvest assay. First a plaque was picked from the last titer assay. This plaque was then added to an epindorf tube with 100 microliters of phage buffer in it. A serial dilution was then done to 10 ^-3, 10 microliters of each dilution was added to. 0.3 milliliters of M. Smegmatis. The solution was then incubated at room temperature for fifteen minutes. Then 4.5 milliliters of top agar was added to each tube and plate onto petri dishes. Next the most diluted plate was flooded with 4 milliliters of phage buffer, incubated at room temperature for two hours. The plate was then inverted in order to collect one milliliter of liquid collected on the plate’s top, which was then placed in an epindorf tube. From that epindorf tube 10 microliters of it was then added to 90 microliters of phage buffer and vortexed. A serial dilution was then completed to 10 ^ -4. Each dilution was then added its own tube with 0.3 milliliters or M. Smegmatis. To those tubes 4.5 milliliters of top agar was then added to each and each were plate on to their own petri dish. The 10^-3 and 10^ -4 plates from the previous harvest assay were used in the next harvest assay. Both plates, the 10^-3 and 10^-4, were flooded with 4 milliliters of phage buffer each. These plates were then incubated for two hours. After the two hour incubation period the

During the second laboratory session, the two TSA plates that were streaked and incubated from the first laboratory session were given back in order to observe the isolated microbial colonies of microbes grown on the aerobic and anaerobic TSA plates. Gram stains were performed using the isolated microbial colonies that had formed on the aerobically and anaerobically incubated TSA plates. The Gram stains were examined using 1000x total magnification and oil. Then, the unknown mixed culture was streaked onto one MacConkey agar plate and incubated aerobically at 37° Celsius for 24 hours. The unknown mixed culture was then streaked onto one Columbia CAN agar with 5% sheep blood plate (CAN) and also incubated aerobically at 37° Celsius for 24 hours.

Using the mixed culture given, each student performed two Gram stains, which is described in the lab manual Microscopy And A Survey Of Microorganisms in Exercise 6 (McPherson pg. 53). During each procedure, every student followed aseptic technique in order to minimize contamination. This process is explained in Exercise 4 (pg. 37). Following the two Gram stains, each slide was viewed using a bright light microscope in order to find out the initial morphologies and different Gram

For this lab we used two microcentrifuge tubes, a pair of vinyl gloves to prevent contamination, a micropipetter, six sterile micropipetter tips, 500 µl of a transformation solution containing CaCl2, seven sterile loops, ice, a timer, a 42°C water bath, a floating rack two colonies of E. Coli bacteria, pGLO plasmid DNA, four Luria Bertani (LB) broth nutrient agar plates (one with LB alone, two with LB and ampicillin, and one with LB, ampicillin, and arabinose), 500 µl of LB nutrient broth, tape, a UV light, and a 37°C incubator.

In order to obtain well-isolated discrete colonies, the quadrant streak and spread technique was used. This allowed dilution of the original microbial material over the entire surface of the plate. As the original sample was diluted by streaking and spreading it, over successive quadrants the number of organism decreases. Usually by the third or fourth quadrant only a few organism were transferred on the by the inoculating loop and theses produce a few isolated

100 µL of bacteria and fungus from freshly prepared culture was taken in the pipette and poured in the middle of the respective petri plate. Remove excess inoculum by lightly pressing the swab against the tube wall at a level above that of the liquid.Using a cotton swab that has already put in UV light, the bacteria and fungus was spread evenly on the surface of the plate so that bacteria and fungus were spread in each corner of the plate and dried for 4-5 minutes Inoculate the agar by streaking with the swab containing the inoculum. Rotate the plate by 60° and repeat the rubbing procedure. Repeat two times. This will ensure an even distribution of the inoculum. Allow the surface of the medium to dry for

With a glass-marking pen preferably black, mark four sterile test tubes. The test tubes should be marked 1A, 2A, 3A, and 4A. First, using a mortar and pestle to grind together a spatula of sand, a 0.5 cm pieces of apple, and 1 ml of distilled water. Then pour this mixture into test tube 1A. Second, using a mortar and pestle to grind together a spatula of sand, a 0.5 cm pieces of potato, and 1 ml of distilled water. Then pour this mixture into test tube 2A. Third, using a mortar and

The first thing to be done is to isolate the bacteria with the goal of obtaining a pure culture. This is done by streaking Trypticase Soy Agar (TSA) plate, Dnase plate as well as Mackonkey using the streak method and