Streaking Lab Report

- Of the following, the most efficient method for sterilization of a bacteriological transfer loop is

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

Next, each tube was opened and 250 µl of transformation solution was added to each and then placed in the ice. While the tubes were on the ice, a sterile loop was used to pick up to 2-4 large bacteria colonies of bacteria from the starter plates and immerse to both +pGLO. The tubes were then placed back on the ice. Once the colonies of bacteria were well placed in the +pGLO solution, the same process was repeated for the –pGLO tube using a new sterile loop.

For this particular assignment, I was given a slant with an unknown bacteria by my lab instructor. There were several procedures performed to identify the unknown bacteria. The first procedure was streaking tryptic soy agar plate (TSY). The purpose of streaking is to identify the bacteria in a sample presenting a pure culture of colony morphologies. After several hours of incubating the streaked plate, visual isolated colonies were present. The steps to streaking are as follows; Flaming an inoculation loop with a Bunsen burner until red hot, then allow to cool before taking the unknown bacterial sample. Streaking four quadrants onto the TSY medium plate by dragging the loop gently through first quadrant then sterilizing the loop by flaming it again. This procedure was continued into the third and fourth quadrant. The second procedure performed was the bacteria smear. The motive of the smear is to have the bacteria ready for a stain after it has been heat fixed on a slide. These steps conclude by first labeling the slide with the my initials, flaming the inoculated loop, then allowing the loop to cool before spreading the unknown bacteria on slide after instilling a drop of distilled water. This process took about 10 minutes for the wet surface to dry before heat fixing. Heat fixing is when a bacteria smear is smeared on a slide and dried at room temperature before passing a slide through a hot flame of a Bunsen burner. Soon after the slide had been heat fixed and

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

You can grow microorganisms in liquid media or on solid media. Most bacteria can be grown in labs as long as the media contain a source of the major nutrients; carbon, nitrogen, sulphur, and phosphorous. They may also need to have other nutrients. These nutrients are made into a broth and the pH and salinity can be adjusted. The nutrient broth is placed in test tubes, which are plugged with cotton wool, capped with foil and then sterilised in an autoclave, at 121c for 20minutes. These tubes are then cooled before they are inoculated. To prepare the streak plates we dipped an inoculating loop into ethanol then placed it in the flame until the loop glowed red. Still holding the inoculating loop by its handle we removed the lid from

The streak plate was a fundamental first test to isolate and grow a pure culture. The streak plate was incubated for 48hrs and grown at 37 degrees. The

The petri dish was sanitized and covered in the agar. The cotton swabs used to swab bacterial samples were dampened using water. The spots around the school were swabbed and brought back to the petri dish to prepare to grow the bacteria. To get the bacteria to transfer off the cotton swab and onto the agar, the cotton swab must be rolled around the entire surface

Samples were collected from the tabletop, the camera, the window and the refrigerator door. The samples, were then placed in the petri dish, in its designated segment. The other petri dish had samples from the mirror, the stethoscope, the doorknob, and the steering wheel of the car.

a. LB+plasmid and LB-plasmid: Both of these plates had a lawn of bacteria. This proves

By subjecting these bacteria through sudden temperature changes or heat shock, a difference in pressure in the outside and the inside of the cell is made, that causes pores, through which the mobile supercoiled plasmid enters. After normalizing the cell temperature, its walls will heal. When the E. coli has now taken up the plasmid with GFP, it will be able to grow in agar plates with ampicillin, an

On August 19th, 2015 this experiment was performed, by 6 separate lab groups.The experiment began by measuring 1 Ml of E. coli into a pipette and pump, then placing the bacteria into a culture medium. The E. coli and medium were then swirled together for a period of 15 minutes, until completely mixed. This mixture was then poured into a petri dish and allowed to solidify for 45 minutes. After the 45 minute solidification time, 5 small paper disks were inserted into the dish. 4 of the disk contained treatments of antibiotic and 1 was left untreated. The

In the first part of the experiment, we plated donor bacteria, which was chloramphenicol resistant, on a Nal plate. Because no donor bacteria would grow on a Nal plate, this was a way of ensuring that the sample of donor E. Coli bacteria was pure.

How do you insert the plasmid inside the bacteria? What process do you use? (1/2 pt)

Turn the starch agar plates upside down and draw two lines with the sharpie on each, dividing each petri dish into four separate sections. Label the sections 1A, 1B, 1C, and 1D on the first petri dish, 2A, 2B, 2C, and 2D on the second, and so on. 2. Pass forceps through the Bunsen burner flame; let them cool for a little bit, then use them to pick up one of the paper discs. Open the culture of Bacillus subtilis, flame the neck of the bottle, and dip the disc in the broth.