What Is 5. 5 Antimicrobial Agar Diffusion Assay

The first result of importance was the result of the Gram stain. The observations of the unknown bacteria from the slant culture after Gram staining showed that the unknown bacteria were Gram negative bacilli (Image 1). After determining the unknown bacteria was Gram negative, an oxidase test was conducted on a sample from the slant culture. The cotton swap with the sample of bacteria did not change color when the oxidase reagent was applied, thus providing a negative result. With a negative oxidase test, further tests were conducted to determine various characteristics of the unknown bacteria. A MR-VP broth was inoculated with a sample from a slant culture of unknown bacteria. After incubation, the methyl red reagent was added to the broth, and the broth turned red, providing a positive result (Image 2). An EMB agar streak plate was inoculated with a sample from a slant culture of the unknown bacteria, and after incubation, growth was found on the plate, providing a positive result (Image 3). A Citrate agar slant was inoculated, and after incubation, growth was found on the media, providing a positive result (Image 4). A Urea agar slant was inoculated, and after incubation, the agar had changed from a peach color to a bright pink color, providing a positive result (Image 5). Using the flowchart (Figure 1) developed from the Table of Expected Results, the lab partners started at the oxidase test. Given the negative result of the oxidase test, the flowchart is

After the incubation period the bacteria was observed for pure colonies. The colonies were sampled and the three streak plate technique was repeated and this sample was incubated for forty eight hours at 37 degrees Celsius. After the incubation of the colonies, a gram stain was performed which is defined in the lab manual.

There were two tubes used in this process: the tube that contained the primary culture and the tube that contained the nutrient agar where the unknown bacteria would grow. First, the inoculating loop was flamed. After removing the caps of both the test tubes, they were flamed to prevent contamination of the unknown bacteria. The inoculating loop was cooled for a few seconds and was then placed into the test tube containing the bacteria. The inoculating loop with the bacteria was placed into the nutrient agar test tube for cultivation. Before the test tubes were capped, they were flamed once again. Also, isolation of the unknown bacteria had to completed. Nutrient agar was placed in the petri dish, and was left to gel for a few minutes. After the agar gelled, the inoculating loop was used to acquire bacteria and streak the unknown onto the plate for

After labeling a new microtube with a marker, the culture tubes were removed from the incubator and observed under the ultraviolet lamp. Using a sterile pipette, the contents of the (+) culture tube were transferred to the 2 milliliter mictrotube and the (-) culture tube was disposed of. The (+) tube was then spun in the centrifuge at maximum speed for 5 minutes. Once it was removed from the centrifuge, a noticeable bacterial pellet had formed at the bottom of the tube and the remaining liquid was disposed of. After the

Crystal violet stains the cell of the bacteria and is placed over the heat fixed slide for one minute then rinsed with H2O. The mordant that binds the stain is gram’s iodine and is recommended to be present on the slide for one minute. Alcohol is used as the decolorizer and is dripped over the recently rinsed slide until the slide runs slightly blue. If the bacterium retains the crystal violet it is due to the thicker peptidoglycan in the cell wall (gram-positive). The bacteria will then be stained with the counterstain safranin for 45 seconds. When crystal violet is not retained in the cell wall of the bacterium it will stain pink (gram-negative). A gram stain was immediately performed with the recently inoculated streak plate. An inoculating loop was used to gather some of the bacteria from an isolated colony. The loop was placed on a new slide and the transferred bacterium to the slide. The slide was dried, heat-fixed, and the gram stain process just described was completed. After microscopic analysis, it was determined the first unknown bacteria was a gram-positive cocci. The second unknown was determined to be gram-negative cocci. With this information a set group of test decided and performed based on the Unknown Bacteria Flow Chart as provided in the lab manual (Pg.) these test and the results are shown in record on the following pages. These are separated for the gram positive and the gram negative

The unknown plate acted as the stock master plate, and was used to aseptically streak the unknown for isolation and was incubated at 37°C for 24 hours. After the incubation, a Gram stain was performed. Using an inoculating loop, a drop of DI water was placed on a clean slide, and a sample of bacteria was aseptically transferred and mixed in the DI water. The slide air dried for a couple of minutes, and was then heat fixed to keep the bacteria on the slide. Using a staining tray to hold the slide, 2 drops of crystal violet dye was placed on the dried bacteria and allowed to sit for 2 minutes (2). After the CV was set, DI water was used to rinse off any remaining dye. The smear was covered in Gram’s iodine for 1 minute. After a minute the iodine was rinsed off and the slide was tipped to the side to allow 2-3 drops of ethanol decolorizer to remove any remaining purple coloring. The slide was rinsed again, and the smear was covered with safranin for 30 seconds. After 30 seconds the slide was rinsed off and blotted dry with bibulous paper (2). The smear was then observed under a microscope to determine gram nature and

After gaining some knowledge about bacteria, we were giving an investigating bacteria growth lab to do. Our objective was to observe the conditions required for bacteria to grow and to test the effectiveness of substances that may be antibacterial, disinfecting, and or sanitizing. My group and I began our procedure by gathering all the bacteria by swabbing our necks and mouths. After this, we inoculated the culture by rubbing the bacteria on the agar, a nutrient rich gel made from sea kelp, on the bottom side of the container where we grow bacteria, the Petridish. We hoped for the results to come back with little or even no colonies and an immense zone of inhibition around the tiny circle cut out of filter paper covered in toothpaste, Neosporin, and Chlorhexidine Gluconate 4% Solution.

Next the tubes were placed in an ice bath, while obtaining a sterile loop to swipe a single colony of E.coli to put into the tube. After gently swiping a colony onto the loop, it was then spun in the +pGLO tube to get it to come off, returned to the ice bath. Next using a different sterile loop, it was swooped it in a container labeled pGLO plasmid DNA and again spun it ONLY into the tube with the solution labeled +pGLO to get it to come off. After about 10 minutes on ice the tubes were then placed into a 42ºC water bath for 50 seconds exactly, and immediately after placed them back into the ice bath. Finally, after 2 more minutes in the ice bath the tubes were separated into 4 containers. 250 ul of +pGLO solution was added to the containers containing +pGLO, LB broth, with ampicillin and +pGLO, LB broth, ampicillin, with arabinose. Also 250 ul of the –pGLO solution was added to the 2 containers containing LB broth, and ampicillin, with LB broth. Using a sterile loop for each plate the solutions were spread out gently and thoroughly on to the containers with agar. After the containers were incubated in 37ºC for at least 24 hours, the results were observed and disposed of (Weedman,

Incubation of both closed tubes took place in an ice bath, insuring the tubes were making contact with the ice, for ten minutes. While the ten minutes was passing five different plates were collected from the TA table: 1 LB plate, 2 LB/Ampicillin plates, 1 LB/Arabinose plate, and 1 LB/Ampicillin/Arabinose plate. Both the +pGLO and the –pGLO were transferred into the 42°C water bath using the foam rack as a holder. The tubes needed to make contact wityh the warm water for 50 seconds. After these fifty seconds were completed the tubes were placed back on ice immediately for another two minutes. The transfer from ice, to hot water, back to ice creates the best bacterial transformation results. Removal of the rack containing the tubes from the ice took place, putting the tubes on the bench top. Using a new sterile pipette, 250 µL of LB nutrient broth were added to each tube, making sure that for the second tube a new pipette was used. A 20-minute incubation at room temperature occurred. Then, using a new sterile pipette for each tube, approximately 100 µL were placed onto the appropriate

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.

A four-quadrant method, also known as a streaking technique, was performed to isolate colonies of the two bacteria. Using an aseptic procedure for organism removal, a heated and sterilized loop was used to inoculate bacteria from the unknown test tube and then used to streak two solid nutrient agar plates, one plate was placed in a 37 degree Celsius incubator and the other plate was placed in the laboratory lockers at room temperature. After a 48-hour incubation period, both nutrient agar plates were observed; the plate incubated at 37 degrees Celsius had the more distinguished bacterial colonies of the two nutrient agar plates. The two colonies that were qualitatively identified were a yellow colony and a clear colony. Both colonies were smeared on separate slants and incubated and a Gram stain was performed to observe morphology. Gram staining is an experimental technique that allows an observer to distinguish between colonies of bacteria based on the composition of a bacteria’s cell wall. The information obtained in a Gram stain indicates whether the bacteria is Gram-positive or Gram-negative based on the ability of its cell wall to retain stain. A gram staining technique includes bathing a smear of bacteria in crystal violet to stain the bacteria purple, following a treatment with iodine and decolorizer, and a counterstaining with safranin. Gram-negative

All it takes is one single cell to start a pure bacterial colony. This lab allows researchers to observe isolated highly concentrated samples of bacteria, to view their traits and to isolate pure colony samples for use in future experiments. Unfortunately successful isolation was not achieved in this lab. Possible sources of error could be that the streaking was too close together resulting in an unsuccessful isolation, the loop was not flamed adequately which permitted carryover of bacteria from one sector to the next or forgetting to let the loop cool before touching the agar. To expand this lab, researchers could collect a single colony from an agar plate and grow it inside a nutrient tube to observe the oxygen nutrient requirements for that specific sample of

For the first step, using clear 2cm wide cellotape and a wooden applicator stick to make a small cellotape flag (2x2cm). Using sterile technique, gently press the sticky side of the flag onto the surface of the culture. Remove and apply a drop of 95% alcohol to the flag, this acts as a wetting agent and also dissolves the adhesive glue holding the flag to the applicator stick. Place the flag onto a small drop of Lactophenol cotton blue on a clean glass slide, remove the applicator stick and discard, add another drop of stain, cover with a coverslip, gently press and mop up any excess stain. Then we can observe under the microscope the sporulation occurred from the fungi

The petri dish lid was raised to insert the loop. The loop was touched to the agar area on the opposite side of the dish. The bacteria on the loop were transferred to the agar. The bacteria were spread in the first sector of the petri dish by moving the loop back and forth across the dish (zigzag motion).

A fresh pipette was used to transfer 0.5ml of broth culture of E. coli, to be inoculated, into a tube of molten agar previously boiled to drive off oxygen. The tube was then rolled to distribute the bacteria and allowed time for the agar to harden. The tube was incubated at 37oC for 48 hours. The procedure was repeated for broth cultures of the bacterium Clostridium sporogenes and B. Subtilis.