Gram Staining Lab Report

During the purification section of this lab, the LB/amp/ara agar plate was examined for well-isolated green colonies and the LB/amp plate was observed for white colonies with space between each other. These colonies were circled on the outside of the plates using a marker. Next, two 15 milliliter culture tubes containing 2 milliliters of nutrient growth media were obtained and labeled “+” and “-“. Using a new inoculation tube, the circled colonies from each plate were scooped out and immersed in their respective culture tubes. Once the bacteria was mixed into the solution, the tubes were sealed and placed horizontally into the 32⁰ incubator for 24 hours.

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

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 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.

Bacteria that can tolerate the high concentration of salt in the media are from the Staphylococcus genus. A sample of unknown A was also used to stab a gelatin test tube, which contains the gelatinase enzyme that breaks down the gelatin and changes the media from solid to liquid. A sample of the same unknown along with a sample of unknown B was then used to stab a citrate test tube each. The citrate test is used to determine whether the bacteria utilizes citrate as a carbon source. If the bacteria used the citrate, the color of the media turns green, but if the citrate was not used, the media remains blue. The color change in the media is due to the presence of the pH indicator bromothymol blue. Afterwards, a sample of both unknown B and unknown C were used to make single line inoculations on both an Eosin Methylene Blue (EMB) and a Salmonella Shigella (SS) plate. Each plate was divided by a a line in half so that unknown B was inoculated on one side and unknown C was inoculated on the opposite side. The EMB plate contains lactose and dyes eosin and methylene blue. The media is used to differentiate between lactose and non-lactose fermenters. The SS plate also contains lactose in addition to bile salts and brilliant green (dye) to select for species of Salmonella & Shigella. The media also contains some

coli culture incubated at 37 ᵒC and the other one for an E. coli culture incubated at 25 ᵒC. To start the lab, one milliliter of the culture, Luria broth, is transferred to a spectrophotometer tube and zeroed. One milliliter of each culture is transferred to a clean cuvette and the optical density at a wavelength of 595 nm is measured. The OD values and times should be recorded. If the OD value exceeds 1.0, one hundred microliters of the culture and nine hundreds microliters of the nutrient broth must be taken and the OD should be measured again. One of the measured cultures is then transferred into an Eppendorf tube.

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 general unknown lab’s experiments focus on biochemical and physiological characteristics of Enteric and Cocci bacteria, in aid of identification. The difference between gram-positive and gram-negative

Many other tests can be used to identify bacteria and they are used when it is best suited as there are many disadvantages in comparison to the gram stain procedure which is a quick procedure and it is not expensive to proceed

In order to observe the morphological characteristics of unknown culture #304 tests were performed. The tests go by the protocols of Brown. Inoculating and incubating two agar slants in two different temperatures 27 C and 37 C was conducted as the first test to determine the optimum temperature for incubation on unknown#304. Motility test was the following experiment using a motility tube to see if the organism was motile or non-motile. A gram stain was done to determine the shape of the organism. An EMB plate was inoculated with the unknown culture as well as a PEA plate. This was used to help determine if the organism was gram-negative or gram-positive.

Most bacteria are classified as either gram positive or gram negative. Gram positive is a purple stain and gram negative will appear pink or red. Gram negative can cause many kinds of infections. One type of bacteria is Escherichia which is a rod shaped bacteria. A Gram stain is used in lab setting to identify what type of bacteria is present. A sample is placed in a nutrient media and incubated. This media will encourage bacteria growth present. This process allows for further testing and to identify what kind of bacteria is present to allow for appropriate treatment.

The key to the process is in the second step, this is when iodine is applied to the specimen. The iodine causes the dye to form crystals that get trapped in the cell walls. In  Gram-positive cells the walls are thicker and the layer of dye saturates the cell wall  more extensively. The third step in the process is to apply alcohol which dissolves the lipids in the outer membrane of the Gram-negative cells. This allows for the violet dye to wash out, leaving these cells colorless.

In the world of microbiology it is vitally important to be able to discern the identities of microorganisms. Not only is it important in a lab setting but as well as in healthcare in general. Properly identify what strain of bacteria a person has will aid in the proper medicine and dose given. Throughout the semester we have learned about different types of bacteria and certain test that can clearly identify them. The purpose of this lab report is to identify a Gram-positive or Gram-negative bacterium. Using all the knowledge of procedures and lab techniques identify the unknown and discuss all the tests you performed.

When the slide for the Gram-Stain was placed under the microscope no organisms were able to be found and conclusion could not be made using that Gram Stain. After the T-Streak had been incubated for 48 hours, it was removed and observed for morphology. The isolate colonies in the morphology observation were yellow, round, had smooth edges, and were flat. The result from the second Gram-Stain using the incubated T-Streak culture was Gram-Positive cocci. When the catalase test was performed, rapid bubbling occurred giving a positive result. Next, once the Fluid Thioglycollate Test (FTM) was performed and had incubated for 24 hours, there was a yellow, milky solution at the top of the solution and some throughout the beaker. The last test performed was a Mannitol Test, and after incubating for 48 hours was removed from the incubator for observation. The test was negative as there was no color change around the

This report represents my individual effort. I did not receive or offer aid to anyone when performing this assignment, nor did I plagiarize any material.