Gram Staining Procedure

To perform this test, a small drop of water is placed on a clean microscope slide. A metal loop that has been properly sterilized in the blue flame and allowed time to cool is used to

An unknown bacterium was handed out by Dr. Honer. The appropriate tests were prepared and applied. The first procedure that was done was the gram stain. Under a microscope, if the gram stain is purple, the bacterium is gram positive, if the stain is red, it is gram negative. The next test was the fermentation tests for glucose, sucrose and

The purpose of this experiment is to distinguish and indentify an unknown bacterium. There are several tests that can help one eliminate and narrow down the options. The most useful test, and the very first one done, is a gram stain. This test will tell whether the bacterium is gram-positive or gram-negative. After the type of gram stain is identified, the tester has a wide array of differentiating tests at their disposal. Based on the results from these tests, and the numerous others that are available, one can accurately establish the identity of an unknown bacterium.

The identification of unknown bacteria are used in labs by microbiologist for the purpose of studying bacteria that cause diseases, diagnosis of diseases and for treatment of the diseases. Therefore, it is important for them to effectively identify the right bacteria that is causing harm to people, animal or environment and treat it successfully. Hence, discussing the different type of test used and the processes of how one could identify the unknown bacteria.

The test tube was labeled with the bacteria identifying number. The cap on test tube was removed, and the lip of the test tube was flamed. Next, the Bunsen burner was used to sterilize the inoculating loop. Then, bacteria were picked up from the working plate with the loop and the agar was inoculated. The loop was then re-flamed. Finally, the plate was placed in the 37˚C incubator and left to sit for 48 hours and any changes in color were observed. A negative result appears green, and a positive result appears blue. This is because it tests for the organism’s ability to use citrate as its sole source of carbon, and if it does then it produces ammonia and ammonium hydroxide which make the medium basic, changing the green agar blue (Leboffe & Pierce,

The oxidation fermentation test was used to differentiate if the organism utilizes lactose, mannitol, glucose and citrate aerobically (oxidation) or anaerobically (fermentation). A methyl red test was performed to determine if the organism carried out mixed-acid fermentation when supplied glucose. A Voges-Proskauer test was performed to evaluate if the unknown was able to ferment glucose into butanediol. A citrate test was performed to determine if the unknown organism was able to break down citrate into ammonia. An oxidase test was then performed to determine if the unknown culture was oxidase positive or negate.

I performed a gram stain on my unknown 7. It appeared to be purple and round shaped. I came to the conclusion that my bacterium is gram-positive, cocci. A gram stain is a differential test. It allows recognizing the difference between two cell walls structures. In a gram-positive cell wall contains a thick peptidoglycan layer covering the plasma membrane (2). The purple staining is retained around the cell

One MR-VP broth was the control tube, which was not inoculated at all. The other MR-VP broth was the treatment broth that was inoculated with the colony 2 stock culture at 30 degrees Celsius for five days. The reason for completing this test first after gram staining was because it was a 5-day test that could be left over the weekend. There were no variables, it was either negative or positive, and there would be no misleading guesses. The plan I had going into this was to perform all the tests possible with no variables or not determined results to reduce the potential for errors. On Monday I will get the control and treatment inoculated test tubes back to determine wither the unknown organism is positive or negative based off of the color in which the broth will

Whenever there is an unknown disease caused by microorganisms, tests are usually made in order to identify the organism causing the disease. There are several tests that need to be made and they include tests such as performing a gram stain, streaking a plate to isolate colonies, inoculating a broth culture, inoculating API strip, and performing oxidase and catalase tests. Having knowledge on how to identify these tests are of high importance in the medical field so it would be to the advantage of those individuals who know how to examine microorganisms and be able to identify it by correctly performing tests on organisms.

The identification of bacteria is important in studies related to diseases. Identifying how bacteria responds to certain biochemical tests can give the experimenter valuable information such as the metabolic activity and morphology of an unknown bacteria. With this information, the scientific community can identify a bacterium and respond appropriately to disease outbreaks This information also helps doctors provide the appropriate diagnosis for patients afflicted with a bacterial disease.

For the Gram stain, I used a microscope slide, wash bottle of water, clothespin, and the reagents crystal violet, Gram’s iodine, ethyl alcohol, and safranin to observe whether the organism was Gram negative or positve. The method was placing and heat fixing a loopful of the unknown organism on the slide. The organisms were then stained with crystal violet for a minute, rinsed off, flooded with iodine for a minute, rinsed off, decolorized with alcohol for 30 seconds, and then finally stained with the counter-stain, safranin, for a minute. I streaked the unknown onto a TSA plate and incubated at 35 C. With a pure colony, we performed a second gram stain procedure and inoculated it into a TSA slant. I used BCP Lactose broth and a loop to perform a BCP Lactose test. The broth was inoculated with a loopful of the unknown and incubated

(4 pts) 3. You are handed two slides that have been prepared with your organism. One has been properly Gram stained and the other has been properly Acid-Fast stained.

Each test performed in the lab on theses unknown bacteria have a very specific significance. With each test performed correctly the lab officer is able to move closer towards a proper identification of the unknown bacteria. After performing Gram staining and deciphering if the unknown was gram positive or negative the lab officer was then able to proceed to the next step of identification. The gram positive unknown’s reaction to the catalase test informs the tester of the Genus theyre working with. This indicates which tests to perform next. The MacConkey agar is a selective medium that only allows the growth of gram positive bacteria confirming the results received from the gram staining procedure. The NaCl growth test indicates whether or not the organism is able to

Half of each tube’s contents are poured into a new test tube each respectively after the tubes are incubated for 1 hour. One set of tubes is tested for:

Image 5. A negative Citrate test on the left and a positive result on the right.