Gram Staining Procedure

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

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

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 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.

The test showed a clear zone around the colonies thus determining that the bacteria can break down starch meaning it was a positive result. With a positive starch hydrolysis result the next task was to perform a VP. The results from this test came back negative because there was no color change in the broth. My next step was to check for a swollen cell. I found that the cell was swollen looking from the spore stain slide.

The ways we can examine organism is by using a microscope. We’ve used a bright-Field microscopy that shines light through a specimen (1). The organism is then magnified as light refracts through the four objective lens 4X, 10X, 40X, and 100X found on the revolving nosepiece. It is then further magnified as we look through the ocular lens at 10X. By multiplying the objective lenses and the ocular lens gives a total magnification of 40X, 100X, 400X and 1000X. Once reaching objective lens 100X oil needs to be put on the slide. By using oil, it increases the lens magnification because oil refracts light just like the lens in the microscope and it has the same refractive index (1).

Picture 3. This picture shows the stock cultures for both colony 1 and colony 2. The left test tube shows the growth from colony 1 on the TSA slant. The right test tube shows the growth from colony 2 on the TSA slant.

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.

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.

Purpose: The MSA streak plate test is used to differentiate between different microbes by their reaction on the specific media. The remaining microbes are Staphylococcus aureus and Staphylococcus epidermidis. On the MSA plate, Staphylococcus aureus turns the MSA plate yellow. Staphylococcus epidermidis turns the MSA plate a darker red (Harley, 2014).

In this test, you use an agar, in an incubator, which acts as a medium to allow the growth of the sample swab you took. This process usually takes about 2 to 3 days. It will show you what type of infection are present, if any, and also can provide valuable information about antibiotics sensitivity.

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: