Gram Negative Or Gram Positive Based On It's Color And Shape

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

The Gram stain was used to determine if the bacteria was gram positive or negative. A negative test shows a pink color and a positive test is a purple color. When a bacterium is negative it is because it has an outer membrane and a thin layer of peptidoglycan that is much harder to stain. A positive bacterium has a thick layer of peptidoglycan and no outer membrane that can be penetrated by crystal violet.

2) Record the shape of the bacteria, the arrangement of the bacteria, and the gram staining characteristics.

This experiment was given to us to utilized previous knowledge learned throughout the semester to identify a gram negative unknown bacterium. We had to first learn the difference between a gram negative and a gram positive organism. We started off with doing gram stains to determine whether it was positive or negative. Based on the gram stains, a gram positive stains purple and a gram negative stains pink. A gram positive stains purple because the cell walls is made of a thick peptidoglycan layer and doesn’t

The drops of crystal violets, approximately 15 drops, were flooded until the smear were all covered and then allowing resting for one to two minutes. After two minutes, the slide was titled over the sink and washed off, with the distilled water bottle, by aiming the stream of water above the smear. The specimen appeared blue-violet when observed with the naked eye. The drops of Gram’s iodine were applied on the slide until covered and then allowed to react for one minute or more. After the time elapsed, the slide was rinsed again with distilled water following immediate drops of Gram stain decolorizer added one drop at a time.

When performing a gram stain you failed to wash your slides with ethanol, but all the other steps were completed correctly. Answer the questions below based on what you would see after completing the procedure incorrectly. (3pts)

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

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

(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.

By using gram staining methods, catalase, oxidase, DNase, hemolysis, antibiotic sensitivity, citrate, and SIM tests, one can determine what bacteria species are present. These tests are extremely important because they can indicate important characteristics of the bacteria species present. In order for these tests to provide conclusive results, positive and negative controls must be used for each test. The gram stain is a very important test that is the first step in separating the bacteria. Knowing if the bacteria is gram positive or gram negative can be highly sufficient for further testing. Some tests will provide better results on gram positive than gram negative and vise versa. The citrate and SIM tests were used for initial identification of the gram-negative bacteria. The indole test is part of the SIM test that determines if tryptophan is breaks down into indole and pyruvate. The citrate test determines if the bacteria can use citrate. For gram positive, further tests were required. Oxidase, hemolysis, DNase, and antibiotic sensitivity were all used in determining the gram-positive bacteria. The oxidase test determines if the

First a thin smear of bacteria, placed on a clean slide, covered with crystal violet stain for 1 minute. Next, completion of a wash and then an iodine solution is added to the smear to enhance staining for 1 minute. Being rinsed again in water and then 95% ethanol for one minute. Washing the violet stain off will differentiate between the two types of bacteria. If the bacteria retains the violet color, it means they are gram positive, while the others without strain are gram negative. The cell wall of the gram positive bacteria has a thick outer layer made up of peptidoglycan and the cell plasma membrane causing the stain to stick. The gram negative bacteria has an outer layer made up of lipopolysaccharides and proteins and then a thin layer of peptidoglycan and the plasma membrane. The lipids from the outer membrane dissolve with the ethanol wash, causing them to lose the violet stain. After the ethanol wash, the smear is washed in water and stained with safranin for one minute allowing for the gram negative bacteria visualization. Before the

These materials varied depending on which flowchart the gram stain led to. For the gram stain, the materials required included: microscope slide, crystal violet, distilled water, Gram’s iodine, 70% ethyl alcohol, safranin, bibulous paper, slide holder, and a Bunsen burner. The gram stain was the first test that was performed for this experiment, after a subculture of the bacteria was made. Gram stains are performed by first heat fixing the sample, the covering the surface with crystal violet (20 sec), washing with distilled water for 5 secs, covering the surface with iodine for one minute, decolorizing with ethyl alcohol (15 secs), wash with distilled water (20 secs), counterstain by covering smear with safranin (1 min), wash with distilled water (5 secs), blot in bibulous paper, and then view under a microscope. After determining that we had a gram positive cocci bacteria, we performed a catalase test which required adding drops of 3% hydrogen peroxides to our sample.

Gram positive bacteria have thick peptidoglycan layer, when alcohol (declorizer) is added it dehydrates peptidoglycan thus causes shrinking and tightening of peptidoglycan. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost.Gram positive bacteria have thick peptidoglycan layer, when alcohol (declorizer) is added it dehydrates peptidoglycan thus causes shrinking and tightening of peptidoglycan. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.

Finally, the slide is washed again and stained with a counterstain, such as safranin. The counterstain will dye the gram negative cell and will not effect the gram positive cell.

Isolation, gram staining and identification of the micro-organism is a basic diagnostic tool in clinical as well as in research. This method is useful in diagnosis and treatment of diseases. The gram staining is simple, least expensive and is rapid. It is useful in counting of total bacteria. Because of the staining, the gram positive and negative bacteria can be viewed under the microscope. The shape, size can be determined. The gram positive bacteria may lose the stain easily. The retention of stain depends on the age of the cell. Old cultures get readily decolorized. Yet, this method is feasible method for diagnosis of the minute