How Gram Staining Is Negative Or Positive, It 's Morphology, And Arrangement Of The Cells

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 colonies were smooth, translucent, and had a white brownish color. The Gram stain resulted in Gram positive cocci. After the Gram stain was completed, the bacteria were streaked on a Mannitol-Salt Agar plate and a Catalase test was performed. After these test were completed a Phenol Red Dextrose Fermentation tube was inoculated, and a SIM Tube inoculated.

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

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 next step of the project included preparing a Gram stain to discover the cell shape, arrangement, and if the bacteria is gram positive or

1) Apply the stain to your first unknown slide and examine it under the microscope.

Then we carefully submerged the nitrocellulose sheet on the transfer buffer for about 5 minutes, and set up the transfer apparatus in the method shown in the lab outline week 2 step 3-6. Afterwards we took apart the transfer apparatus and detached the nitrocellulose sheet. Then we put the nitrocellulose sheet for 5 minutes into the blocking buffer, and we left nitrocellulose sheet in a container till week 3. The second part of week 2 was staining half our PAGE gel from week 1. Which consisted of pouring deionized water and use the shaker machine for 5 minute intervals, and repeating the the process three times. Then the Fresh Coomassie stain was added to the gel and was rinsed (UFV BIO 202 Lab,

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.

The chart below shows the biochemical tests of the gram stain below. Test performed Purpose Materials used Observation Results Gram stain test Gram reaction to specimen Crystal violet, iodine, alcohol safranin Pink/purple colored Gram positive cocci/ gram negative rods After the gram stain showed the specimen as Gram positive cocci, and Gram negative rods, more tests are necessary. Test performed Results Enterotube II Gram negative rod: Citrobacter

Gram staining is a technique used to determine if the bacteria is Gram positive or Gram- negative. Gram staining procedure uses crystal violet stain, iodine moderator, alcohol decolorizer and safarin counter stain. In Gram- negative bacteria the primary stain will be washed out with the decolorizer and it will be stained with the counterstain. Whereas in Gram-positive bacteria the primary stain will not leave the cell wall. This difference comes from difference in the structure of the cell wall that retains the stain.

Figure #1 This picture show the gram stain of unknown #3. The image is under 1000x magnification using oil immersion technique. Gram (-)

Escherichia is a genus of aerobic gram-negative rod-shaped bacteria of the family Enterobacteriaceae that form acid and gas on many carbohydrates, such as dextrose and lactose, but not acetone, which include occasional pathogenic forms, including some strains of E. coli which are normally present in the human intestine as well as other forms which typically occur in soil and water (Webster). Escherichia coli is a gram-negative bacilli that rarely varies in shape and size and when stained often resemble safety pins because the ends of some bacilli stain more densely than does the middle; which is a characteristic called bipolar staining which is common in enteric gram-negative bacilli (ASM). Gram negative cells have a thin cell wall layer and will stain red to pink. The staining process is the same as Gram positive, requiring four steps: applying a primary stain, adding a mordant, then rapid decolorization and completing with a counter stain. Applying the alcohol for decolorization dissolves the outer membrane and leaves small holes in the thin peptidoglycan layer through which the crystal violet-iodine diffuse. The gram-negative bacteria is colorless after the decolorization; therefore adding safranin

Comparing the cellular morphology of table 2 to their respective pure culture in table 3. B. cereus presented with a white colony, round colony morphology, smooth margins and flat elevation in both pure culture and isolated culture; E. coli presented with a Yellow colony, round colony morphology, smooth margins, and flat elevation; S. aureus presented with a White colony; round colony morphology, smooth margins, and raised elevation.

The Gram’s staining procedure is still used today and is the method that forms the basis for identifying bacteria. In order to perform this procedure, you will need a Bunsen Burner or a Tirrill burner, alcohol- cleaned microscope slide, and water. You will also need The following reagents: Crystal violet, Gram’s iodine solution, 95% alcohol, and safranin. These stain reagents are important because they are used to determine the Gram reaction for microorganisms’ identification. Crystal violet will stain the bacterial cell, and Iodine will bind the stain. The alcohol differentiates bacteria retaining or not the crystal violet within the cell wall, and the safranin will be used as a counterstain to stain the bacteria. You are now ready to start you process.

Another purpose of this experiment is to stress the importance of knowing the identity of a microorganism. Knowing the species of microorganism present in a sample provides a