The Unknown Bacteria Plate Chosen

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 Unknown Bacteria 36/Bacteria # 2 on a TSA plate was examined by the naked eye and under a dissecting microscope. Bacteria # 2 was approximately 3 - 4 mm in diameter. They were circular in form with an entire margin and a flat elevation. The colonies were rough (granular), translucent, and white brownish color with black granules. The Gram stain resulted in a Gram negative rod. After the Gram stain was completed, the bacteria were streaked on an Eosin -Methylene Blue Agar plate and an Enterotube II was 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

This test is used to detect if the bacteria contains any deoxyribonuclease activity. Because no color change was observed from blue to clear my unknown bacteria displayed a negative result.

Micrococcus luteus was first isolated and discovered in 1929 by Alexander Fleming. Is a gram positive bacteria that is commonly found on human skin, water, soil and dust. Since it is an obligate aerobe and thrives in the presence of oxygen, it is also found in the human respiratory tract. It takes the shape of cocci and is often clumped irregularly, in tetrads, or groups of four. It is also non-motile and does not form endospores. Although it has a specific optimal temperature range of about 37 to 45 degrees Celsius, it is halophilic. Micrococcus luteus is also catalase positive, meaning that it produces an enzyme that protects it from any harmful oxygen metabolism by-products (Bergey, 1923).

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

Hypothesis: If the bacteria turns out pink/red then the gram test is negative. Null Hypothesis: If the bacteria turns out pink/red then the gram test is not negative. Purpose: Identify the three morphological types of bacteria and use the Gram’s test to examine the bacteria infection by the stain technique. Differ between plant and animal cells and the different cellular components of eukaryotic cell.

This staining procedure was developed in the 1882, by a Danish bacteriologist, Hans Christian Gram. This technique identifies differences in a bacteria’s cell wall. It utilizes crystal violet (primary stain), Gram’s iodine (mordant), and safranin (counterstain). Most bacteria fall into two categories, Gram positive or gram negative. Gram positive bacteria have a thick layer of peptidoglycan, and techoic acids in their cell walls. This thick peptidoglycan layer retains the crystal violet stain, and displays the cells as a purple color under the microscope. Gram negative bacteria cell walls have significant differences from gram positive bacteria. Gram negative cells have a thin peptidoglycan layer, and do not have techie acids. They also have an outer membrane that is similar to the phospholipid bilayer of a cell membrance. These differences allow the crystal violet stain to rinse away from them, then they retain the safranin stain and appear reddish/pink under the microscope. Although it is very useful, it is not the only tooled needed to identify a bacteria. Some bacteria, are gram-variable, displaying both positive and negative reactions. Other bacteria, such as Mycoplasmas, do not have a cell wall, rendering this test ineffective. Finally, it does not provide enough information alone to identify a

In gram positive, the cell wall is mostly made up of peptidoglycan; therefore, it stains purple due to the absorption of the primary stain. In gram negative, the cell wall has a thin layer of peptidoglycan and a lipopolysaccharide layer allowing the counter stain to have an effect causing a pink color. To gram stain a bacterium the steps of simple staining are used with a mordant like crystal violet, however it is not dried off until the end of the process. The slide must be rinsed between every step of the gram staining process after crystal violet is used. A mordant is then placed on the slide for one minute to strengthen the color of the stain. Alcohol or acetone is used to decolorize the stain allow it to sit for ten to fifteen seconds. A counterstained is used to add color back to the colorless effect of the decolorizer. The safranin must stay on a slide for one minute before the final rinse, then dried on blotting paper to be seen under a microscope. It can finally be distinguished as a gram positive or gram

The Gram stain is a chemical preparation which allows bacteria to be classified into Gram positive and Gram negative by their ability to retain the stain. Bacteria which keep the purple stain are Gram positive and those that loose the stain or turn a pink colour are Gram negative. This procedure is based on the differences in the cell walls structure. The bacterial cell walls will all contain peptidoglycan but the gram positive bacteria the peptidoglycan layer will be thicker. Gram negative, as well as having a thinner peptidoglycan layer, will also have lipopolysaccharides covering the peptidoglycan layer causing less of the stain to be retained.

Bacteria can be divided into two types of species, gram positive bacteria and gram negative bacteria. To determine whether a certain type of bacteria was classified tests were conducted by Hans Christian Gram; who discovered the differences within the cell walls of the bacteria. Gram positive bacteria have a thick wall which consists of the protein peptidoglycan. This is opposite to gram negative bacteria which has a thin cell wall, which fold over each other (Wells, 2017). They retain a much thinner peptidoglycan wall between two membranes. The membrane surrounding the cytoplasm and the outer membrane. (Wikipedia, 2017). Both gram positive and gram negative bacteria both stain, gram positive retains violet dye. Gram positive bacteria don't retain the dye and turn red or pink (Diffen, 2017). Due the difference in cell wall thickness gram negative bacteria is harder to inhibit, compared to gram positive bacteria.

The gram negative and gram positive bacterias are comparable in many different aspects. Both the gram negative and positive are located on the inner most plasma membrane, however the gram positive attains a thick peptidoglycan cell wall while the gram negative has a think cell wall. The outer membrane for the gram positive bacteria is absent, however the the gram negative has an outer membrane made up of lipo polysaccharide and contains a present protein. The gram positive is also tougher to treat with antibiotics and stains a purple/violet while the gram negative stains a red/pink. The gram stains are simple methods used to identify types of infectious bacteria. Teichoic acid is present in the gram positive bacteria but absent in the negative bacteria. Lipid content is also very low in the gram positive in comparison to a fair amount of lipid in the outer membrane of the gram negative bacteria.

Gram Stain: The Gram Stain test was performed on our unknown bacteria and the results were negative. Meaning that the cell wall of the bacteria did not retain the purple dye, therefore it classified it as Gram-negative (-). After observing the slide under the microscope, (Figure 1 below) our bacteria’s morphology was identified as rod-shaped/bacillus.

Classified as gram-positive or gram-negative based on the thickness of cell wall. Gram-positive bacteria stains purple under the dye while Gram-negative bacteria stains pink

A complex between crystal violet and iodine (CV-I) is formed within the cell. The structure that determines the Gram reaction is the cell wall structure and not that shape. Bacillus cereus and Staphylococcus aureus are stained purple in the Gram staining as they have a high amount of peptidoglycan which forms the outer layer of the cell. This thick peptidoglycan layer is able to trap the purple CV-I complex even after alcohol treatment. Escherichia coli is stained pink in the Gram staining and it is a Gram-negative bacteria. Gram-negative bacterias usually have a thin peptidoglycan layer compared to Gram-positive bacterias. The peptidoglycan layer is located between the plasma membrane and an outer membrane containing lipopolysaccharide and this outer layer is dissolved during the alcohol treatment which results in a loss of the CV-I complex, hence the pink safranin counterstain is trapped by the peptidoglycan layer (ASM, 2004).