Alexander Flemming: The Antibacterial Enzyme

This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

Crystal violet stains the cell of the bacteria and is placed over the heat fixed slide for one minute then rinsed with H2O. The mordant that binds the stain is gram’s iodine and is recommended to be present on the slide for one minute. Alcohol is used as the decolorizer and is dripped over the recently rinsed slide until the slide runs slightly blue. If the bacterium retains the crystal violet it is due to the thicker peptidoglycan in the cell wall (gram-positive). The bacteria will then be stained with the counterstain safranin for 45 seconds. When crystal violet is not retained in the cell wall of the bacterium it will stain pink (gram-negative). A gram stain was immediately performed with the recently inoculated streak plate. An inoculating loop was used to gather some of the bacteria from an isolated colony. The loop was placed on a new slide and the transferred bacterium to the slide. The slide was dried, heat-fixed, and the gram stain process just described was completed. After microscopic analysis, it was determined the first unknown bacteria was a gram-positive cocci. The second unknown was determined to be gram-negative cocci. With this information a set group of test decided and performed based on the Unknown Bacteria Flow Chart as provided in the lab manual (Pg.) these test and the results are shown in record on the following pages. These are separated for the gram positive and the gram negative

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).

Enzymes are a key aspect in our everyday life and are a key to sustaining life. They are biological catalysts that help speed up the rate of reactions. They do this by lowering the activation energy of chemical reactions (Biology Department, 2011).

A microbiologist named Vincent Fischetti was intrigued with phages that he showed how lysins could potentially help mice overcome throat infection. Here, Fischetti came into conclusion that lytics could be use in a therapeutic way. According to Fischetti, the enzymes can be frozen or dried for a long time and still keep their activity.---with the example of the

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.

After the multitude of tests performed, it was determined that the bacterial unknown was Staphylococcus aureus. The gram stain slide was positive. The morphology and arrangement was grape like cocci clusters. On the glucose fermentation test the bacterial unknown tested positive for acid and negative for gas. The oxidase test was negative. The bacterial unknown tested positive for catalase. In the litmus milk medium a K or alkaline reaction was observed. It tested negative for urea hydrolysis. The bacterial unknown tested positive for nitrite. On the Kigler 's iron agar media it tested negative for gas, positive for glucose, positive for lactose, and negative for hydrogen sulfide. On the SIM medium media it tested negative for indole, negative for hydrogen sulfide, and negative for motility. The bacterial unknown tested negative for methyl red. It tested negative for Voges-Proskauer. It tested negative for citrate. It tested

AIM – The aim of the experiment is to determine the relative effectiveness of several anti-microbial substances on developing pathogens. (E. coli)

The unknown bacteria plate chosen was plate #2. It was identified to be Micrococcus luteus. It is a gram positive, Coccus bacteria that is commonly found in dust, water, soil, and the air. M. luteus also thrives in the human mouth and upper respiratory tract. Sir Alexander Fleming discovered it in 1928 before he identified penicillin. It is part of the normal flora on human skin as well as other mammals. Since it is part of normal flora it is normally not pathogenic, but can become opportunistic in an immune-deficient person. It has been known to cause septic shock, UTI’s, and even pneumonia. Micrococcus luteus is both urease and catalase positive. It does not utilize tryptophan for indole production. It is a facultative anaerobe. Mobility is not present for this bacterium. Starch is also not hydrolyzed and oxidase is not present.

In 1918, Alexander Fleming returned to St. Mary’s when the war had ended. Later in 1921, Alexander Fleming had discovered an important bacteriolytic substance which he named Lysozyme. Fleming had thought

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

Cell wall of bacteria contains rigid and complex structure which maintains the cytoplasm of cell gives structural integrity and prevents osmotic lysis. Both Garm positive and Gram negative bacteria contain a carbohydrate complex structure called Peptidoglycan also called Muerin. The layer is thicker in Gram positive bacteria whereas in Gram negative bacteria the peptidoglyacan layer is thinner; moreover this is protected by an outer membrane layer called Lipopolysaccharide (LPS). The Peptidoglycan consists of two monomers called N-Acetylglucosamine(NAG) and N-Acetylmuramic acid(NAM). These two carbohydrate containing monomers are linked by glycosidic linkages. A pentapeptide bond attaches to NAM which gives crosslinkage and rigidity to the layer.

The zone of inhibition, the circular area lacking bacterial growth around the test disk, was used to study the effectiveness of the antibacterial solution. The compounds used in the experiment were 10% solutions of Febreze, Clorox, Anti-bacterial soap, moisturizing soap, Bactine, and Hydrogen peroxide.

I inoculated an Eosin-Methylene Blue (EMB) agar plate and a MacConkey agar plate using Alderson’s streak plate technique (Alderson, 2015, pp. 64-65). Next, using Alderson’s procedure for aseptic techniques for inoculation (Alderson, 2015, p. 53) along with the procedures for each individual test I conducted the biochemical tests on 1 lactose Durham broth (Alderson, 2015, p. 267), 1 glucose Durham broth (Alderson, 2015, p. 267), 1 TSIA slant (Alderson, 2015, p. 267), 1 SIM agar (Alderson, 2015, p. 268), 2 MRVP broths (Alderson, 2015, p. 268) and 1 citrate agar (Alderson, 2015, p. 268). I also tested my microbe’s ability to produce oxidase (Alderson, 2015, p. 267). Finally, I tested for the presence of catalase (Aryal, 2015, para.