Identification of Salmonella Typhimurium and Staphylococcus Aureus Using Microbial Metabolic Testing

Identification of Salmonella typhimurium and Staphylococcus aureus using microbial metabolic testing.

Anthony Evans

Abstract:

        Microbial metabolic test are often used in the process of identifying unknown bacteria. To determine the identity of unknown mixed bacteria culture 5, master plates were created to so that isolates could be used in testing. Initially Gram stain tests were used to distinguish the to bacteria from one another. Multiple metabolic test followed, resulting in the identification of one unknown as Staphylococcus aureus based on a positive Gram stain, a positive Glucose test, a positive Nitrate test, and a positive Dnase Test. The other bacteria in the culture was identified as Salmonella typhimurium based on a negative Gram stain, a negative Sucrose tets, a positive Glucose test, and a negative β-galactosidase test.

Introduction:

        Staphylococcus aureus is an immotile, spherical shaped, Gram positive bacteria. S. aureus’ Gram positive status indicates that it has a thick peptidoglycan layer in its cell wall that is used for protection from the environment. S. aureus strains are typically found in cluster. The reason for this is that this organism reproduces asexually by budding, but the two daughter cells never fully detach from one another.(Bierne, 2012) The optimum growth temperature for this organism is 37 degrees Celsius, and it will grow between 10 and 49 degrees Celsius.

        S. aureus is a facultative anaerobe that utilizes organic carbons, from sources such as glucose, to obtain its energy. The pH range that this organism can live in falls between 4.8 and 8.0. S.aureus can live in environments with up to 20% salinity, and is characterized by its ability to secrete catalase, which means that this organism mediates the transformation of hydrogen peroxide to water and free oxygen.(Humphreys, 2012) It is also known to secrete DNase, an enzyme that breaks down DNA so that the organism can use the carbon for energy. This can be a severe issue in the cases in which S.aureus enters another organism.(Menichetti, 2012)

        The most common environment for these organisms is the human skin and in the nose, where the organisms are typically harmless. When the tissues that S.aureus usually live in are breached, the organism has the opportunity to infect the host, which is detrimental because its optimum growth temperature is the same as the body’s resting temperature.(Grosset-Janin, 2012) This infection can be minor, such as a case of acne, or major and cause diseases such as pneumonia. The only way to be rid of this organism is through the use of antibiotics.(Humphreys, 2012)

        Salmonella typhimurium is a motile, Gram negative bacteria. S.typhirium’s Gram negative classification indicates that the organism has an outer membrane made of lipopolysaccarides that is utilized in protecting it from the environment.(Garai, 2012) The rod shape of this organism results from its reproductive process through asexual binary fission. The optimum growth temperature for S.typhimurium is 37 degrees Celsius, but it can live in temperatures that range from 15 to 45 degrees Celsius.(Garai, 2012)

        S.typhimurium is classified as a facultative anaerobe, which indicates that it can live in oxygenic and anoxygenic environments. In the area of energy production, S.typhimurium prefers organic carbon as its carbon source.(Schmitz, 2006) This organism can be found in pH environments that fall between four and nine, and can also survive in up to 20 percent salinity concentration.(Saldana, 2012) A distinguishing factor of S.typhimurium is that it has the ability to reduce nitrites to nitrates. It can also utilize citrate as its sole carbon source.

        In terms of pathogenicity, this organism can be very dangerous. The typical environment for this organism is the lumen of the intestinal tract. The toxicity of this organism is primarily due to its outer membrane, which is meant to protect the organism from the environment.(Shirron, 2009) S.typhimurium is known to cause gastroenteritis and typhoid fever in humans. It can also affect other animals, such as mice. Salmonella is most widely known for being a food borne illness, but this organism will be denatured at temperatures above 70 degrees Celsius, so it is important to cook ones food.(Saldana, 2012)

Materials and Methods:

        First two TSA plates were obtained and labeled with the experimenters initials the date and the title master plate. Next a mixed culture labeled unknown number 5 was obtained. Unknown mixed culture 5 contained two of the following fourteen organisms: Alcaligenes faecalis, Bacillus subtilis, Bacillus thuringiensis, Citrobacter fruendii, Enterobacter aerogenes, Enterococcus faecalis, Eschericia coli, Klebsiella pneumonia, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens, Staphylococcus aureus, and Staphylococcus epidermidis. A Bunsen burner was lit, and an inoculating loop was held in the flame. The inoculating loop was then allowed to cool, and was then dipped into the mixed culture. The sample then was Quadrant streaked onto both TSA plates. Quadrant streak protocol can be found on page 9 of Anthony Evans lab notebook. The 2 master plates were then incubated at 37⁰C for 22.5 hours, and then moved into a 4⁰C fridge until they were needed for experimentation.

        Once both master plates presented individual colonies, one organism was deemed organism A and another organism B. First organism A was bacterial smeared (lab notebook, 5), and then that procedure was repeated for organism B. Next a Gram stain (lab notebook, 6) was performed on organism A, and repeated for organism B. A positive Gram stain resulted in a purple stained organism. A negative gram stain resulted in a pink stained organism. Based on these test results, a flow chart of metabolic test to determine the identity of the unknown was created.

        In the case of organism A, the Sucrose test was performed (lab notebook, 25), and the results were obtained. A positive Sucrose test resulted in a yellow solution in the Sucrose tube, with a possibility of a bubble in the Durham tube. A negative Sucrose test resulted in a red solution in the Sucrose tube. Based on the results obtained by the Sucrose test, the next test performed on organism A was the Glucose test (lab notebook, 25). A positive Glucose test resulted in a yellow solution in the Glucose tube, with the possibility of a bubble in Durham tube. A negative Glucose test resulted in a red solution in the Glucose tube. The Glucose test results then led to the performance of the β-galactosidase test (lab notebook, 25). A positive β-galactosidase tube resulted in a yellow solution upon the addition of sodium bicarbonate. A negative β-galactosidase test resulted in no color change in the β-galactosidase tube. The results of the β-galactosidase test resulted in the identification of the unknown organism.