The process of identifying an unknown bacteria in a sample is an essential procedure that allows for the effective treatment and prevention of various disorders (Kellenberger, 2001). Furthermore, bacteria are the leading causative agent of food poisoning in most homes and food processing industries (Kellenberger, 2001). This reality underlies the importance of correctly identifying unknown bacteria. It requires a systematic process not dissimilar from a differential diagnosis often conducted by physicians. In a differential diagnosis, a proper diagnosis is systematically arrived at by eliminating other competing diagnoses based on the available evidence (Cook & Decary, 2020).
In this microbiology experiment, a similar process was undertaken. Specifically, an unknown bacterium was presented and required identification through a series of systematic tests and techniques. This was aided by comparing the unknown species to different potential bacteria, as described in Bergey’s Manual, among other sources. This experimental process allowed for the eventual identification of Staphylococcus Aureus.
Materials and Methods
In this section, the procedure used to identify the unknown bacterium will be outlined. As will be described, elimination was Staphylococcus Aureus the main strategy used to identify organisms when conducting such tests. Moving from one test to another eventually narrows the scope until a single bacteria is found.
For correct identification of the unknown bacteria in the laboratory, the gram stain test was done by placing a sample of the solution on a slide and passing it over a heat source. The primary stain was added to the slide sample and incubated for approximately one minute. Also, add gram’s iodine to the sample and rinse with acetone for about three seconds the finally rinse with water. The addition of a secondary stain, safranin, and incubation for about a minute helps to determine the gram of the bacteria.
Secondly, a mannitol salt agar medium consisting of 7.5% sodium chloride in a test tube with the unknown bacterium helps to determine the amount of oxygen required for the growth of the bacteria. The main aim of this medium is to act as a selective compound to favor the growth of various bacteria while inhibiting that of others. The mannitol salt and DNAse test require HCL. The test tube should be observed for growth and pigmentation.
Consequently, hemolysis is the partial or complete breakdown of red blood cells y releasing hemoglobin. The red color from the surrouding medium is cleared forming a green-yellow zone. Therefore, the addition of CAN agar, a type of blood agar, helps in the hemolysis of the solution. Since several bacteria utilize citrate, various colonies of the gram-positive bacteria tubes were incubated at 35 degrees for about four hours and later observed for clot formation. In addition, the coagulase test involved the use of urea broth media for the identification of the genus.
For the catalase test, growth from a TSA slant on a glass slide is used and illuminated properly to ensure proper observation in case there is the production of bubbles. Lastly, biochemical characterization is carried out by the use of carbohydrate fermentation tests such as glucose, sucrose broth, mannitol broth, and lactose broth with the addition of phenol-red as a pH indicator.
When carrying out the gram stain test, the sample on the slide turned purple suggesting that the bacterium is gram-positive. This means that the cell wall is made up of a thick peptidoglycan layer. The bacteria were in clusters of cocci-shaped grape-like structures dividing in more than one plane. In addition, they had large, round, and golden-yellow colonies. On carrying out the spore stain test with safranin solution, the inside and outside of the cells remained red indicating the absence of endospores.
For the capsule test, the solution manipulated a basic stain portraying the absence of capsules. Consequently, the cells were non-motile, made ATP and grow in the absence of oxygen (Pollitt and Diggle 2015). The cells grew in the concentrated sodium chloride media indicating that they can survive in media with less or no oxygen. This means they are facultative anaerobes. The tubes used for the citrate test did not form clots, meaning the bacterium does not contain citrates.
When the hemolysis test was carried out, there was total clearing around the colony caused by the breakdown of red blood cells in enzymes suggesting that the result was beta hemolysis. Since urease is important for pH balance and viability in urea-rich conditions under weak acid stress, it was incorporated in determining the unknown bacteria (Zhou et al. 2019). The result showed that the cells produced a weak acid. There was production of several bubbles in the catalase test, which indicates that a catalase activity took place. The incorporation of the carbohydrate fermentation test on different sugars led to the change of the phenol-red indicator to yellow for glucose, lactose, sucrose, and mannitol. This is an indication that the bacteria trigger fermentation of the four sugars.
The gram-positive cells were divided into the shapes they formed and the sample collected formed cocci shapes with grape-like structures. After the spore formation test, the test sample did not form endospores, therefore, the flowchart reads non-spore-former. Additional capsule tests performed led to a conclusion of no capsule formation for the sample bacteria. In the consideration of the motility of the bacteria, the outcome was a non-motile bacterium.
Consequently, the bacteria increased in size in the sodium chloride solution meaning it did not require oxygen for growth. After the addition of CAN agar, there was total clearing around the colony meaning the bacteria were beta-hemolytic. Lastly, carbohydrate fermentation of mannitol, glucose, lactose, and sucrose all led to an acidic solution and production of a gas meaning that the bacteria triggers fermentation and its genus is Staphylococcus aureus.
The Medical Significance of Staphylococcus Aureus
Staphylococcus aureus is one of the essential bacteria that cause diseases in human beings. Staphylococcal infections are suppurative, producing abscesses filled with pus and destroyed leukocytes surrounded by necrotic tissues (Taylor 2020). It is the primary cause of skin and soft tissue infections and one of the most severe known to cause serious disorders such as blood infections, pneumonia, and joint infections. In addition, S. aureus causes food poisoning when contaminated food is ingested into the system with an enterotoxin-producing strain.
Various skin infections caused by the bacteria heal without medical intervention, while others require incision and drainage of the site of infection and administration of antibiotics by a medical practitioner. Serious infections such as pneumonia require medical attention and treatment with intravenous antibiotics. Staphylococcal pneumonia is now a more frequent infection caused by the bacteria globally. The breach of the skin and mucosal membrane barriers leads to the bacteria gaining access to the underlying tissues and the blood leading to infections. People who have weak immune systems are vulnerable to infection by the bacteria.
Problems and Future Suggestions
The major problem associated with this project was that the bacteria are resistant to several reagents used and the experiment required additional reagents to help determine the specific type of bacteria present. Several future suggestions to ease carrying out such a project are additional knowledge on the specific reagents that can be used to determine specific bacteria. In addition, more research should be carried out on the resistance of bacteria to specific reagents and how it can be reduced to enhance the collection of more accurate results (Sutejo et al. 2017).
Cook, C.E., & Decary, S. (2020). Higher order thinking about differential diagnosis. Brazilian Journal of Physical Therapy, 24(1), 1-7. doi:10.1016/j.bjpt.2019.01.010
Kellenberger, E. (2001). Exploring the unknown: The silent revolution of microbiology. EMBO Reports, 2(1), 5-7. doi:10.1093/embo-reports/kve014
Pollitt, E. J., Crusz, S. A., & Diggle, S. P. (2015). Staphylococcus aureus forms spreading dendrites that have characteristics of active motility. Scientific Reports, 5(1), 1-12.
Sutejo, S. V. H., Amarantini, C., & Budiarso, T. Y. (2017). Molecular detection of Staphylococcus aureus resistant to temperature in milk and its products. In AIP Conference Proceedings. AIP Publishing LLC.
Taylor T.A., & Unakal C.G. (2020 Aug 23). Staphylococcus aureus. In: StatPearls. Treasure Island (FL): StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK441868/
Zhou, C., Bhinderwala, F., Lehman, M. K., Thomas, V. C., Chaudhari, S. S., Yamada, K. J., & Fey, P. D. (2019). Urease is an essential component of the acid response network of Staphylococcus aureus and is required for a persistent murine kidney infection. PLoS Pathogens, 15(1).