Posted at 10.15.2018
The reason for this lab was to identify unknown bacteria ethnicities using various differential lab tests. The identification of the unknown civilizations was achieved by separating and differentiating possible bacteria based on specific biochemical characteristics. If the tests performed determined specific enzymatic reactions or metabolic pathways, each was found in ways to help discover those specifics and identify the unknown cultures. To start differential screening of anonymous microorganisms, the Gram stain technique was performed. The fine art of Gram staining is important since it allows students, as well as microbiologists, to distinguish between bacterias and the individuals cell. Along with disclosing the dissimilarities between bacteria and the individual cell, Gram stain, also divided bacterial skin cells into two different groupings known as Gram positive and Gram negative. While disclosing the id of cells, Gram staining also points out differences between different types of bacterias and figures specific to them. Bacterias are usually composed of either cocci, or sphere shaped, bacillus, or pole molded, and spirillum, which are helical and curved.
More recent developments have aided in identifying the morphology and recognition of microorganism. One known method is the 16S rRNA gene sequencing. 16S rRNA gene sequences contain hypervariable areas which provides species-specific signature sequences ideal for bacterial identification. Because of this, 16S rRNA gene sequencing is becoming widespread in medical microbiology as a rapid, accurate alternative to phenotypic methods of bacterial id (Smit and etl 2007).
In this product, students also centered on the pathogens of the respiratory tract and gathered the flora of the throat by inoculating the sample onto a bloodstream agar media. Trojans are distinctive brokers that cause inflammation of the neck (pharynx) and due to this there are a number of bacterias that can cause pharyngitis. The three most usual causes of pharyngitis are Streptococcus pneumonia, Haemophilus influenza, and Moraxella catarrhalis. Less common, but more serious is streptococcal pharyngitis, or strep neck, induced by Streptococcus pyogenes. The pharynx has a standard flora, a community of microorganisms that commonly inhabit the pharyngeal tissue. This test was designed to show the occurrence of hemolytic microorganisms and their capability to ruin red blood cells. Even though hemolysis is a common attribute among upper breathing pathogens, there are different types of hemolysis (Maxell 2008).
The differential lab tests used to identify the first unknown culture were Catalase ensure that you Mannitol Sodium Agar for the gram-positive and the Simmons Citrate Slants, Sugar Fermentation and the Lactose test for the gram-negative microorganisms. The mysterious #102 culture was exposed utilizing the Catalase test, Mannitol Salt agar, and Lactose test.
For the laboratory that centered on the respiratory tract, students used sterile swabs to gather saliva from the tonsialary region of the mouth area. The material accumulated was inoculated on the blood media and dilutions were created from this material to determine the population density. The testing performed on the mysterious bacteria ethnicities were all used to look for the individuality of the bacteria. Each of the tests performed provided some key information about the bacterias involved and how it functions. Students compiled a dichotomous key upon undertaking any differential screening. A dichotomous key is employed to organize the phenotypic characteristics of organisms so that they can produce a systematic way of figuring out unknown microorganisms. This key is set up based upon the known characteristics of organisms. Therefore, it only helps in the recognition of organisms which have been previously described (Maxwell 2008). The developed dichotomous key segregated the unknown examples into Unidentified A and Unknown B by differentiating the microorganisms by their cell wall structure composition and morphology, using the Gram stain technique. After separating the organism by whether they were gram-positive or gram-negative different test were advised for each and every group independently. The assessments performed and what constitutes a negative and positive test are the following.
The Catalase test was performed only on gram (+) bacteria, as this test would not help in differentiating the gram (-) bacteria because all of the possible undiscovered gram (-) bacteria were catalase positive. This test is employed to find the presence of catalase, which helps to breakdown harmful hydrogen peroxide created from the travel of high-energy electrons directly to air. Catalase is examined for with the addition of hydrogen peroxide to the culture, and looking for the development of gas bubbles. If gas bubbles appear immediately, the culture is catalase positive. However, if no bubbles are found, the culture is negative for catalase (Difco Laboratories, Initials. 1964).
Mannitol Salt Agar is both a selective and differential media used for gram (+) cocci. It is selective for sodium tolerance and differential for mannitol sweets fermentation. It also contains phenol red, which functions as a pH sign, turning yellow under acidic conditions. The agar is most often used for selecting S. aureus. Expansion and a yellow color change are positive test outcomes. No progress is a negative test result (Difco Laboratories, Initials. 1964).
For the organisms that were labeled as gram-negative other testing were performed to differentiate between these teams as a whole as well.
The Phenol Red Broth test can be used to determine fermentation reactions when differentiating microorganisms. This broth was found in the Blood sugar Fermentation test and in the Lactose test. Phenol Red Broth is used as a control for fermentation studies or as basics for the addition of carbohydrates. A pH signal is employed to detect acid solution creation. The medium transforms from red to yellow when organic acids are produced. The inverted Durham tube will gather gas, if produced, during the fermentation reaction. To perform testing inoculate the pipes of media with a heavy inoculum for 48 hours at 37C and keep carefully the tube's caps loose. If acid exists a yellow color will form and if the liquid is displaced in the Durham pipes this is clear evidence of gas creation (Difco Laboratories, Initials. 1964).
The Simmons Citrate Agar test is utilized for gram-negative bacteria based on citrate utilization. This medium consists of inorganic salts in which an ammonium sodium was the only nitrogen source and citrate being the only carbon source to distinguish between Escherichia coli and Enterobacter aerogenes. This press was revised, by Simmons in 1926, with the addition of agar and bromthymol blue and microorganisms that expand well on this medium can handle metabolizing citrate. Slants were inoculated with a light inoculum that grew from a real culture and incubated for 48 hours at 37C within an aerobic atmosphere. A (+) effect reveals an intense blue color inside the slant and a (-) response shows no change in color (medium remains dark green), indicating no development (Difco Laboratories, Initials. 1964).
The results for our throat swab unveiled no development on our 10-4 dilution dish and slight development on our 10-3 dilution dish. The students' bloodstream agar media shown no growth whatsoever and can be characterized as Gamma (ґ) Hemolysis, which signifies that there surely is no hemolytic activity.
The student's results for the Undiscovered A revealed an optimistic test for Glucose fermentation and Lactose, but a poor consequence for Citrate. The citrate test was negative which continued to be green. Bacteria that is able to survive and utilize the citrate, convert ammonium phosphate to ammonia and ammonium hydroxide, which alkalinize the agar, turning it blue. Thus, the change of the medium to blue is a positive citrate test. No color change is negative. The broth from glucose fermentation and lactose fermentation transformed from green to yellowish indicating an optimistic result.
For the Unknown B, that was determined gram-positive, both the Catalase ensure that you Mannitol phenol broth test exposed positive. The Mannitol salt agar test was determined ineffective for the student's undiscovered solution therefore the TA decided to test the unknown against its broth solution for greater results. For Unknown #102, the results were positive for both the Catalase and Mannitol phenol broth solution test.
The willpower of the mysterious identities was achieved using the many differential tests. By performing each test and monitoring the results, the unknowns were able to be determined using previously known bacterias data and the utilization of the dichotomous key. Unknown A was decided to be Escherichia coli, Unknown B was established to be Staphylococcus epidermis, while unfamiliar #102 was identified to be Staphylococcus aureus platinum.