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The purpose of this lab was to identify unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. To initiate differential testing of unknown microorganisms, the Gram stain procedure was performed. The art of Gram staining is important because it allows students, as well as microbiologists, to differentiate between bacteria and the human cell. Along with revealing the differences between bacteria and the human cell, Gram stain, also divided bacterial cells into two different groups known as Gram positive and Gram negative. While revealing the identity of cells, Gram staining also explains differences between different types of bacteria and shapes specific to them. Bacteria are usually composed of either cocci, or sphere shaped, bacillus, or rod shaped, and spirillum, which are helical and curved.
More recent advances have aided in identifying the morphology and identification of microorganism. One known method is the 16S rRNA gene sequencing. 16S rRNA gene sequences contain hypervariable regions which can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid, accurate alternative to phenotypic methods of bacterial identification (Smit and etl 2007).
In this unit, students also focused on the pathogens of the respiratory tract and harvested the flora of the throat by inoculating the sample onto a blood agar media. Viruses are distinctive agents that cause inflammation of the throat (pharynx) and because of this there are a number of bacteria that can cause pharyngitis. The three most common causes of pharyngitis are Streptococcus pneumonia, Haemophilus influenza, and Moraxella catarrhalis. Less common, but more severe is streptococcal pharyngitis, or strep throat, caused by Streptococcus pyogenes. The pharynx has a normal flora, a community of organisms that commonly inhabit the pharyngeal tissues. This experiment was designed to show the presence of hemolytic organisms and their ability to destroy red blood cells. Even though hemolysis is a common characteristic among upper
respiratory pathogens, there are different types of hemolysis (Maxell 2008).
The differential tests used to identify the first unknown culture were Catalase test and Mannitol Salt Agar for the gram-positive and the Simmons Citrate Slants, Glucose Fermentation and the Lactose test for the gram-negative organisms. The unknown #102 culture was revealed by using the Catalase test, Mannitol Salt agar, and Lactose test.
Material & Methods
For the lab that focused on the respiratory tract, students used sterile swabs to collect saliva from the tonsialary region of the mouth. The material collected was inoculated on the blood media and dilutions were made from this material to determine the population density. The tests performed on the unknown bacteria cultures were all used to determine the identity of the bacteria. Each of the tests performed provided some key information about the bacteria in question and how it functions. Students compiled a dichotomous key upon performing any differential testing. A dichotomous key is used to organize the phenotypic characteristics of organisms in an attempt to produce a systematic way of identifying unknown organisms. This key is assembled based upon the known characteristics of organisms. As such, it only helps in the identification of organisms that have been previously described (Maxwell 2008). The developed dichotomous key separated the unknown samples into Unknown A and Unknown B by differentiating the organisms by their cell wall structure and morphology, using the Gram stain procedure. After separating the organism by whether they were gram-positive or gram-negative different test were suggested for each group separately. The tests performed and what constitutes a positive and negative test are as follows.
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 unknown gram (-) bacteria were catalase positive. This test is used to detect the presence of catalase, which helps to breakdown toxic hydrogen peroxide produced from the transport of high-energy electrons directly to oxygen. Catalase is tested for by adding hydrogen peroxide to the culture, and looking for the production of gas bubbles. If gas bubbles appear immediately, the culture is catalase positive. However, if no bubbles are observed, 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 salt tolerance and differential for mannitol sugar fermentation. It also contains phenol red, which acts as a pH indicator, turning yellow under acidic conditions. The agar is most often used for the selection of S. aureus. Growth and a yellow color change are positive test results. No growth is a negative test result (Difco Laboratories, Initials.1964).
For the organisms that were classified as gram-negative other tests were performed to differentiate between these groups as a whole as well.
The Phenol Red Broth test is used to determine fermentation reactions when differentiating microorganisms. This broth was used in the Glucose Fermentation test and in the Lactose test. Phenol Red Broth is used as a control for fermentation studies or as a base for the addition of carbohydrates. A pH indicator is used to detect acid production. The medium turns from red to yellow when organic acids are formed. The inverted Durham tube will collect gas, if produced, during the fermentation reaction. To perform testing inoculate the tubes of media with a heavy inoculum for 48 hours at 37°C and keep the tube's caps loose. If acid is present a yellow color will form and if the liquid is displaced in the Durham tubes this is clear evidence of gas formation (Difco Laboratories, Initials.1964).
The Simmons Citrate Agar test is used for gram-negative bacteria on the basis of citrate utilization. This medium consists of inorganic salts in which an ammonium salt was the only nitrogen source and citrate being the only carbon source to differentiate between Escherichia coli and Enterobacter aerogenes. This media was modified, by Simmons in 1926, with the addition of agar and bromthymol blue and organisms that grow well on this medium are capable of metabolizing citrate. Slants were inoculated with a light inoculum that grew from a pure culture and incubated for 48 hours at 37°C in an aerobic atmosphere. A (+) reaction reveals an intense blue color inside the slant and a (-) reaction reveals no change in color (medium remains dark green), indicating no growth (Difco Laboratories, Initials.1964).
Table 1: This table shows the results for all of the tests performed on Unknown A,
Unknown B and Unknown #102. The results noted N/A were not performed on
Gram (-) rod
Gram (+) cocci
Gram (+) cocci
No color change slant remained green (-)
Manitol Phenol Red broth
(+) broth color changed from pink to yellow
(+) Yellow zones/ Yellow zones on TSA
The results for our throat swab revealed no growth on our 10-4 dilution plate and slight growth on our 10-3 dilution plate. The students' blood agar media displayed no growth at all and can be characterized as Gamma (Î´) Hemolysis, which indicates that there is no hemolytic activity.
The student's results for the Unknown A revealed a positive test for Glucose fermentation and Lactose, but a negative result for Citrate. The citrate test was negative which remained 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 conversion of the medium to blue is a positive citrate test. No color change is negative. The broth from glucose fermentation and lactose fermentation turned from pink to yellow indicating a positive result.
For the Unknown B, which was determined gram-positive, both the Catalase test and Mannitol phenol broth test revealed positive. The Mannitol salt agar test was determined ineffective for the student's unknown solution so the TA decided to test the unknown against its broth solution for better results. For Unknown #102, the results were positive for both the Catalase and Mannitol phenol broth solution test.
The determination of the unknown identities was achieved using the various differential tests. By performing each test and tracking the results, the unknowns were able to be identified using previously known bacteria data and the use of the dichotomous key. Unknown A was determined to be Escherichia coli, Unknown B was determined to be Staphylococcus epidermis, while unknown #102 was determined to be Staphylococcus aureus gold.