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Biological systematic is considered to be the study of the diversity of life on the planet earth, and the relationships among living things through time. Systematics is often used synonymously with taxonomy and scientific classification. However, taxonomy is the describing, identifying, classifying, and naming of organisms. Classification is focused on categorizing organisms within specific groups that show their relationships to other organisms. Systematics uses taxonomy as a means to understand organisms. ("Systematic biology," 2003) Some bacteria can be identified by visual observation using microscopy, but the best identification usually requires further tests, many of them biochemical. Diagnostic laboratories use various biochemical media to isolate and identify bacteria. Most bacteria of medical importance can be grown on artificial culture media. Culture media can be either selective or differential media and are used to isolate or identify particular organisms. Selective media allow certain types of organisms to grow, and inhibit the growth of other organisms. Differential media are used to differentiate closely related organisms or groups of organisms. The presence of certain dyes or chemicals in the media, allow the organisms to produce characteristic changes or growth patterns that are used for identification or differentiation. (Abedon, 2006)
The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria and archaea. In addition to these, mitochondrial and chloroplastic rRNA are also amplified. Universal PCR primers are used to amplify the 16S rRNA gene to provide phylogenetic information.Comparative analysis of the 16S rRNA sequences is done with the help of primers, called universal primers.In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain 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.
Respiratory microorganisms exist in the normal flora of the human body. The pharynx also has a normal flora that only live n the pharyngeal tissues. Some organism such as Streptococcus pyogenes is not part of the normal flora and can cause the common strep throat. Hemolytic organisms, which are organisms that can destroy red blood cells, can be isolated and the type of hemolytic activity can be determined. Beta hemolysis is observed by the clear zone around the colonies that are grown on the blood agar. Alpha hemolysis is observed by the green halo that forms around the colonies. Gamma hemolysis will not produce a halo and represent that there is no hemolytic activity occurring.
Materials and Methods:
The procedures for respiratory microorganisms were to have a partner sit down and say ah so that they tonsilary region can be swabbed by making a rolling motion up the tonsilary region. Next the media is inoculated by rolling the swab across the first third of the blood agar plate and then discard the swab. Then flame an inoculating loop and streak for isolation. The plate then is placed in a candle jar in order to produce an anaerobic incubation environment. Results can be read during next lab to determine hemolytic activity. Another swab is also taken following the procedures just described and is used to determine the population density of organisms recovered from the throat.
The dichotomous key is used in order to identify an unknown organism through a series of test. The first test used to determine the identity is the Gram Stain. This will place the organism in the gram positive or gram negative category. An isolated colony is placed on a slide and heat fixed. The colony then is flooded with Crystal Violent for 60 seconds, and then rinsed. Next it flooded with Iodine for 60 seconds, then rinsed. Next the decolorizer is applied and left on for no more than 20 seconds, then rinsed. Finally the safranin is applied and left for 45 to 60 seconds and then rinsed. Gram positive organisms will appear purple and gram negative will appear pink. From this identification a series for test can be performed based on the dichotomous key.
The first test for the unknown gram negative organism would be the lactose test using the Phenol Red Broth. Phenol Red Broth Base is used for the determination of fermentation reactions in the differentiation of bacteria. This test is best performed on gram positive organisms. Performing this procedure consists of inoculating the tubes of media with growth from a pure culture using an inoculating loop. The tubes are then incubated with loosened caps for 18-48 hours. The results of positive carbohydrate fermentation are organic acids which, in the presence of phenol red, produce a color change in the medium from red to yellow. Negative results will retain the red color. (Difco Laboratories, 1964)
Lactose positive organisms would then be identified using the Citrate test. The citrate test is performed only on gram negative bacteria and is used to determine the ability of an organism to use citrate as its sole carbon source. Bacteria that possess citrate-permease are able to do this. The medium used for this test contains bromthymol blue dye, which is green at neutral pH, but blue at a basic pH. The procedure is to take a colony and streak the slant of the citrate media. Bacteria that can 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. (Difco Laboratories, 1964) Negative results will give the identity of the unknown organism.
The first test for the gram positive second unknown organism is based on shape. Rod shaped organism will be identified based on the catalase test. The catalase test was performed only on gram positive bacteria and 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, 1964)
Catalase positive gram positive rods will then be identified by performing the casein test using skim milk. Skim milk used for the cultivation and differentiation of microorganisms based on the coagulation and proteolysis of casein. Skim Milk is a source of lactose and casein and other nutrients required for the growth of lactobacilli. It can be tested on gram positive or gram negative organisms but distinguishes Clostridial species based on their ability to enzymatically degrade proteins to peptones or coagulate milk. It may be used to detect the stormy fermentation produced by Clostridium perfringens. Heat the medium in a boiling water bath for 2-5 minutes with caps loosened and cool to room temperature with caps tightened. Inoculate tubes using a calibrated loop or sterile disposable pipette. Incubate tubes, with tightened caps for clostridia and loosened caps for other organisms, and read at intervals for 7 days to observe growth. Clostridium perfringens produces stormy fermentation, Escherichia coli produces acid, and Lactobacillus rhamnosus also produces an acid. (Difco Laboratories, 1964) Negative or positive results of the casein will identify the unknown organism in question.
The first test for the gram positive third unknown organism is based on shape. Coccus shaped organisms will further be identified using the catalase test. The catalase test was performed only on gram positive bacteria and 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, 1964)
Catalase positive gram positive coccus shaped organisms then can be identified using the mannitol salt agar media. Mannitol salt agar is a selective medium used for the isolation of pathogenic staphylococci and would only be used on gram positive organisms. The medium contains mannitol, a phenol red indicator, and 7.5% sodium chloride. The high salt concentration inhibits the growth of most bacteria other than staphylococci. The procedures for using this medium are to obtain isolated colonies and streak onto the medium. Incubate plates, for 18-24 hours in an aerobic atmosphere. On MSA, Staphylococcus aureus produces small colonies surrounded by yellow zones. The reason for this change in color is that S. aureus ferments the mannitol, producing an acid, which, in turn, changes the indicator from red to yellow. The growth of any other Staphylococcus will produce small to large colonies with red zones. Micrococci will produce large white to orange colonies. The growth of other types of bacteria is generally inhibited. (Difco Laboratories, 1964) to distinguish between the S.aureus (gold) and S.aureus (white), the color observed on the TSA will be gold or white.
Results for the respiratory microorganisms were that gamma hemolysis activity was occurring and it was observed by no halo being produced around the colonies grown on the blood agar plate. This is a good indicator that S.pyogenes is not present .Too few colonies were observed when attempting to determine the population density of organisms recovered from the throat, by only observing 25 colonies.
The results for identification of the first unknown were gram negative. The results for the Phenol red lactose were positive. The results of positive carbohydrate fermentation are organic acids which, in the presence of phenol red, produce a color change in the medium from red to yellow.
Phenol Red Broth (Lactose)
A citrate test was performed next. The results for the citrate test were negative which remained green. Bacteria that are 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 negative results provided the identification of unknown one.
Unknown two was gram positive and shape was used to divide the organisms. The unknown was rod shaped and a catalase test was used to further identify the unknown. The unknown was catalase positive due to formation of bubbles once hydrogen peroxide was added to the smear. Catalase positive organisms then can be identified based on the casein media. The results for casein were negative, showing that the unknown microorganism does not have the characteristics needed for coagulation and proteolysis of casein. Unknown two can now be identified based on the results of this media.
Unknown three was gram positive as well and from observance under the microscope appeared to be coccus shaped. A catalase test was then performed according to the dichotomous key. Positive results were observed due to the formation of bubbles immediately when culture smear interacts with hydrogen peroxide. The unknown was then placed in a mannitol salt agar in which it produced no growth. This allowed for the identification of unknown three.
Mannitol Salt Agar
The first unknown gram positive organism was identified as E.coli. Escherichia coli is a Gram negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some, such as O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine. E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily-manipulated or duplicated through a process of metagenics, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology and microbiology.
The second unknown gram negative organism was identified as C.diptheria. C. diphtheriae is an aerobic gram-positive bacillus.Toxin production occurs only when the bacillus is itself infected by a specific virus carrying the genetic information for the toxin. Only toxigenic strains can cause severe disease.Culture of the organism requires selective media containing tellurite. If isolated, the organism must be distinguished in the laboratory from other Corynebacterium species that normally inhabit the nasopharynx and skin.There are three biotypes of C. diphtheriae which are gravis, intermedius, and mitis. The most severe disease is associated with the gravis biotype, but any strain may produce toxin. All isolates of C. diphtheriae should be tested by the laboratory for toxigenicity. Susceptible persons may acquire toxigenic diphtheria bacilli in the nasopharynx. The organism produces a toxin that inhibits cellular protein synthesis and is responsible for local tissue destruction and membrane formation. The toxin produced at the site of the membrane is absorbed into the bloodstream and distributed to the tissues of the body. The toxin is responsible for the major complications of myocarditis and neuritis and can also cause low platelet counts called thrombocytopenia and protein in the urine called proteinuria.
The third unknown gram positive organism was identified as Micrococcus. Micrococcus is a genus of bacteria in the Micrococcaceae family. Micrococcus occurs in a wide range of environments, including water, dust, and soil. Micrococcus has a substantial cell wall, which may comprise as much as 50% of the cell mass. The genome of Micrococcus is rich in guanine and cytosine. Micrococci often carry plasmids that provide the organism with useful traits. Micrococci have been isolated from human skin, animal and dairy products, and beer.