Respiratory microorganisms include numerous types of bacteria, some which are found in the normal flora and some of which are pathogenic. Normal flora is the mixture of organisms regularly found at any anatomical site. In the experimental Laboratory, two swabs of the tonsil area of each lab members were taken and were used to determine two things. The first swab tested to see if the bacteria in the respiratory environment are hemolytic or not. The second swab was used to determine the population density of organisms that are present on a swab from the throat. It was important to keep aseptic technique through out the lab so that airborne bacteria were not incorporated in the growth of the plates. After seven days of incubation, the blood agar was observed and growth was seen on the plate. The colonies were various shapes and sizes and the color ranged from white to a shade of gray. The pour plate of the 10-4 was the only one that produced a countable plate, more than 30 CFU but less than 300 CFU. The plate had 202 colony forming units that can be calculated to be 2.42 x 106.
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The purpose of this lab was to give an introduction to the Unknown experiment that was to be conducted over the next few weeks where unknown sets of bacteria were assigned to each student and with in a few tests, the microorganism was to be determined. The reason the respiratory tract experiment was an introduction because an application of making a dichotomous key and using tests to identify a given organism can be made. Rapid and accurate identification of microorganism used many methods that has progressed over the years. Culturing the organisms collected is one of the simplest, oldest, and most common way to determine the organisms, but it takes a few days to observe the results. Polymerase Chain Reaction is a speedier way of identifying an organism by extracting DNA from the sample and performing PCR. Comparison of the bacterial 16s rRNA gene sequence has emerged as a preferred genetic technique. 16s rRNA makes up the bulk of the 30 s subunit. It is important for subunit association and translation accuracy. 16s rRNA is a type of RNA that is involved in the production of protiens. The machinery of protein synthesis does not vary much from one organism to another, so the RNA that assists protein production does not vary much. 16s rRNA is used as a comparison of certain location on a molecule.
A vial of two microorganisms were given to each student where the first step was to isolate the two organisms by streaking a plate with the broth followed by picking up a single colony from the plate and applying to a second TSA plate as well as adding a isolated colony to a broth where the microorganisms are grown. At this time a Gram Stain was conducted on both organisms where it was determined that organism A was a Gram negative and organism B was a Gram positive. Once each microorganism was grown on an isolated plate, the first test was conducted. To better organize this paper each organism will be discussed separately.
Organism A, which was determined Gram negative, was a red pigmented organism when grown on a TSA a plate. It was also determined that it best grew at 25 degrees Celsius. The first test that was conducted was the Oxidase test. This test was chosen to be executed first because it is quick to determine weather it is oxidase negative or positive and a second test can be easily conducted. Oxidase test is used to identify organisms that have the enzyme cytochrome C oxidase. Enterobacteriaceae are oxidase negative and Pseudomadaceae are oxidase positive. Cytochrome C oxidase delivers electrons to oxygen, reducing it to water, from the electron transport chain. The oxidase test will show if tetramethyl-p-phenylenediamine, the electron donor (or reducing agent) turns purple when it is oxidized. Cytochrome C oxidase is reduced by if present. The donor molecules will turn dark purple when it is oxidized if the organism is oxidase positive. In the case of Organism A, no change occurred, the organism is deemed Oxidase Negative. Along the Dichotomous Key, B. capacia and P. aeruginosa were not an option for this organism since these organisms are Oxidase positive. Once organisim A was determined Gram negative and Oxidase positive, a Citrate test was conducted. Simmons Citrate Agar, is used to differentiate gram-negative bacteria that utilize citrate as their sole carbon source. If the bacteria can utilize citrate, its enzyme citrase will hydrolyze citrate into oxaloacetic acid and acetic acid. The oxaloacetic acid is hydrolyzed into pyruvic acid and CO2. The CO2 will react with the media and produce an alkaline compound, Na2CO3. This will turn the bromthymol (a pH indicator) from green to blue, indicating a positive reaction. E.aerogenes is citrate positive while E. coli is citrate negative. The test was performed by inoculating a slant agar with a pure culture growth, a light inoculum, and incubating the tube for 5 days at 37 degrees Celsius. The results were drastic and showed the original green agar had turned blue giving a positive result. It can be applied that organism A cannot be Proteus vulgaris or E. coli. The next test to be administered was the MR-VP test, particularly the VP test. MR-VP (Methyl Red-Voges Proskauer) media are used to identify which fermentation pathway is used to utilize glucose. The mixed acid fermentation pathway allows bacteria to ferment glucose and produce several organic acids; lactic, acetic, and formic. This will keep the pH below 4.4. When the methyl red indicator is added, a red color appears due to the high acid content. If the methyl red turns yellow, it indicates that the mixed acid pathway has not been used . The other pathway, 2,3 butanediol fermentation pathway will produce an alcohol as an end product rather than an acid. To test this, ÎÂ±-naphthol and KOH is added to the two tubes and shaken vigorously. After they sit for one hour, the presence of acetone will indicate a positive reaction by showing a reddish-to-pink color change. The negative result will be yellow. E.aerogenes is MR negative (yellow) but VP positive (red), while E. coli is MR positive (red) and VP negative (yellow). To perform this test, a light inoculum is used to inoculate MR and VP tubes from 18-24 hour pure cultures. The tubes are then Incubated aerobically at 37 degrees Celsius for two days. The media test in-lab for VP was performed on organism A. The tube was inoculated and 15 drops of solution A, provided be the instructor, was added to the VP tube and 5 drops of solution B. The tubes were read every 10 minutes for 60 minutes in order to observe the reaction. After 60 minutes A color change was observed resulting in a positive VP test. This conclusion led to crossing-out the option of the organism being C. freundii. According to the prepared dichotomous key, the organism can either be E. areogenes or S. marcescens. Knowing that the organism is pigmented red when grown on a TSA plate at 25 degrees Celsius, Organism A can be accurately identified as S. marcescens.
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Organism B was isolated and plated to show white colonies, stained to be Gram positive, and grew best at 37 degrees Celsius. This first test that was conducted was the Catalase test. Catalase, is used to identify organisms that can produce the enzyme Catalase which breaks down hydrogen peroxide into oxygen gas and water. Staphylococcus ssp. and Micrococcus ssp. are catalase positive. Enterococcus ssp. and Streptococcus ssp. are catalase negative. A positive result is indicated by the formation of bubbles, gas production, as soon as several drops of hydrogen peroxide are dropped on the organism. This test was chosen for the same reason as the Oxidase test, it is quick and easy and a second test can be conducted right away. For organism B, hydrogen peroxide and the organism produced gas and so it can be identified as a Catalase positive organism. Knowing that the unknown organism B is Catalase positive can eliminate the option of the organism being E. faecalis or L. acidophilus. Once the organism was concluded to be Catalase positive, a mannitol test was conducted.
Mannitol Salt Agar is used to selectively isolate and enumerate staphylococci from clinical and nonclinical materials. Mannitol contains peptones and beef extract, which provide nutrients such as nitrogen, carbon, sulfur, and trace elements. With a 7.5% sodium chloride concentration, bacteria other than the staphylococci are inhibited. When the mannitol is fermented, the phenol red indicator changes color. A positive result is good recovery with yellow growth (S. aureus). A negative result is no growth (E. coli, E. aerogenes, and P. mirabilis). The test was performed by plating a pure culture on a mannitol salt agar and incubated at 37 degrees Celsius for 48 hours in an aerobic environment. The agar showed growth with a yellow halo around the streak on the plate. It can be determined that the organism is Mannitol Positive, eliminating M. roseus and S. epidermis as an option for unknown organism B. The next test to be conducted, according to the dichotomous key, is the Lactose test. Lactose test is tested by inoculating a tube of Phenol red broth with a lactose carbohydrate base. To determine if the organism is positive or negative the Phenol Red acts as an indicator for acid and therefore the organisms ability to grow in the presence of Lactose is tested.
Phenol Red broth base with lactose, will show if the organisms can ferment lactose that has been incorporated into a basal medium. This can be used on gram-positives and gram-negatives. The product of this reaction will be acid production or acid and gas production along with a color change in the media. A positive reaction of acid and gas is yellow, while a negative reaction (which might have growth but no acid production) will be red/pink. To perform the test, heavy inoculums is used to inoculate tubes from an 18-24 hour growth of pure culture. The tubes are then incubated with loosened caps at 37 degrees Celsius for 48 hours in an aerobic environment. For organism B, the broth turned to a bright yellow giving a Positive reaction to Lactose. Following the prepared dichotomous key, B. Subtilis and C. diphtheria identifications can be eliminated due to their inability to ferment lactose. The only two organisms left that this unknown bacteria could be, is S. aureus gold or S. aureus white. Based on the white colored colonies that are formed on TSA plates, it can be accurately confirmed that the given bacteria is S. aureus white.
In the midst of identifying the first two organisms, a third organism was introduced. The same procedure were followed, but this time the given broth of bacteria was already isolated. The organism was then stained to know that the organism was differentiated as Gram positive. Like the previous organism B, a Catalase test was conducted giving a negative result with no gas production. The next step to follow was the Indole test. The indole test uses SIM media, it is used to differentiate enteric organism that can produce sulfide and indole. It has several ingredients that help indicate these reactions. Sodium thiosulfate and ferrous ammonium both indicate if hydrogen sulfide a black precipitate, has been produced. The casein peptone has tryptophan, which will allow organisms to feed and produce indole which is detected after other reagents are added. The organism is stabbed only two-thirds of the way down in the center of tube. The tubes are incubated at 37 degrees Celsius for 48 hours in an aerobic environment. To test for indole, 3-4 drops of Kovacsââ‚¬â„¢ reagent is added and watched for a red color change. For the Final unknown organism, the solution turned red concluding two things, the unknown is Indole Positive, and it is Enterococcus Faecalis.
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Preparing and following a Dichotomous key is great practice for testing and identifying an unknown organism if an organism was found in the environment. The identification of the three unknowns was achieved using the various differential tests. By performing each test and tracking the results, the unknowns were identified using previously known bacteria data.
Slonczewski, J.L., and Foster, J.W., 2009. Microbiology An Evolving Science, p. 647-651. Michael Wright (ed.), W.W. Norton & Company, Inc. New York.
Slonczewski, J.L., and Foster, J.W., 2009. Microbiology An Evolving Science, p. 646. Michael Wright (ed.), W.W. Norton & Company, Inc. New York.
DifcoTM & BBLTM Manual of Microbiological Culture Media, Mary Jo Zimbro (ed.) and David A. Power (ed.), Becton, Dickinson and Company, Sparks, Maryland. Various pages