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The final and culminating project in the laboratory was the identification of given unknown organisms. By way of various procedures learned throughout the semester, the necessarily skills for bacterial identification were reproducible and understood. Although a one credit hour course in microbiology barely provides the time for complete comprehension or an adequate learning environment due to time restrictions, a lot of material was covered in one semester and the fundamentals of the lab were learned. This final project touched on procedures like aseptic techniques to Gram stains to microscopy to dichotomous keys. Through the combination of these skills, despite a compressed timeframe, most students successfully identified their unknown organisms.
As learned in the beginning of the semester, bacteria can generally be classified through basic characteristics like shape or arrangement. Some bacteria are cocci shaped while others are bacilli. Some organisms are typically found isolated from other cells, while others are generally grouped together in bunches or in long chain-like formations. The familiarity with common organisms and their structural characteristics can be a solid foundation for organism identification. However, one of the most common differentiating characteristics for bacteria is the recognition of a species as Gram-negative or Gram-positive through the Gram stain. Most organisms are described first and foremost by their Gram classification. Then, through a series of biological tests, microorganisms can be further classified and differentiated from one another. Most species can be correctly identified through the combination of a Gram stain, morphological characteristics and biological tests.
Although we did not directly study more complex or advanced procedures in the lab, there are alternative widely practiced methods for organism identification. One such method makes use of 16S rRNA. This specific RNA is involved in protein production, and due to its predictable location in organisms, it basically resembles a fingerprint for species identification (The Center for Environmental Bioinorganic Chemistry, 2010). To date, there are a total of over 10,000 published names in a database used to match 16S rRNA sequences (Euzeby, 2010).
In addition to working on unknown organisms in the final weeks of lab, we also did experimentation on bacteria of the upper respiratory tract. While several types of bacteria are common in the normal flora of the pharynx, there are several pathogenic species that can cause pharyngitis and the inflammation of the throat. One of the steps in identifying harmful bacteria of the pharynx is to test for hemolytic organisms using specially designed media. Beta Hemolysis is demonstrated in the destruction of red blood cells and breaking down of hemoglobin. Alpha Hemolysis shares an equal destruction of red blood cells, but not a complete breakdown of hemoglobin. Lastly, Gamma Hemolysis indicates no hemolytic activity (lab handout).
Materials and Methods:
The procedures for the respiratory lab were fairly simple and straightforward, despite the fear of gag reflexes kicking in and empty vomiting sounds erupting throughout the room. Swabs were used to collect samples from the tonsilary region of each student. One sample was used to inoculate a blood agar plate, which was then incubated in an anaerobic environment. The plate was streaked using a quadrant streak method. A second swab was swirled inside a test tube of sterile water to dislodge any present organisms. The water was then diluted using serial dilutions and poured onto plates with nutrient agar.
The process and procedures for identification of the unknown organisms began with the production of a dichotomous key. A dichotomous key is used to help differentiate between various microorganisms based on their characteristics (metabolic, morphology, etc.). First, I set up a table and researched each organism in order to compare the organisms within their classification as Gram-positive or Gram-negative. Most Gram data is very easy to obtain through a variety of microbiological literature and resources. The following is a shortened version of my table. I highlighted the tests that proved to be best at differentiating between the organisms. The lactose test was chosen as the first test for both sets due to its simplicity and ability to split the organisms into near equal parts.
Next, after developing this table, I put together a corresponding dichotomous key. The dichotomous key is simply a better visual representation of the data in the tables above, but also easier to follow as a literal pathway for reaching conclusions regarding identification. In the end, I was able to produce a dichotomous key that effectively narrowed down the organisms and used three or fewer tests in doing so (with the exception of the pigment test for Staphylococcus). I tried to focus on using tests that were simple and quick in order to reach a conclusion regarding my organisms in the least amount of time and with the least amount of work.
The following two images are the two halves of my dichotomous key, separated into Gram-negative and Gram-positive for the simplicity of viewing within the document. The Gram-positive side consists of the Phenol Red test for lactose fermentation, the Milk Agar test, the Catalase test, the Mannitol Salt test, the Nitrate Reduction test, the SIM test and a simple pigment test on TSA agar. The Gram-negative side consists of the Phenol Red test for lactose fermentation, the Simmons Citrate test, the Oxidase test, the Methyl Red test and the Nitrate Reduction test.
In total, there were 10 tests included in my dichotomous key for the identification of 15 possible organisms. Each test is performed differently and reveals specific characteristics about the organisms tested. The following is a list and description of each test included:
The Gram stain is the most fundamental biological test and at the top of most dichotomous keys. As a result, a misinterpreted Gram stain can have significant effects on the identification of unknown organisms. The procedure begins with the inoculation of a sample onto a slide. The slide is air-dried and then heat fixed. Crystal violet dye is then added to the slide and allowed to set for 60 seconds before being rinsed thoroughly with water. Next, iodine is added and set for 60 seconds as well. The slide is then rinsed with water. Decolorizer is added to the slide for approximately 10 seconds and then rinsed with water. Finally, the safranin dye is added to the slide and allowed to set for 60 seconds before the final rinse. The slide is then blotted dry and observed under a microscope. Red or pink cells indicate a negative result, while purple or violet cells indicate a positive result.
The Phenol Red Broth (for lactose) is used to determine lactose fermentative reactions. Lactose is embedded in the medium and causes certain bacteria to produce acid and/or gas. This helps to differentiate between species. A pH indicator, phenol red, changes the color of the medium from red to yellow when acid is produced. Otherwise, the medium stays red. Also, if gas is produced, it is visible on an inverted Durham tube, which is placed inside the test tube within the medium. The main procedure itself is a simple inoculation of the prepared broth. A specific broth is designed for lactose as opposed to the Phenol Red Broths for glucose and sucrose fermentation (BD, 2010).
Milk Agar (Casein):
Skim Milk Agar can be used to differentiate between microorganisms based on the coagulation and proteolysis of casein. Since skim milk is a source of lactose and casein, which are valuable for the growth of lactobacilli, this media can be used to differentiate organisms that utilize lactose and casein. The test is performed by inoculating an organism onto a prepared Milk Agar plate. As the organism grows, a clearing around the culture represents a positive result. This implies that the organism is utilizing the casein. If no clearing exists, the organism is negative (BD, 2010).
The Nitrate Reduction test is used in identifying aerobic and facultative anaerobic bacteria. Certain organisms can reduce nitrate to nitrite. The nitrite then reacts with two added reactants, sulfanilic acid and N, N-dimethyl-alpha-naphthylamine, to produce a red color. This is considered a positive test for nitrate. A Durham tube is also used in this procedure. If gas is present in the tube and the organism is not a fermenter, the test is said to be positive for denitrification (which is an midway point toward complete nitrate reduction) (BD, 2010).
The Catalase Test is a good differential test for bacteria, examining the presence or absence of the enzyme catalase. Many microorganisms produce catalase in order to inhibit the development of hydroxyl radicals from hydrogen peroxide. Catalase is beneficial in the sense that it coverts hydrogen peroxide to water and oxygen gas instead of the hydroxyl radical (lab handout). The biological test for catalase is quick and easy. A large sample of the desired organism is inoculated onto a slide. Then a few drops of hydrogen peroxide are added to the slide. An immediate formation of bubbles indicates the organism's ability to produce catalase and is a positive result. No formation of bubbles, even under a microscope, represents a negative result (lab handout).
The Mannitol Salt Agar is commonly used to differentiate between Staphylococcus species. Most other organisms are unable to grow on this agar as a result of the high sodium chloride concentration. The Staphylococcus species are differentiated by their ability to ferment mannitol. Normal inoculating procedures are used to plate this test. Staphylococcus species will appear as different colors due to the pH indicator (BD, 2010).
SIM Media tests for sulfide production, indole formation and motility. It differentiates enteric bacilli and aids in the identification of the Enterobacteriaceae on the basis of these results. The ingredients within the medium help indicate three activities that can take place. A black precipitate is formed when hydrogen sulfide gas reacts with the ferrous ammonium sulfate in the medium. This blackening along the stab line indicates a positive test for H2S production. Certain microorganisms attack the tryptophan in the medium, resulting in indole production. 3-4 drops of Kovacs' reagent are added after incubation and a red color indicates a positive indole test. Motility is the last activity monitored. Growth that has developed away from the stab line in the medium indicates a positive for motility. This medium is extremely useful as it serves multiple purposes and differentiates in various ways through one procedure (BD, 2010).
The Simmons Citrate media is designed to differentiate gram-negative bacteria due to the utilization of citrate. Microorganisms that are able to use citrate for metabolism (gram-positive) grow well on the Simmons Citrate media. A positive reaction will exhibit growth in addition to an intense blue color in the slant media. A negative reaction will remain green in color and exhibit no growth. The test is performed by simply inoculating prepared slants with the desired organisms (BD, 2010).
MR positive organisms produce high acidity during fermentation of dextrose and produce a red color by overcoming the phosphate buffer. The MR test is performed by adding 5 drops of methyl red indicator to the tube and viewing the results immediately. A positive result for the MR test is recognized by a red color at the surface of the medium, whereas a negative result would display yellow (BD, 2010).
The Oxidase Test differentiates bacteria based on the presence of cytochrome C oxidase. Bacteria typically use cytochrome C oxidase as the final member of the electron transport chain. The final electron acceptor is oxygen for aerobic organisms, and the oxidase donates electrons to oxygen. In the oxidase test, tetramethyl-p-phenylenediamine is a reducing agent that changes color when oxidized. An oxidized agent turns deep purple/black, but a reduced agent is clear with no color (lab handout). There are two different methods to performing the Oxidase Test. The first involves a special Oxy-Swab. After touching the tip against the edge of the bacterial culture, pressure against the cotton tip will cause a potential color change after 10-30 seconds. A positive test will show a dark purple or black color. No color change indicates a negative test. The second method involves a special Dry-Slide. Microorganisms can be inoculated onto the squares of the slide using a wooden probe. The slide will change color to deep purple/black very quickly if oxidase is present. Otherwise a negative result is assumed (lab handout).
Two different experiments were done related to respiratory organisms. The serial dilutions that were poured onto plates were counted. The following table represents the data from our table.
Brandon: 96 CFU = 9.6 x 105
Disha: 242 CFU = 2.42 x 106
The colony forming units (CFU) were counted and then multiplied out to obtain the total number of viable cells in the original sample (on the swab). The second respiratory experiment involved the blood agar plates. Due to time constraints and the pressure to get specific tasks done related to our unknown organisms, no results were obtained regarding the blood agar plates. (Just for the record, I do not believe the respiratory lab should have been included with week 12. The stress was significant with the unknown organisms alone. The respiratory experiment could have easily been added to a previous week.)
For my unknown organisms, the first procedure involved plating the culture for isolation. Upon plating the organisms, one plate was incubated at 25° and the other at 37°. More growth was recorded on the 37° plate. Large, pale yellow colonies were noted. Small colonies, white in color, were hidden and slightly noticeable under the larger colonies. The 25° plate showed two distinct colony sizes. The colonies were all still pale yellow in color. For reference sake, I identified the smaller colony as Organism A and the larger colony as Organism B.
The next step was to Gram stain both organisms. Due to poor stains, the results were inconclusive. As a result, I proceeded with further tests due to the time constraints while still working on obtaining a solid Gram stain result. In the end, I did finally get a good Gram stain. Organism A was identified as Gram-positive and Organism B was identified as Gram-negative. The following tables indicate the results from the tests performed on both organisms, as well as the Organism C.
Organism A: cocci shape, grouped in clumps, Gram-positive
Organism B: rod shape, mostly isolated, Gram-negative
Organism C: rod shape, mostly isolated, Gram-negative
Phenol Red (Lactose):
Organism A: turned yellow, no gas = A (positive)
Organism B: turned yellow, gas produced = A/G (positive)
Organism C: turned yellow, gas produced = A/G (positive)
Organism A: no bubbles formed = negative
Organism B: no bubbles formed = negative
Organism A: slant remained green = negative
Organism B: slant remained green = negative
Organism C: slant turned blue = positive
Organism A: no color change to red = negative
Organism A: majority of tube turned black = positive
Since no Gram tests were done on the respiratory organisms and they were simply observed, no conclusions can be made regarding the individual species present. However, the data from the pour plates indicates that a large quantity of normal flora is present in the upper respiratory tract.
For the unknown organisms A and B, the order and selection of tests were adjusted as necessary due to the initial faulty Gram stains. Double tests were done on both organisms in some instances to help with direction in the dichotomous key by way of simple process of elimination. Also, the Methyl Red test was unnecessary with Organism C because C. freundii was eliminated due to the rod shape of the organism. One other note relates to the SIM test. Due to the time crunch, the test did not resolve properly and gave a false negative for Organism A. It was determined that a supplemental Bile Esculin test could be performed, which gave a better indication of the organism.
Organism A was identified as Enterococcus faecalis. E. faecalis is a facultative anaerobic, cocci-shaped (hence the name), Gram-positive organism (Fraser, 2010). The organism is also recognized as alpha hemolytic and it can grow well in 40% bile or 6.5% NaCl (Gladwin and Trattler, 2008). While the organism can grow in many environments, it is commonly found in the normal flora of human intestines. As such, it is one of the most commonly cultured species from the human body (Fraser, 2010).
Organism B was identified as Escherichia coli. E. coli is a very common organism studied in the laboratory, as evidenced by our semester. The organism is rod-shaped and Gram-negative. It typically resides, innocently, in the colon. Although E. coli rarely causes disease on its own, the interaction with other organisms and other virulence factors can result in diarrhea, urinary tract infections, neonatal meningitis and sepsis (Gladwin and Trattler, 2008).
Organism C was identified as Enterobacter sp. The Enterobacter species are rod-shaped, Gram-negative and highly motile. They are normal flora found within the human intestinal tract. On occasion, they are responsible for infections in the hospital environment (Gladwin and Trattler, 2008). However, this bacterial genus rarely causes disease in healthy individuals (Fraser, 2010).