Introduction:

Biological organisms are classified uniformly in order to easily categorize and identify organisms. This classification, or taxonomy, uses the genus name followed by the species name, in Latin. By having a universal method of identifying bacteria allows for all scientists from any part of the world to identify the same species in an identical manner allowing for a precise of classification. Bacteria are distributed throughout the world in almost every conceivable habit. Bacteria are unicellular microorganisms, with variable shapes and nutritional needs. They lack a distinct nucleus and occur singly or in chains or clusters and form distinct colonies. Bacteria are classified on the basis of many characteristics. Morphological and physiological features such as cell shape, motility, formation of spores and other distinguishable structures, and reaction to Gram stain is a good start in identifying bacteria. Other staining techniques such as Acid Fast stain are also useful in determining species. More important in identification of a genus and species of bacteria are biological tests, including the determination of the types of nutrients a cell can use, the products of its metabolism, and the response to specific chemicals. Other factors that can assist in identification of bacteria are their ecological habitats and more advanced methods such as genetic and molecular composition. Using various techniques one is able to distinguish and ultimately assign then genus and species of the unknown bacteria.

Methods:

Gram Staining: The Gram stain separates bacteria in two distinct classes and is also useful in distinguishing morphology. Through this technique one is able to identify bacteria as either gram positive or gram negative. The gram-negative bacteria have thin peptidoglycan layers that do not hold the primary stain, rather the secondary stain, staining red in color. On the contrary, gram-positive bacteria have a teiochoic acid shield and a thick peptidoglycan layer which hold in the primary stain, staining violet in color. Through an enhanced observation of the morphology of the bacteria’s cell shape is a cocci (spherical), bacillus (rod), or spirilla (spiral). Catalase test: During Aerobic respiration the formation of hydrogen peroxide can form by the reactivity of hydrogen and oxygen. This product is lethal to most cells unless a Catalase enzyme is present to break down the hydrogen peroxide. Most aerobic bacteria have this enzyme while anaerobic are deficient, therefore using other compounds or final electron acceptors. In this test we are exposing the bacteria to hydrogen peroxide. If the bacteria are aerobic, it will have the Catalase enzyme and bubbles should form, signifying the bacteria’s ability to break down the hydrogen peroxide into water and oxygen. If bubbles do not form it is clear indication that the bacteria is anaerobic and does not have the ability to breakdown the hydrogen peroxide into water and oxygen. If bubbles do not form it is a clear indication that the bacteria is anaerobic and does not have the ability to break down the hydrogen peroxide. Glucose Fermentation: By using fermentation tubes one is able to indicate whether the bacteria ferments glucose to drive ATP synthesis or not. The end produces of glucose fermentation is hydrogen gas, carbon dioxide and organic acids. The fermentation tubes is used to detect if these end products are present. These tubes are to be capped and contain carbohydrates, peptone, phenol-red (acid-base indicator) and an inverted smaller tube. Glucose fermentation that results in an acid end product turns the phenol-red indicator yellow due to decreased pH levels, designating glucose fermentation. If gas is present due to fermentation of glucose, some of the gas will be trapped in the inverted tube in the fermentation tubes. Yet after a prolonged period of time there may be an exhaust of the carbohydrate supply causing the bacteria to begin growing oxdatively on the peptone, therefore neutralizing the media and turning the indicator back to the original red color. Therefore, only after a 24 hour time period, positive bacteria that ferment glucose should have gas in the inverted tube and a yellow median. PEA agar (phenylenthyle alcohol agar): The PEA agar selects for gram-positive bacteria, inhibiting growth of Gram-negative bacteria on the PEA plates. TSA agar : This is a medium in which all bacteria grow, it was use to grow our bacteria before we used it for the individual tests.

Results: During this experiment, testing and observations were made to determine unknown bacteria – unknown #56. The first few tests were broad attempts to determine between hundreds of types of bacteria that could be possible. Further testing was used to focus down the possibilities of the types of bacteria present. Eventually the actual genus and species name of the unknown bacteria’s can be identified. The bacteria was inoculated on to both EMB and PEA plates and incubated for seven days. The gram positive bacteria that were in the culture grew on the PEA plate, while the gram negative bacteria grew on the EMB plate. After incubation, a gram stain was performed on the bacteria that grew on these plates. The bacteria that grew on the PEA plate held the violet colored primary stain (crystal violet), concluding that the bacteria were indeed gram positive.

When doing the gram stain it is possible to determine certain distinct morphological characteristics. It is difficult to distinguish whether the bacteria cell shape is cocci or bacillus, yet with further investigation it is conclusive that the bacteria is cocci and arranged in grape-like clusters. After incubation a gram stain was performed on the bacteria that grew on these plates. The bacteria that grew on the PEA plate held the violet colored primary stain (crystal violet), concluding that the bacteria were indeed gram positive. After growth on PEA plate was present, a TSA plate was inoculated with the bacteria. After the incubation period was over, a small amount of bacteria, that had grown on the TSA plate, were then transferred to a slide and exposed to hydrogen peroxide (Catalase test). The bacteria immediately begun to bubble, indicating a positive Catalase test and aerobic bacteria. After indicating that the gram positive bacteria are aerobic a glucose fermentation test was done in order to indicate if the bacteria ferments glucose to drive ATP synthesis. A glucose fermentation test tube was inoculated with the bacteria. After 24-Hour incubation period the teacher’s Assistant pulled the tube out of the incubator. The test produces no gas in the inverted smaller test tube and no color change from red to yellow media, indication a negative glucose fermentation test. There was some uncertainty as to where it was truly a negative result on this part of the lab, so for a more conclusive answer this test was run again just to make sure. The second time around the test came back negative so we continue on with the lab. Indicating that the gram negative bacteria did not ferment glucose, a pigment test on TSA (Trypticase Soy Agar) was done in order to indicate what bacteria it was. A TSA medium was inoculated with the bacteria. After 24 hours of incubation period the teacher’s assistant pulled the tube out of the incubator. The test was then looked to see the color change. A red color would indicate Micrococcus roseus or Micrococcus luteus.
There was an obvious color of yellow in the test tube to indicate that our organism was
Micrococcus luteus. Discussion: The results for this experiment seemed to have very little ambiguity. The only major problem that was encountered was determining if the gram-positive bacteria were cocci or bacilli. The possible explanation for this ambiguity was due to the fact that the bacteria were not left to grow for about a week causing the cell shape to be distorted. Yet through further investigation it was determined that the gram-positive bacteria cell shape is cocci. Other possible problems that could have arose due to lack of growth time, might be that the gram positive bacteria could gram stain red, as if it is a gram negative bacteria, instead of violet. This is due to the degradation of the thick peptidoglycan layer allowing for the primary violet stain to decolorize and have the secondary red, safranin, stain appear. The continual use of the old culture when doing the oxidase test, unlikely yet possible, could have altered the result of the oxidase test, due to a change in the bacteria’s chemical makeup. Other possibilities that may have caused an incorrect interpretation of the oxidase test could be the importance of waiting for a minute or two for the color change that indicates a positive oxidase test. This small detail may be easily glanced over leading to the identification of the wrong bacteria. Over incubation is also an important factor when determining the results for such tests such as glucose, mannose, and mannitol fermentation. As discussed earlier, after a prolonged period of time there may be an exhaust of the carbohydrate of interest causing the bacteria to begin growing oxidatively on the peptone, therefore neutralizing the media and turning the indicator back to the original red color. Therefore, only after a 24-hour time frame a positive fermentation result should have gas in the inverted tube and a yellow median. If the teacher’s assistants were not responsible enough to remove the tubes from the incubator after 24 hours the results may lead one to assume a negative fermentation result instead of a positive result.

It is clear that all sorts of contamination can occur at any time during this three week long lab. To decrease the likelihood of contamination the aseptic technique should clearly always be used. In addition to the aseptic technique, a decrease in the length of time to determine the bacteria and between each experimental test, may also help decrease contamination. Through this experiment it is conclusive that the gram-positive bacterium is Micrococcus luteus. This bacteria has no known pathogenic effect The only thing it seems to do is causes fish to rot. It also seems to be an opportunistic pathogen. It seems to favor people that are already sick or have already had a low immune factor. There is something about M. lutes that separates it from the other bacteria and that is the fact that it is the only one that is positive for growth on inorganic nitrogen agar1. With this in mind one can get further the experiment and take our unknown sample and plate it onto an inorganic nitrogen plate let if incubate for the proper amount of time and see if there is growth on the plate. Secondly M. lutes is also susceptible to lysozyme, while other species are slightly resistant to lysozyme1. With that in mind you can make a experiment to test for growth on an agar with different concentrations of lysozymes on it. From the data you can help narrow which might be M. lutes and which are not. Using the test preformed in the lab I was able to determine that my unknown was in fact M. lutes.