Unknown organisms can be identified by using techniques that target specific and defining properties of organisms. In the identification of unknown bacteria, the gram stain can be used to identify whether bacteria are gram positive or gram negative. Gram positive bacteria, which have a thick peptidoglycan layer, stain purple because precipitate from the gram stain reaction remain within the peptidoglycan layer. Gram negative bacteria stain pink or red because the compounds in the gram stain technique remove the outer membrane from bacteria. The secondary stain, safranin, is used to stain the cell wall of the gram negative bacteria. Other stains, such as the Endospore stain and Acid Fast stain, target other cellular components. The Endospore stain works by using dyes and heat to penetrate the protein coat of the endospore, while the acid fast stain uses carbolfuchsin dye to target mycolic acid of Mycobacterium bacteria. Differential test and selective media can also be used to identify unknown bacteria. Selective test only allow certain species of bacteria to grow, inhibiting all others. Differential test target a specific property of organisms to distinguish the colony from other bacteria.
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Stains and media are used as components of dichotomous keys. A dichotomous key is a chart that uses a series of test to identify a type of bacteria. Each test or stain is used to separate organisms based on phenotypic characteristics. To use a dichotomous key, the first test of the key, usually a gram stain, is performed on a sample of bacteria. The result is used to determine the next test that should be performed, until a terminal branch is reached that identifies the unknown organism.
Figure 1 Dichotomous key used for the identification lab.
Three organisms, Unknown A, Unknown B and Unknown 104, were tested for identification. Various types of media were used in the dichotomous key, and results were recorded in the lab notebook as positive or negative. Additionally, any distinguishing properties of the bacteria that help to identify the organism were also recorded.
The Citrate Simmons test is a differential test for gram negative bacteria. Bacteria that can utilize sodium citrate as the exclusive carbon and energy source, along with dihydrogen phosphate (NH4H2PO4) as the single nitrogen source grow in this media. If the media turns blue, the organism is able to use citrate as a carbon and energy source. Bacteria that cannot use citrate as a carbon and energy source will not increase the pH, and the media will remain green in color (Difco Laboratories).
Phenol red is a differential test that shows if a particular bacteria species is fermentative. It uses carbohydrates, peptone, phenol red as an indicator, and a Durham tube to collects gas, if produced by fermentation. If the organisms can ferment the sugars and produce acidic products, the color of the media will turn yellow. If gas is produced, an air bubble will form in the Durham tube. If the bacteria cannot utilize the carbohydrates, the bacteria may be able to utilize the peptone in the media, which then becomes fuchsia or red colored (Difco Laboratories).
Methyl Red-Voges Proskauer medium, a differential test, utilizes two different types of reagents to test for the type of fermentation a bacterial species uses. The methyl red pH indicator is used to test for bacteria that undergo the mixed acid fermentation pathway. The majority of products are acidic, which reduces the pH in the media and allows the methyl red indicator to change the media to red. The Vogues-Proskauer test is used to test for bacteria that use the butylene glycol, or butanediol fermentation pathway. The diacetyl, which is made while the bacteria are in the media fermenting the sugars, produces a red color, which is indicative of a positive VP test (Difco Laboratories). For methyl red, a positive test is red in color; negative tests are yellow in color. For the VP test, a red color within five minutes of adding the reagent is a positive result (Difco Laboratories).
The Nitrate (NO3) test is a differential test that shows if an organism can reduce nitrate to nitrite using the enzyme nitrate reductase. This test is usually used on gram negative aerobic or facultative anaerobic bacteria. If the organism is able to reduce nitrate, which is in the form of potassium nitrate in the media, to nitrite, the nitrite will react to the sulfanilic acid (reagent A). The product of the reaction will then react to N, N-dimethyl-alpha-naphthylamine (reagent B) and produce a red color, which is considered a positive result (Difco Laboratories).
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Zinc dust, or reagent C, is used as a final step if the solution does not turn red. Zinc reduces any nitrate that was not reduced by the enzyme nitrate reductase. The nitrite produced by the zinc reaction then reacts to the reagent A and B that was previously added, forming a red coloring. If the solution does not turn red, then the nitrate was reduced completely into molecular nitrogen and ammonia. This type of reaction is called a complete positive. The Durham glass can collect nitrogen gas, which is a typical end product of the dentrification pathway (Difco Laboratories).
After incubation, if gas is observed, then nitrate was reduced to nitrate gas, and the test is positive. Ten drops of both reagent A and B are added, and a red color formation within two minutes is a positive result. If the color does not change, then 20mg of reagent C is added. A red result is a negative result, while no color change within 5-10 minutes is a positive result (Difco Laboratories).
The skim milk test is a simple differentiating test that shows whether a bacterial species can break down casein, which suspends in milk and gives milk its white color. Bacteria that can break down the casein protein into smaller peptides or amino acids use the enzyme casease, which works by breaking down casein outside the cell. This allows the cell to bring in the material and utilize it. In most tests, colonies are inoculated onto a plate, followed by the addition of the skim milk. Any clearing is considered positive (Difco Laboratories).
SIM media is an abbreviation for sulfur reduction, indole production, and motility. It is a differential test that tests for the three properties previously listed. Bacterial sulfur reduction can produce hydrogen sulfide gas via the enzyme thiosulfate reductase. In the media, the sulfur source, sodium thiosulfate can be reduced by the bacteria. If hydrogen sulfide gas is made, it reacts with the ferrous ammonium sulfate in the media and produces a black precipitate. Therefore, black colored media are positive for sulfur reduction. Indole can be produced by bacteria that utilize tryptophan via the enzyme tryptophanase, which is a composition of the casein peptone within the media. To test for the presence of tryptophanase, three to four drops of Kovacsâ€™ reagent is added. If a red color forms, then the bacteria are indole positive. Lastly, motility can be observed if growth is formed growing out from the stab area or if the media is cloudy in texture (Difco Laboratories).
Eosin Methylene Blue agar is both a differential and selective media for gram negative organisms. The methylene blue and Eosin Y dyes both inhibit certain gram negative bacteria from growing and are indicators of fermentation of lactose and/or sucrose. Bacteria take up both the products of fermentation and the dyes. Because fermentation lowers the pH, colonies that ferment the carbohydrates are dark blue to black in color, due to the blue-black complex in their cell walls.
Mannitol Salt agar is a selective media for staphylococci and differential for mannitol fermentation. The media contains a concentration of 7.5% sodium chloride, which partially or completely inhibits the growth of organisms other than Staphylcocci. Other organisms can grow in the solution, but they grow very weakly. Phenol red indicator turns yellow when mannitol is fermented, due to the acidic byproducts of fermentation. Mannitol is the only sugar in the medium. Nonfermenting organisms would turn pink, due to the breakdown of peptone within the solution (Difco Laboratories).
The Catalase test identifies organisms that can produce the enzyme catalase. All bacteria, when utilizing the electron transport chain, undergo the risk of creating hydroxyl radicals or other agents that can damage the cell. Some species are able to create the enzyme catalase, which is needed to protect the cell against hydrogen peroxide or its radicals. In order for these compounds to occur, flavoproteins in the ETC react with O2 to make oxidized flavoproteins and hydrogen peroxide. Hydrogen peroxide can also form hydroxyl radicals. Catalase, which is not a ubiquitous enzyme, catalyzes hydrogen peroxide conversion to water and oxygen gas (Difco Laboratories). After flooding 1-2 drops of hydrogen peroxide onto a slide, bubble formation indicates a positive result. If no bubbles are seen, even after looking under a microscope, the bacteria are catalase negative. The bubbling is the result of oxygen gas formation (Difco Laboratories).
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For Unknown A, the gram stain streak plate contained very small white colonies. After gram staining, the colonies stained pink and were rod shaped. The EMB test produced around 3 mm of growth. The bacterial growths were black in color against the burgundy colored media. The citrate slant, which was inoculated with bacteria from the EMB media, was green colored throughout the tube.
For Unknown B, colonies were yellow in color and three times larger than the colonies in unknown A. After gram staining, the colonies stained purple and were cocci shaped. The phenol red test with lactose turned yellow and produced gas. The catalase test produced bubbles. However, the bubbles were slow to form and few in number. In salt mannitol media, the bacteria grew and produced bright yellow colonies.
For Unknown 104, colonies were off-white in color and small, around the same size as unknown A. After gram staining, the colonies stained purple and were cocci shaped. The phenol red test with lactose turned yellow and did not produced gas. The catalase test produced many bubbles immediately after hydrogen peroxide application. In salt mannitol media, the bacteria grew and produced pink colonies.
For Unknown A, the bacterial colonies were gram negative. The Eosin Methylene Blue test was positive, meaning that the unknown can ferment lactose and/sucrose. This rules out Pseudomonas aeruginosa, Burkholderia capacia, Proteus vulgaris, and Serratia marcescens. The citrate test was negative. Out of the remaining three organisms, Escherichia coli was the only organism that was citrate negative. Therefore, Unknown A is E. coli.
E. coli is found in mammals, both in the lower intestine and feces, and in areas of extremely high temperatures, such as on the edge of hot springs. Some strains of E.coli can be pathogenic. E. coli O157:H7, for example, produces a toxin that is similar to shigella toxin after ingestion of contaminated food. Other strains can cause urinary tract infections, mastitis, diarrhea, and even kidney failure. Other species are beneficial to mammals, and aid in digestion, absorption of nutrients, and vitamin k production. Additionally, some species can function as water purification indicators, due to their greater population in human feces than other microorganisms. In biotechnology, recombinant E. coli is used to produce proteins and enzymes, most notably human insulin for diabetes patients. Other uses for E. coli include rapidly producing human abeta sequence in order to combat Alzheimers disease (Slonczewaki and Foster 2008).
For Unknown B, the bacterial colonies were gram positive. The Unknown test was Phenol red positive, and produced gas. These results rule out Bacillus subtilis, Microcococcus roseus, and Corynebacterium jeikeium, which do not ferment lactose. The catalase test was positive, ruling out E. faecalis and Lactobacillus acidophilus. These organisms do not produce the enzyme catalase. To distinguish between the last two possibilities, Staphylococcus aureus and Staphylococcus epidermis, the salt mannitol test was used. The bacterial growth was yellow in color, making the Unknown B S. aureus.
S. aureus is found on the skin of humans, particularly in the mucous membranes of the nose. It is part of the natural flora of humans, but can become pathogenic if it enters a wound in the skin. The bacteria can cause multiple diseases, including mild skin infections, toxic shock syndrome, osteomyelitis, and a plethora of other diseases. Current research on S. aureus includes building a protein expression index and creating a super drug for the developing strains, many of which are drug resistant (Slonczewaki and Foster 2008).
For Unknown 104, the bacterial colonies were gram positive. The Unknown test was Phenol red positive, and did not produce gas. The results rule out B. subtilis, M. roseus, and C. jeikeium, which do not ferment lactose. The catalase test was positive, ruling out E. faecalis and L. acidophilus. These organisms do not produce the enzyme catalase. To distinguish between the last two possibilities, S. aureus and S. epidermis, the salt mannitol test was used. The bacterial growth was pink in color, making the Unknown B S. epidermis.
S. epidermis lives in the human body, mostly on the skin. S. epidermis is a pathogenic bacterium that can cause infection on biomaterials, such as prosthetic valves. The bacteria attach to medical devices and forms biofilms. Because of the structure of the biofilms, bacteria inside the biofilm are resistant to antibiotics. This allows bacteria to break off and form biofilms and infection throughout the body. In biotechnology, S. epidermis is used to flavoring material for food by synthesizing esters, which give flavor to many types food. In addition, new techniques and compounds are being tested to combat the bacteriaâ€™s virulent biofilms (Slonczewaki and Foster 2008).