The differential tests used to identify the unknown cultures were oxides, catalane, accost and sucrose fermentation, Keller/iron agar, nitrate reduction, gelatin hydrolysis, starch hydrolysis, amanita salt, MR..-UP, citrate, bile esculents, indolent, areas, Dense, and coagulate. Material & Methods The tests performed on the unknown bacteria cultures were all used to determine the identity of the bacteria. Each of the tests performed provided some key information about the bacteria in question and how it functions.
Not all of the tests were performed on every culture, however, as some of the tests were used only for gram (+) or gram bacteria, while others were even more pacific and used only for Cisco bacteria. The tests performed and what constitutes a positive and negative test are as follows. The oxides test was performed only on gram (-) bacteria and was used to test for the presence of stockroom oxides. Living bacteria were placed on a paper towel and saturated with a carcinogenic reducing agent.
Within seconds the reagent, acting as an artificial electron acceptor, will turn purple if oxidized stockroom oxides is present, indicating a positive test. If no color change is observed, no stockroom oxides is present and the test is negative. The catalane test was performed only on gram (+) bacteria, as this test would not help in differentiating the gram (-) bacteria because all of the possible unknown gram (-) bacteria were catalane positive. This test is used to detect the presence of catalane, which helps to breakdown toxic hydrogen peroxide produced from the transport of high-energy electrons directly to oxygen.
Catalane 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 catalane positive. However, if no bubbles are observed, the culture is negative or catalane. Lactose and sucrose sugar fermentation were tested using a broth containing the respective sugar compound, phenol red, and inverted Durham tubes. The broths were inoculated with the unknown bacteria cultures and incubated for growth.
If fermentation of the sugar molecules was carried out, the pH in the tube would be lowered, and the phenol red would be converted to yellow under the acidic conditions. Thus, the conversion of the originally red broth to yellow signifies a positive test, indicating the bacteria can ferment using either lactose or sucrose. Fifth broth remains red, fermentation on these sugars was not possible and the test is negative. The production of gas by the fermentation was monitored using the inverted Durham tubes. If gas was produced during the fermentation process, the Durham tube would contain a bubble about 2/3 it’s Size.
The iron agar slant was used to test for the fermentation of glucose and lactose, as well as the production of HAS. Gas production was also monitored, looking for fissures produced by production of gas during fermentation. The conversion of the originally red slant and butt of the agar to yellow indicates that glucose orientation took place. HAS could have been produced by the reduction of tessellate contained in the media. Production of this compound will cause a reaction to occur with ferrous sulfate and will result in the production of a black precipitate.
Thus, the culture is positive for HAS production if a black precipitate is seen and negative if one is not observed. Nitrate reduction was tested for by inoculating a nitrate broth with the unknown cultures, and allowing growth to take place. An inverted Durham tube was used again to test for the production of any gas. Adding 10 drops of both Lilliputian acid and -naphthalene to the medium if the first test to see if nitrite is present. If nitrite is present, the medium turns red, indicating a positive test. However, if the medium does not change, a second test is performed to see if nitrite was further reduced.
In this second test, zinc powder is added to the broth to catalyst the reduction of any nitrate present to nitrite. If nitrate is present when the zinc is added the reduction of this compound will cause the medium to turn red, from the previously added reagents. Red medium on the second addition indicates nitrate was not reduced and a negative test result. However, if the medium does not change after the addition of the zinc, the unknown is positive for nitrate reduction, as the nitrite has just been further reduced, preventing its detection.
A gelatins test was performed using nutrient gelatin to determine the ability of the bacteria to produce gelatins. The gelatin was inoculated with the unknown cultures and incubated. The tubes were then placed on ice to reform the warm gelatin. If the gelatin remained solid after inoculation, the bacteria did not produce the gelatins enzymes to liquefy the agar, constituting a negative test. However, if the gelatin was liquefied, the enzymes were secreted and the bacteria were positive. A starch test was performed by plating the unknown cultures on starch agar to see if the bacteria could hydrology the starch in the medium.
Iodine was the dropped on the cultures to react with the starch and form a dark brown color. The observance of a yellow halo around the culture indicates that the starch was hydrolysis and the bacteria were positive for starch hydrolysis. No halo constitutes a negative test. Amanita salt agar is both a selective and differential media used for gram (+) coco. It is selective for salt tolerance and differential for maintop sugar fermentation. It also contains phenol red, which acts as a pH indicator, turning yellow under acidic conditions. The agar is most often used for the selection of S. Russ. Growth and a yellow color change are positive test results. No growth is a negative test result. Methyl red (MR..) is a test used to detect organisms capable of overcoming an added phosphate buffer in the medium to lower the pH of the broth. The test was performed only on gram (-) bacteria, as it mostly tests for enteric who can o this by performing mixed acid fermentation. After inoculation, the broth is incubated for 5 days and then methyl red is added. A red broth is a positive test result, whereas a yellow or orange broth is a negative test result.
Vogues-Prosperous (UP) is a test used to detect organisms that ferment but quickly convert their acid products to action. Addition of the UP reagents (KOCH and -naphthalene) oxidized action to dedicate, which in turn reacts with guanidine nuclei to produce a red color. A positive UP test is red on top of the medium. No color change is negative. The citrate test was performed only on the gram (-) bacteria and was used to determine the ability of an organism to use citrate as its sole carbon source. Bacteria that possess citrate-permeates are able to do this.
The medium used for this test contains biorhythms blue dye, which is green at neutral pH, but blue at a basic PH. Bacteria that are able to survive and utilize the citrate, convert ammonium phosphate to ammonia and ammonium hydroxide, which alkaline the agar, turning it blue. Thus, the conversion of the medium to blue is a positive citrate test. No color change is negative. Bile esculents is a selective and differential media used to select for interconnect and group D streptococci, as they are resistant to bile.
The test was performed only on gram (+) bacteria as the agar slant contains bile salts, which inhibits gram (+) (except interconnect and group D streptococci) and sodium aside, which inhibits gram (-). Interconnect and group D streptococci can grow well on the medium and will usually darken it in 18-24 hrs. Thus, a slant that has not been darkened after 3 days is negative and a darkened slant is positive. The indolent test was performed only on the gram (-) bacteria using SIMI medium ND was used to detect the hydrolysis of thyrotrophic. Indolent will be released if the bacteria culture is utilizing thyrotrophic.
Adding five drops of Kava’s reagent to the culture and looking for the appearance of a red ring at the top of the broth test this. The red ring indicates a positive test. The areas test was performed only on the gram (-) bacteria, testing for the breakdown of urea. Urea hydrolysis to ammonia by areas-positive organisms will overcome the buffer in the medium and change it from orange to fuchsia. Rapid areas-positive bacteria will turn the broth pink within 24 hours. Areas- negative bacteria will produce no color change or turn the broth yellow.
The Dense test was used only for gram (+) Cisco, but more specifically to differentiate S. Epidermis from S. Erasures. Gram (+) bacteria cultures were grown on a Dense test agar plate with methyl green to determine if the unknown produced this enzyme. A positive test results in a clearing around the growth, producing a pink halo. A negative result shows no change in the agar around the growth. The coagulate test was performed again only on the gram (+) Cisco to try to identify S. Erasures. This test is used to identify bacteria that produce a coagulate enzyme.
The unknown bacteria culture was added to rabbit plasma in an offender tube. The coagulation, or solidification, of the plasma within 24 hours indicates a positive test result. If the plasma does not coagulate, the test is considered negative. Results Table 1 : This table shows the results for all of the tests performed on Unknown 7 and Unknown F. The results labeled N/A indicate tests that were not performed on the unknown culture. Unknown 7 Unknown F Gram Stain Gram (+) cardiogram (-) rod Oxides N/A Not Purple (-) Catalane Bubbled (+) N/A
Lactose Fermentation Yellow (+)No gas Yellow (+)Gas Sucrose Oversimplification (+)No gas Yellow (+)Gas Keller/lord Slant Growth, Agar Yellow black precipitate HAS production (-) Growth, Agar Yellowed precipitate HAS production (+) Nitrate Reduction Red after first testes gas (+) Red after first testes gas (+) Gelatin Hydrolysis Solid agar (-) Solid agar (-) Starch Hydrolysis No Yellow Halo (-) No Yellow Halo (-) Amanita Salt Growth on slant and buttonhole agar (+) N/A MR.. N/A Broth turned Red (+) UP N/A NO red Citrate N/A Growth, Blue Agar (+) Bile Esculents Light brown agar color change (-) N/A
Indolent N/A Green Ring on top (-) Areas N/A Peach colored broth (-) Dense Pink Halo (+) N/A Coagulate Turned Solid (+)N/A Unknown 7 is Staphylococcus Erasures. Unknown F is Cacciatore friendlier. Discussion The identification of unknown 7 was determined fairly easily. Once the unknown had been identified as a gram (+) Cisco using a gram stain, three specific tests were used to narrow it down to either S. Epidermis or S. Erasures.
The three test used were catalane, starch, and nitrate reduction. Of all the possible gram (+) bacteria only those two staphylococcus cultures were positive or catalane, negative for starch and positive for nitrate reduction. Two tests, Dense and coagulate, were then specifically performed to differentiate the two possible staphylococcus bacteria. Since the unknown culture 7 was positive for both Dense and coagulate, the culture was highly suspected to be S. Urges. The remaining tests that were performed supported this identification, especially the positive test result seen for maintop salt agar. Amanita salt agar is typically used for the isolation and differentiation of pathogenic staphylococci, principally S. Erasures. The identification of unknown F was a little more difficult as more dependency was placed on the results of two tests. Unknown F was identified as a gram (-) rod using the gram stain.
Based on the positive test results observed for the lactose, sucrose and glucose fermentation, the bacteria identification was limited to four possibilities. The production of the black precipitate indicated that HAS was produced, limiting those possibilities to either Cacciatore friendlier or Kielbasa pneumonia. These two bacteria could then be separated using the MR..-UP and areas tests. As unknown F was found to be MR.. Positive and UP active, the culture was suggested to be Cacciatore friendlier.
The negative results of the areas tests also support this identification. The remaining tests that were performed on unknown F supported this identification, without any contradictions. Conclusion The determination of the unknown identities was achieved using the various differential tests. By performing each test and tracking the results, the unknowns were able to be identified using previously known bacteria data. Unknown 7 was determined to be Staphylococcus Erasures, while unknown F was determined to be Cacciatore friendlier.