Process and Techniques of Finding Unknown J

Abstract: As part of a focal study within Microbiology, many scientists have endeavored to identify the many species and types of microbes found in different environments. In an effort to categorize, understand and distinguish one microbe from another, scientists developed tests to show unique characteristics of microbes. This experiment enlists these tests, such as PCR, Simple and Gram staining, anaerobic growth tests, IMViC, Catalase, Oxidase, selective and differential media to identify an unknown microorganism. The Unknown organism studied was labeled “J” and found to be a gram negative, rod shaped bacteria that does not produce endospores. The selective and differential agars produced no growth on the MSA agar plate showing that the bacteria did not favor a salty environment of the Mannitol salts and showed an acidic by product in the selective and differential media of MacConkey’s Agar. The bacteria showed to metabolize sugars but did not produce any gaseous byproducts. After 16s rRNA was processed and run through a PCR, electricphoresis was used to run the RNA out on a gel in order to sequence the RNA which was then compared in a database. Furthermore, the Unknown J did not produce or metabolize starch. The bacteria did not react with the Citrate, KEY oxidase. When compared to other known bacteria tested, unknown J proved to be Escherichia coli.
Introduction:
Identifying bacterial causes for certain diseases that have affected the population or identifying growth on old bread is not new to science. Joseph Lister developed one of the first ways to separate the desired unknown bacteria from other bacterial colonies in 1867. Lister’s aseptic technique, used in both science and healthcare, destroys the microbes that are growing on the tools before they are utilized (Carolynm, 2009).This technique applies to microbiology and allows the microbe of interest to grow so individual colonies, or a manageable amount of colonies can be studied. Due to the development of the aseptic technique, scientist are able to separate the desired bacteria so it could be studied and allows for new tests to be developed differentiating different species of bacteria and further developing multiple ways to detect unique and identifiable traits within the microbe. The light microscope allows scientists to look at the morphology of the different organisms at different phases. Simple staining and gram staining determines if the microbe of interest is a gram positive or gram negative along with other more defined traits seen after the microorganisms have been stained. Furthermore, different agars are used to select for certain traits within each microbe, for example, the MacConkey’s agar which is both selective and differential. Endospore staining and capsule staining are performed on the bacteria, determining the presence or absence of spores or capsules, further defining the unknown microbe. After the staining, fermentation pathways are observed using different tests such as carbohydrate fermentation, ONPG test, triple iron agar test, starch hydrolysis test, anaerobic growth, IMVic tests (Indole test, Methyl red, Voges-proskauer, & Citrate utilization), the catalase test, and oxidase test. These were all done to determine the pathways in which the unknown bacteria used and compare them to the known bacteria that were being tested. The final test run on the bacteria was the PCR on the unknowns RNA and comparing the RNA sequence obtained from the PCR to the Bernie’s Manual. This was to see how accurate the test were at indicating what bacteria the unknown specimen was and the ability for the groups to be able to keep the unknown from becoming contaminated.
Methods:
For the unknown, the first and most important thing to do was learn the aseptic method of transferring the culture into a new sterile container to continue the growth process without contamination, which would allow for the results to be accurate in indicating which microorganism the unknown is. After that technique is learned, the ability to see and tell the differences between the different morphologies it important. To accomplish this, the different bacteria’s and the unknown were looked at under the light microscope at different phases and magnification. Next, staining was done to see what initial group the unknown mirrored. The first staining to be done was the simple staining, which allowed for a better look at the cultures morphology. This simple staining was done by adding using an inoculated loop to transfer the desired specimen to a sterile slide before adding methylene blue to the slide for a minute before rinsing for 30 seconds and blotting dry so it could be observed under the microscope. The gram staining was done first by taking an inoculated loop of desired specimen and placing on sterile slide than adding crystal violet stain for 30 second, rising with water for 5seconds, gram iodine for a minute, rinsing for five seconds, decolorize for 15 seconds, rinsing for 5 seconds, safranin for a minute, and finish with another rinsing for 5 seconds before blot drying and looking at it under the microscope. The next staining done was for endospore, which was started with an inoculated loop placing desired specimens on a sterile slide. The slide was then put over a container of boiling water and malachite green was applied to keep the culture moist while is heated for 5 minutes. The slide was then allowed to cool and was rinsed with water for 30 seconds before safranin was added for a minutes before being rinsed again and dried before viewing. After that, different Medias were used to select and differentiate the unknown and the other bacteria. The two agars used for this was MacConkey which select for gram negative while being differential for fermentation, Mannitol salt is selective for organisms that can grow in its harsh conditions, and the starch agar that selects for the ability metabolize the carbohydrate.. The plates were sections out with labeling before inoculated loops added the desired culture in the spread plate technique. The plates were allowed to grow for a week before observations were taken. Next the ability for the unknown organism to metabolize sugars was looked at in a series of test. The first test done was looking at carbohydrate fermentation and OMPG. For this test, inoculated loops were used to move desired specimens to individual suspension of 1.5 mL and mixed before the carbon tablet was placed in the tube and incubated for a week. The next sets of fermentation test done were Indole test, Methyl red, Voges-proskauer, & Citrate utilization. These were done by using an inoculated and putting the desired specimens into 1.5 mL of water and mixing. The corresponding tablets were dropped into each tube (one each) and observed after two hours than again a week later. Following that, the triple sugar iron agar test was used. The slats were first streaked with an inoculated loop that was used to move the desired organisms to the tube, that it was also stabbed so the bacteria would be in the butt of the tube as well. The bacteria’s were then allowed to be incubated for a week before observations were taken. The catalase test was then used to further identify the unknown. This was done by using an inoculated loop to transfer desired bacteria to a sterile slide and adding 3% H2O2 before observing reaction. Lastly, the oxidase test was used to see the biochemistry within the bacteria. This was down by placing some of a strip of the oxidase paper into the slat and seeing if the color changed within 30 seconds. The Very last thing done was the running of the PCR. This was done by taking 1 mL of broth and 1.5 mL of unknown that is centrifuge at 12,000 rmp for five minutes before decanting and adding 1 mL of sterile water and let boiled for 15 minutes before it is allowed to cool. At this point, .2 mL of pcr are added, than the Taq is added and mixed before it is put on in the gel and ran for 30 minute. A picture was then taken and the data was recorded and compared.
Results:
Lab Test | Results | | Escherichia coli | Unknown J | Stain Tests | Endospore Stain | does not produce spores | Stain showed no proof of spores present in either stain performed | | Gram Stain | Gram Negative | Stained Pink on the slide - Gram Negative bacteria | | Methyl Blue | Rod Shaped Bacteria | Showed bacillus Shaped Bacteria, but some looked like they could potentially be coccus | Differential and Selective Agars | MacConkey's Agar | Purple plate, all over growth | Purple, pink plate, all over growth | | Mannitol Salt Agar | No Growth | Very, Very, Very little growth, Red plate | Sugar Metabolism | Mannitol | Tube turns yellow but no bubbles produced | Week 1: pink and No reaction Week 2: Yellow and no reaction | | Dextrose | Tube turns yellow but no bubbles produced | Week 1: Orange and no reaction Week 2: Yellow and no reaction | | Lactose | Tube turns yellow but no bubbles produced | Week 1: Orange and no reaction Week 2: Yellow and no reaction | IMViC Metabolism tests | Methyl Red | Red | Red | | Indole | Red | Red | | VP | Yellow and dark at top | Yellow but darker at top | | Citrate | Negative | Negative | β-galactosidase | Reaction is produced | Week 1: No reaction Week 2: Yes | Carbon Iron Agar | Gas is produced and there is color change from red to yellow and forms a small amount of precipitant | Gas: yes, Color: Red to Yellow, Precipitation: Yes a small amount in the butt of the tube | Catalase | Reaction is produced | Yes, bubbles produced | KEY Oxidase | No reaction | No | Starch Presence | No reaction, does not produce starch, halo or change color of dye | No starch presence, no halo and the dye remained blue | * Table 1: This table goes over the test done on the unknown and E.coli.

* Figure 1 is a picture of the PCR gel after electrophoresis for unknown after weeks of use.

* Figure 2 is the PCR gel of the unknown before use,

AGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGgtaacaggaagc agcttgctgctttgctgacgAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAAC-------GTCGCAAGCA--------CAAAGAgggggaccttagggcctcTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCAACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCNGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAagggagtaaagttaatacctttgctcat-TGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGcccttgagg--CGTGGCTTCCGGANNTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGG-CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGATGAGAATgtgccttcgggaaCCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGgtccggcCGGGA-ACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTagcttaacttcgggag-----GGCG * Figure 3 is the sequence of the PCR Genome for the unknown The data from the different test of the unknown were compiled into a data table than compared against the known bacteria until for similarities. These similarities were than check by the RNA sequencing done in the PCR gel that was ran to see if the fight conclusion were found or if the unknown sample was compromised. The results indicate through the different test, that the unknown is E. coli. This was backed up by the PCR gel that RNA sequenced showed that the unknown was E. coli. All results for the test done on the unknown expect for the PRC, can be seen in a side by side comparison to E.coli. For staining, the test on the unknown does not show signs of endospores, that it is a gram negative bacterium, and has a bacillus coccus shape. The staining on the E. coli showed no indication of endospores, that it is a gram negative bacterium, and that it is rod shaped. For the different media growth, the unknown had growth all over on the MacConkey plate, it was able to metabolize and grow on the starch plate, and very little growth on the Mannitol salt plate. The different media plates for the E. coli showed that the there was growth all over the MacCockey plate, it was able to metabolize and growth on the starch plate, and there was no growth at all on the mannitol salt plate. For the sugar matabilism test, the unknown had not reaction with mannitol, no reaction with dextrose, and no reaction with lactose. For the sugar metabolism for E. coli showed that there was no reaction with mannitol, no reaction with dextrose, and no reaction with lactose. For the IMViC test for the unknown, the Indole test was positive, Methyl red was positive, Voges-proskauer was negative but dark at the top, and Citrate utilization was negative. In mirroring results, E. coli showed that the Indole test was positive, Methyl red was positive, Voges-proskauer was negative but dark at the top, and Citrate utilization was negative. For the β-galactosidase test was positive for both the unknown and E. coli. For the triple sugar iron agar test, the unknown and E.coli, both produced gas, the top of the slants color changed to yellow, and there was a small amount of color change in the butt of the tube. In the catalase test, both the unknown and E. coli had a reaction which produced gas bubbles. Lastly, in the Oxidase test, either the unkown or E. coli had a reaction. For the PCR gel, the metric ruler was use to find the weight of the different segments of the unknown which can be seen in figure 1. These measurements were than compared to the PCR gel of the unknown that was taken before the semester started, which can be seen in figure 2. The measurements on the two in comparison the metric ruler were not exact since the original had a mark at 100, 600, 1000, and one that is higher that the ruler, while the PCR done at the end of the testing had marks at 100, 800, and one above were the metric ruler reaches. The second step for the with the PCR was getting the genetic sequence of the unknown which can be seen in figure 3. This was done by putting the sequence into seqtool.sds.edu and picking RNA and The computer identified the sequence as E. coli based off the Bernie’s Manual.
Discussion:
The unknown bacterium was labeled J and a series of tests were performed over consecutive weeks in order to determine unique characteristics that would help to identify the organism. The bacterium was continuously inoculated into new plates or slants to use the following weeks. These transfers, potentially, could have contaminated the sample, but the integrity was continually checked using morphological and physiological data collected during the initial observation of the bacterium. Without a microscope the bacterium were a white, opaque color slightly rounded and smooth when grown on a slant or on a plate. Under a microscope, the physiological traits observed were a slight coco-bacillus rod type clumped together. Once the gram stain was performed the cell showed a distinct red or pink tint indicating that the bacteria was gram negative, due to complications with the first test which rendered questionable results, the test was done again in which the result was as stated above. The bacteria cell was then tested for endopores by staining and the bacteria showed that there were no endospores produced from the bacteria when it was exposed to a stressful environment. These characteristics were consistent with that of the known E.coli characteristics. The second grouping of tests compared the reaction of the unknown bacterium to different environments, the first McConkey’s agar and the second Mannatol Salt Agar. McConkey’s agar is both differential and selective. The only bacterium that grows on the plate is Gram negative and differentiates between different lactose fermentations. The results reinforced the finding that the unknown organism was a gram negative and that the lactose fermented produced the acid by-products required to keep the plate a dark purple. Mannitol Salts provided an extreme environment selecting for the bacteria only able to adapt to salty environment. After inoculation and incubation, the plate showed very little growth, and the two small colonies seen did not seem to share the same physiology as they were more of a solid opaque but was smaller than any of the previous colonies observed on the agar plates. This coupled with the metabolism tests showed that mannitol, dextrose and lactose could be digested, however there was no gas produced within the reaction. These findings solidified further the similarities between the unknown microorganism and E. coli, and furthermore, the bacteria did not produce starch or a halo when tested with Gram iodine. The final step in identifying the unknown was to sequence the 16s rRNA of the unknown and compare it with the known sequence of E. coli. The rRNA was run through a PCR in order to increase the amount of RNA which was then run in an electrophoresis showing the sequence of the RNA. This was compared to the known sequence found in E. coli and was, relatively, close in comparison. There were problems with this test however as it had to be restarted because the agar was made incorrectly the first time. Furthermore, the ruler that was added to the electrophoresis allowing us to measure the length of the strand and by that the sequence of the RNA did not run as far as the other tests and was difficult to see even with the light. After the electrophoresis was run, the sequence was then compared using a computer program to create a tree linking all other unknowns with Unknown J. In comparison, all tests showed a great similarity with E. coli bringing about the same results in each test. Some problems with this experiment resulted from the detailed techniques or improperly conducted tests; these however were more easily rectified as most problems were solved by performing a new test. Furthermore, the results from each test supported the results of other tests strengthening the accuracy behind each. Keeping the bacterial culture pure was difficult but seemed to be achieved as each test followed and coincided with the known results of E. coli. Works Cited
Carolynm. 2009. Aseptic Technique Gowning, Gloving, Scrubbing. Study mode. http://www.studymode.com/essays/Aseptic-Technique-Gowning-Gloving-Scrubbing-247782.html