Basic Local Alignment Search Tool (BLAST)
The Basic Local Alignment Search Tool (BLAST) was the final portion of this lab report. BLAST is provided courtesy of the National Center for Biotechnology Information (NCBI). After running the five chosen tests, students were given the two 16S rRNA sequences that were associated with their assigned culture. The BLAST results served to confirm or disprove the hypothesis of what the unknown may be. The BLAST program compared the given 16S rRNA sequence to a database of known sequences and searched for similarity. (10) This search tool is capable of comparing nucleotide or protein sequences to find statistical significance between the matches. A perfect match to the unknown sequence is indicated by a
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There were two different types of colony forming units present on the TSA plate after incubation. The one chosen to be used for identification of unknown 28 was the larger of the two colonies. The chosen colony was roughly 1mm in diameter and were round in shape. The color was white and slightly waxy. The colonies were convex in elevation and had smooth margins. When compared to the controls in the laboratory, the unknown colony was considerably smaller in size. The Staphylococcus epidermidis control colonies were larger in size and lacked round margins . Although the color between the control and unknown was similar, S. epidermidis had colonies with irregular margins and plateau …show more content…
After appropriate incubation, sulfanilic acid (reagent A) and dimethyl-α-napthylamine (reagent B) were both added to the test tube. A color change should have been observed after approximately one minute if the unknown was positive for nitrate reduction (1). However, no color change was present which indicated no nitrate reduction. As a confirmation test, powdered zin was added to the tube. There was an immediate color change to red after the addition of zinc. The color change from zinc indicated that there were still intact nitrates within the broth, thus, no reduction had occurred. The positive control for nitrate reduction was E. coli which changed to a red color after the addition of reagents A and B. The negative control was M. luteus which had
This test is used to detect if the bacteria contains any deoxyribonuclease activity. Because no color change was observed from blue to clear my unknown bacteria displayed a negative result.
This experiment was conducted to find the genus and species of an unknown bacteria prescribed by the lab teacher, which was unknown bacteria GA3 in my case. Identification of unknown bacteria techniques are used on an every day basis to figure out what type of bacteria it is and to find the best method of how to treat a patient with this bacteria (1). All five “I’s” of Microbiology were used in the testing for the unknown culture. Inoculation was used several times to put the unknown culture into agar plates or into biochemical test tubes. After Inoculation of these tubes or plates, they always were placed into the incubator for further growth and development. Isolation was used to make sure we got the correct bacteria we were testing for. After each further isolation, we gram stained the culture and inspected the culture under a microscope to further help in the identification process of the unknown bacteria. Multiple tests were done on the unknown culture to make sure we were confident in what kind of bacteria the unknown was.
In this experiment, the soil sample that was used in the first experiment was used for even further characterization. A colony was chosen from one of the group member’s bacteria and was amplified with PCR and had gel electrophoresis conducted in order to obtain a sequence. The purpose of this was to figure out the DNA base sequence of the 16S RNA gene from the group member’s colony that was chosen. Then, the BLAST website would be used to determine what the unknown organism was based on the sequence. Additionally, each of us performed a line streak with Streptomyces, E. coli, and S. epidermidis in order to test for the presence of antibiotics.
The possible organisms in this lab included: Serratia marcescens, Escherichia coli, Bacillus subtilis, Sarcina lutea, Micrococcus luteus, Pseudomonas fragi, Staphylococcus epidermidis, Alcaligenes faecalis, Micrococcus roseus, and Clostridium sporogenes.1 In start of the elimination process, the two nutrient agar plates that were made by unknown tube number two were observed and characterized by their colonies. Gram positive: Plate A was incubated at 37 C and had a colony appearance of bright yellow dots on the nutrient agar. This eliminated the possibilities of S. marcescens, E. coli, B. subtilis, P. fragi, S. epidermidis, A. faecalis, M. roseus, and C. sporogenes. S. marcescens colonies are red; M. roseus colonies are orange; E. coli and A. faecalis colonies are beige; B. subtilis, C. sporogenes, and S. epidermidis colonies appear in white.2 This left
Lab Day 1: After receiving my unknown bacteria, I streaked a TSA plate and incubated at 37°C for 48 hours. I then picked a single colony from the plate with my sterile loop and streaked a TSA slant and labeled it “Working Stock”. I did the same with another TSA slant and label the second one “Back-up Stock”. This would be the samples I used to complete the following procedures through the next four weeks to determine my unknown bacteria.
Nitrate reduction was tested for by inoculating a nitrate broth with the unknown gram (-) culture, and allowing growth to take place. Adding 2 drops of both sulfanilic acid and α-napththylamine 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 catalyze 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. The result that yielded was positive on the first step.
A highly conserved gene will be used to identify a prokaryotic species isolated from the body. Fundamental lab techniques will be also explored and utilized, such as amplifying using PCR, cloning, and transforming the gene into a host cell. DNA electrophoresis and specific substrate plating will serve as analysis check points. The final product will be sequenced and compared to similar species to observe phylogenetic relationships.
The purpose of this lab was to identify an unknown bacteria culture using differential tests. The identification of the unknown culture was accomplished by identifying the bacteria based on its specific metabolic characteristics and morphology. It is suggested that culture 11 is a sample of Enterobacter aerogenes.
Many species’ genes have not been sequenced, there could be multiple matches found once more species are added to the genomic database.
All DNA tests were changed in accordance with 100 ng/μl. A 1000 ng (1 μl) aliquot of every example's DNA was utilized for a 50 μl PCR response. The 16S all inclusive eubac-terial preliminaries 530F (5'- GTG CCA GCM GCN GCG G) and 1100R (5'- GGG TTN CGN TCG TTG) were utilized for increasing the 600 bp area of 16S rRNA qualities. HotStar Taq Plus Master Mix Kit (QIAGEN Inc.) was utilized for PCR under the accompanying condi-tions: 94°C for 3 min took after by 32 cycles of 94°C for 30 sec; 60°C for 40 sec and 72°C for 1 min; and a last prolongation venture at 72°C for 5 min. A second-ary PCR was performed for FLX (Roche, Nutley, NJ) amplicon sequencing under the same condition by using designed special fusion primers with differ-ent tag sequences: LinkerA-Tags-530F and LinkerB-1100R. The resultant individual sample after parsing the tags into individual FASTA files was assembled using CAP3. The ace files generated by CAP3 were then processed to generate a secondary FASTA file containing the tentative consensus (TC) sequences of the assembly along with the number of reads in-tegrated into each consensus. The TC was required to have at least a 3-fold coverage. The resulting TC FASTA for each sample was then evaluated using BLASTn (Altschul et al., 1990) against a custom data-base derived from the RDP-II database (Cole et al., 2005) and GenBank website (http://www.ncbi.nlm. nih.gov/). The sequences contained within the
The Unknown Bacteria 36/Bacteria # 2 on a TSA plate was examined by the naked eye and under a dissecting microscope. Bacteria # 2 was approximately 3 - 4 mm in diameter. They were circular in form with an entire margin and a flat elevation. The colonies were rough (granular), translucent, and white brownish color with black granules. The Gram stain resulted in a Gram negative rod. After the Gram stain was completed, the bacteria were streaked on an Eosin -Methylene Blue Agar plate and an Enterotube II was inoculated.
However, nitrate reductase can further reduce nitrite to ammonia or molecular nitrogen, which does not react with the reagents and does not produce a colour change. To verify nitrate has been reduced past nitrite, zinc powder is added to the medium. If the medium does not change colour after the addition of zinc powder, then the nitrates in the medium have been reduced further. If the bacterium does not use nitrate, then the addition of zinc powder will reduce the nitrates in the medium to nitrite, changing the medium to a pink or red colour.
66). The two TSA plates were incubated both aerobically and anaerobically because it displayed how oxygen affected the growth of each organism. Both aerobically and anaerobically incubated plates displayed identical white, convex elevated isolated colonies with entire margins and glossy, convex elevated isolated colonies with entire margins. Therefore, this procedure did not eliminate any of the possible organisms due to the fact that S. aureus, S. epidermis, E. faecalis, E. aerogenes, P. vulgaris, and P. aeruginosa all grow under aerobic and anaerobic
Gram positive staphylococcal and streptococcal species are significant human pathogens and are responsible for at least “a third of all bacterial infections”. These are significant considering that a majority of the species are found in the normal microflora of humans and are opportunistic pathogens. As they are found in the normal microflora of each individual, isolating them is made possible through the employment of various culture based methods and chemical tests. To isolate, identify and characterise species of the medically relevant bacteria I took samples from my nasal mucosa, oropharynx, dental plaque and together with a combination of culturing on different types of agar medium, chemical tests such as catalase, optochin, bacitracin,
EST sequences with vector sequence were edited using Phrap “cross-match” application. Contig Assembly Program 3 (Cap3) was used to assemble the sequences obtained from sequencing for analysis while Consed/Autofinish software was used to control the sequence assembly. All sequences were assembled separately into contigs. BLAST of sequences was conducted to determine the gene homology in order to connect their functions. Unique sequences were analysed for biological characteristics as well as functional annotation using program BLAST2GO. New genes can then be identified eventually.