In the experiment, five periods are done to accomplish the goal of identification of the unknown microorganism. The first period aims to isolate the genomic DNA from the unknown microorganism using a Q1Aamp DNA mini kit, leading to the second period being the amplification of 16s rDNA consensus sequence by PCR, particularly using the primers RW01 and DG74. The unknown sample is then taken for gel electrophoresis to confirm and purify the amplified 16s rDNA fragment, done in the third period of the experiment. Once the running of the gel is completed, a cut of the 370bp PCR fragment is taken, and is put for purification of 16s rDNA fragment by the QIAquick gel elution kit, allowing the DNA to elute at the bottom of the microcentrifuge tube …show more content…
These sequences can then be aligned precisely, to indicate any differences to be easily measured. Therefore, genes that encode the rRNA or rDNA have been extensively determine taxonomy, phylogeny, and to estimate rates of species divergence among bacteria. The function of the 16s rDNA is highly conserved among various bacterial species, hence, random sequences change accumulated during the evolution, and to be used to identify different bacterial species. To go in more depth, according to the article, Then and Now: Use of 16s rDNA Gene Sequencing for Bacterial Identification and Discovery of Novel Bacteria in Clinical Microbiology Laboratories, written by various authors including Woo PC, Lau SK, and many other contributors, “In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has plated a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories.” The identification of bacteria by the 16s rDNA sequencing is important in particular for the cases of bacteria with unusual phenotypic profiles, rare bacteria, slow growing bacteria, uncultivable bacteria, and culture-negative bacteria infection. In addition, provides clinicians choose appropriate antibiotics and determine the duration of treatment and infection control procedures. Concluding, that the comparison of 16S rDNA sequence can exhibit evolutionary relatedness among
I identified the genus and species of an unknown bacterial culture, #16, and I applied the following knowledge of morphologic, cultural and metabolic characteristics of the unknown microorganism according to the laboratory manual as well as my class notes and power point print outs. I was given an incubated agar slant labeled #16 and a rack of different tests to either examine or perform myself; the tests are as follows: Gram Stain; Nutrient Gelatin Test; Carbohydrate Fermentation; Dextrose, Lactose and Sucrose; IMVIC tests; Citrate, Indole, Mythel-Red and Vogues Proskauer test; as well as a Urease and TSI Test. Materials and Methods/Results Upon receiving the Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed a sterile drop of water on the slide and used an inoculating loop, flame sterilized, I took a small sample of the unknown growth in my agar slant and smeared it onto the slide in a dime sized circle and then heat fixed it for ten minutes.
Solid A was identified to be sodium chloride, solid B was identified to be sucrose, and Solid C was identified to be corn starch. Within the Information Chart – Mystery White Solid Lab there are results that distinguishes itself from the other 4 experimental results within each test. Such as: the high conductivity and high melting point of sodium chloride, and the iodine reaction of corn starch. Solid A is an ionic compound due to its high melting point and high electrical conductivity (7), within the Information Chart – Mystery White Solid Lab there is only one ionic compound which is sodium chloride, with the test results of Solid A, it can be concluded that is a sodium chloride. Solid B was identified as sucrose due to its low electrical
1 / 3 BIO3001 Shinhye Jeon (Heather) Professor Wahlert November 21, 2017 Identifying of Spores Belonging to the Division Pterophyta by Utilizing Phylogenetical method 1. Abstract
The purpose of the Unknown White Compound Lab was to identify the unknown compound by performing several experiments. Conducting a solubility test, flame test, pH paper test, ion test, pH probe test, conductivity probe test, and synthesizing the compound will accurately identified the unknown compound. In order to narrow down the possible compounds, the solubility test was used to determine that the compound was soluble in water. Next, the flame test was used to compare the unknown compound to other known compounds such as potassium chloride, sodium chloride, and calcium carbonate. The flame test concluded that the cation in the unknown compound was potassium. Following, pH paper was used to determine the compound to be neutral and slightly
Forensic Science Introduction: Someone in a restaurant has suddenly fallen ill and a mystery powder has been discovered with the victim. As the chief investigator, your duty is to identify the mystery substance through a lab. In this lab, it will consist of five known compounds and one unknown compound. Your job is to distinguish which one out of the five substances is the mystery powder. To figure out the mystery matter you will have to compare their physical and chemical properties and match them with the appropriate compound.
Unknown 10b is Staphylococcus epidermidis. According to Bergey’s Manual Staphylococcus bacteria are gram positive spherical cells that occur singly, in pairs or in irregular clusters. Unknown 10b was gram positive, spherical and occurred in clusters. Bergey’s Manual also says the bacteria grow well in high salt concentrations. Unknown 10b grew well on the mannitol salt agar. The optimum growing temperature is 30-37 degrees Celsius (Bergey’s Manual). Unknown 10b grew best at 37 degrees Celsius. The lab manual and past lab results confirmed all other test results. Unknown 10b was only able to use gamma lysis, it was unable to ferment mannitol and had no coagulase activity. When comparing to past labs it is confirmed that Unknown 10b is Staphylococcus epidermidis.
In this lab project, the microbiology students were given 2 unknown bacteria in a mixed broth each broth being numbered. The goal of this project is to determine the species of bacteria in the broth. They had to separate and isolate the bacteria from the mixed broth and ran numerous tests to identify the unknown bacteria. The significance of identifying an unknown bacteria is in a clinical setting. Determining the exact bacteria in order to prescribe the right treatment for the patient. This project is significant for a microbiology students because it gives necessary skills to them for future careers relating to clinical and research work.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
Introduction The main purpose of accomplishing multiple tests on an unknown organism was to pick a random unknown tube and identify which microorganism it was. One of the important things about these different tests is to make sure the tests are performed correctly and the results are interpreted correctly or else the unknown organism will come out as a mess. One instance where it is important to interpret the tests is in the medical field. If a patient is sick because of an unknown organism, samples from the patient can be taken out and the lab can perform tests on it and interpret the results correctly to identify the exact type of microorganism in order to treat the patient with antibiotics and treatment. In the end, this will help scientists cure diseases, help make treatments for specific organisms, and help the world be a healthier place one step at a time.
There were three bacteria that were studied during this experiment. Staphylococcus is a type of bacteria that is often found on the body of human beings and animals. It is found on the skin and hair as well as in the noses and throats. Staphylococcus can cause food poisoning when it is exposed to food and contaminated because the food is not properly refrigerated. (Food Safety) These bacteria are Gram-positive with a spherical shape that often group into clusters, much like grapes. (Bacteriology) The only way to kill these bacteria is by cooking and pasteurization. Bacillus cereus is a type of bacteria that produces toxins that can cause two types of illnesses. One type causes diarrhea and the other causes nausea and vomiting. These bacteria are found in foods and multiple rapidly at room temperature. (Food Safety) Bacillus cereus is also Gram-positive and is rod-shaped and very large. It can spread easily to many types of foods such as plants, eggs, meats, and dairy products. (MicrobeWiki) Escherichia coli is bacteria found in the gastrointestinal tract and are able to produce a toxin that can produce serious infections. This bacterium can be acquired by consuming contaminated food or water. (European Centre) E. coli is often used to help your body break down and digest certain foods. It becomes dangerous when the bacteria go from the intestines into the blood. (Kids Health) Escherichia coli, unlike any other bacteria that was studied, is Gram-negative. It is also rod shaped and is about average size. (Mansfield)
An unknown broth culture labeled # 17 was supplied by the lab instructor to identify the bacteria. We prepared a SOP by incorporating laboratory techniques that were considered as determinative tests in the dichotomous keys (appendix B and appendix C). The first two steps we performed to form the basis of identification were Gram’s staining and plate streaking of the sample. The supplied broth culture was streaked out simultaneously on one of each Trypticase Soy Agar (TSA), Mannitol Salt Agar (MSA) and MacConkey Agar (MAC) plate. The streaking was performed using the quadrant streak method described in the lab manual (Leboffe & Pierce, 2010). After observing the colony morphology and growth pattern, we further
Briefly, cells obtained from a well isolated colony on an agar plate were resuspended in 50 l of cell lysis solution (.05 M Tris (pH 8), 1 mM 0.5M EDTA, 1% Triton X-100) contained in a 0.2ml PCR tube. The cell solution was lysed by incubation at 94℃ for 10 minutes in a T100 Thermal Cycler (Bio-Rad Laboratories, Irvine, CA). After centrifugation at 10000 × g for 5 minutes, 5 l of the supernatant was used as DNA template for amplification by the Polymerase Chain Reaction (PCR). Reaction mixtures consisted of DNA template, .25 µM each of 8F and 1492r primers (ref??), and 2X GoTaq Green Master Mix (Promega, Fitchburg, WI) in a final reaction volume of 25 µl. Amplification consisted of an initial denaturation step of 94oC for 5 min, followed by 30 cycles of 94oC for 1 min, 50oC for 1 min, and 72oC for 1 min 30 sec, with a final extension of 72oC for 5 min. Electrophoresis on 1% agarose gel under standard conditions was used to visualize an approximate 1600 base pair amplicon. DNA sequencing of the amplified 16S rRNA gene was performed by MCLabs (South San Francisco, CA) followed by analysis using the Basic Local Alignment Search Tool (BLAST) to make genus and species
It is used in many labs and only requires the DNA in question, primers that anneal to the beginning and end of the target genes, Thermus aquaticus, Taq DNA, a heat stable DNA polymerase and all four of the deoxyribonucleate triphosphates. There are three steps in the PCR reaction denaturation, hybridization and DNA synthesis. During these steps the DNA is separated or denatured into two strands, hybridized, where the two single strands are complimentary paired to the respective primers, and then the DNA is synthesized with Taq DNA. This is considered one cycle, and it can commonly take 50 cycles to amplify enough DNA to be used. When the PCR is completed a gel electrophoresis is run. The PCR product is put in a specially formed agarose gel that will allow electricity to flow around the gel and DNA and force the DNA to travel down the gel resulting in white bands depending on their electronegativity. When the DNA is transformed from plasmid into the yeast we use salmon sperm to protect the nucleus from becoming degraded and the plasmid lost. This increases the efficiency of the DNA because the sperm DNA will adhere to the yeast cell wall and allow the plasmid to bind to the