Sample collection and Growth:
Sterile cotton swab was used to collect a sample from two objects, as well as leaving out a petri dish to open air. One object was unwashed hands and the other object was the back of a cell phone. The two samples were transferred to two separate sterile petri dishes, these petri dishes where all labeled with initials, lab section, and place collected from. All three petri dishes where given to TA who proceeded to place them in an incubator that was at 37° C and allowed to grow to form colonies.
Examination and Selection of colonies:
By using aseptic technique, which consisted of using gloves and wiping area with disinfectant, we were then able to inspect the growth on the three dishes, without removing the lids. We compared what we saw to samples we looked at and took notes on the week prior. A small live culture of the unknown bacteria was then made from a petri dish of our choosing. The colony had to be isolated and fairly large. In a 1.5 ml micro centrifuge tube 400 µl of sterile water were mixed with about half of our selected colony, which was transferred by using the broad end of a sterile toothpick and taking from our petri dish.
PCR Amplification & Purification of 16s rRNA gene:
In a new
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1.5 ml tube, half (200 µl) of the live culture made was transferred using a micropipette. A cap lock was placed on the new tube and was then incubated in a 95°C heat block for 10 mins, this was done to release DNA. Obtain and label two PCR tubes containing 25 µl of 2x PCR Master Mix one “B” and the other “C”. One will serve for bacterial DNA and the other as negative control, respectively, and paced on ice. After incubation heated tube was centrifuged. The “B” and “C” tubes then had contents added, to the “B” tube 20 µl of a universal 16s RNA Primer Mix and 5 µl of Bacterial DNA from heated tube. To the “C” tube 20 µl of the universal Primer Mix were added along with 5 µl of sterile dH2O. Both tubes were then placed in a thermocycler with the following profile: 1X: 94°C, 3 min 30X: 94°C, 30 sec; 50°C, 30 sec; 72°C, 45 sec 1X: 72°C, 5 min PCR tubes were removed from thermocycler and then spun briefly in a micro centrifuge. The “B” tube contents were transferred to a new 1.5 ml micro centrifuge tube while tube “C” was stored for later use. 250 µl of buffer BB were added to the new “B” tube. Using a p1000 the contents of the “B” tube were transferred to a spin column, and centrifuged for 30 secs in the Eppendorf 5430. The flow through was discarded from the spin column, 200 µl of buffer WB were added, and it was centrifuged for 30 secs. This step was repeated once more. The contents of the spin column were transferred to a new 1.5 ml tube. 25 µl of buffer EB were added to the center membrane of the spin column and let stand for one minute, the 1.5 ml tube and spin column with buffer were then centrifuged together, the 1.5 ml tube contained the DNA and was saved. Microscopic Examination: A clean microscope slide along with the live culture that was prepared were obtained. 20 µl of live culture were added to the center of the slide, then a cover slip, with a thin line of Vaseline on the edges was placed on the drop. The bacteria was examined with a compound microscope at magnification 400X. KOH String test and Plating on Test Media: Petri dish from where our colony came from and clean microscope slide were obtained. A 50 µl drop of 3% KOH was deposited onto the slide. Using the broad end of a sterile tooth pick the remaining (half) of the selected colony was removed and mixed in with the drop of KOH, it was stirred for 60 secs, then tested for formation of string by lifting the toothpick slightly. This was used as the gram test. Two petri dishes were obtained, one containing EMB (purple) the other PEA (white). 5-10 sterile beads were placed in the petri dishes with medium, then live culture was resuspended and equal amounts of it were distributed to both dishes. The live culture was spread throughout the petri dishes shaking the beads, when live culture is spread out enough beads are poured into alcohol. Petri dishes were placed upside down on a rack and incubated at 37°C for 1-2 days. Gel Electrophoresis: 4.5 g of agarose were added to 30ml of 1X TBE buffer in a flask to make a 1.5% agarose solution. The mixture was heated until the agarose in the solution melted, then ethidium bromide stain was added to the flask. The chamber for gel electrophoresis was assembled, and mixture in flask was added and allowed to set, and 1X TBE buffer was added until gel was covered. PCR tubes were obtained, both the one with DNA and negative control. Two .5 ml tubes were obtained one “B” had 6 µl of purified 16s rRNA PCR products and 4 µl of Blue Juice added to it, the other “C” had 12 µl of the control PCR reaction and 4 µl of Blue Juice added to it. A tube of DNA size standards that contained 5 DNA fragments of known sizes was obtained and was used as a marker to know the size of our band of base pairs. 10 µl from each tube (two tubes of bacterial DNA, one from each group and two tubes of negative control, one from each group, and one tube of DNA size standard) were taken and loaded into the gel slots. The gel ran for about an hour and then a picture was taken and examined and compared to the DNA size standard to know the length of the fragment. Further examination of Gram-positive and Gram-negative bacteria: Gram-positive bacteria had more test conducted on them, the catalase test and the MSA growth test.
For the catalase test a clean microscope slide was placed in a petri dish, fresh bacterial cells were smeared on the center of the slide, then a drop of 3% hydrogen peroxide was administered onto the bacterial cells, and formation of bubbles was observed. MSA test was conducted by obtaining a petri dish containing MSA medium and .5 ml tube. A sample of the bacteria along with 200 µl of sterile water are mixed in the tube, 5-10 sterile beads and 50 µl of the newly made bacterial solution are added to the MSA petri dish and the solution is spread out using the beads. The petri dish is then incubated for 2 days at 37°C, and evaluated for
growth. Gram-negative bacteria had Catalase test and Oxidase test, the catalase test was conducted the same way that it was for gram-positive bacteria. For oxidase test a dry oxidase slide and oxidase-negative, oxidase-positive colony containing petri dishes were obtained. There are four separate test areas, two for the controls and two for the bacterial samples. Colonies from the bacterial sample and controls are transferred, using aseptic technique, to their designated area, the slide is then incubated at room temperature for 20 secs and examined. Cycle Sequencing Reaction: For sequencing reaction a clean PCR product with size about 500bp, and a .2 ml PCR tube are necessary. In the .2 ml PCR tube there are 6 µl of Big Dye Master Mix, to this 2 or 4 µl depending on DNA bp size, and 2 µl of ddH2O (if 2 µl of Big Dye were used). This tube was placed in a thermocycler with the following program: 1X: 96°C, 1 min 30X: 96°C, 10 sec; 50°C, 5 sec; 60°C, 2 min Hold: 4°C When finished, products were cleaned in CCG and sequenced in ABI 3730. All methods previously mentioned from (Holbrook & Leicht, 2014)
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.
The isolate possesses some enzymes required for hydrolytic reactions. Hydrolytic enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Hydrolytic enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been
The first day an unknown sample was assigned to each group of students. The first test applied was a gram stain to test for gram positive or gram-negative bacteria. The morphology of the two types of bacteria was viewed under the microscope and recorded. Then the sample was put on agar plates using the quadrant streak method for isolation. There were three agar plates; one was incubated at room temperature, the second at 30 degrees Celsius, and the third at 37 degrees Celsius. By placing each plate at a different temperature optimal growth temperature can be predicted for both species of bacteria.
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 purpose of this study is to analyze the activity of the enzyme, catalase, through our understanding
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.
This indicated that there was no lysis of red blood cells. By looking at the plate, there was no change in the medium. Next an MSA test was done and the results showed that there was growth but no color change. This illustrates that the unknown bacteria could tolerate high salt concentrations but not ferment mannitol. The MSA plate eliminated Streptococcus pneumonia and Streptococcus pyogenes as choices since the bacteria can’t grow in high salt concentrations.
The purpose of this laboratory is to learn about cultural, morphological, and biochemical characteristics that are used in identifying bacterial isolates. Besides identifying the unknown culture, students also gain an understanding of the process of identification and the techniques and theory behind the process. Experiments such as gram stain, negative stain, endospore and other important tests in identifying unknown bacteria are performed. Various chemical tests were done and the results were carefully determined to identify the unknown bacteria. First session of lab started of by the selection of an unknown bacterium then inoculations of 2 tryptic soy gar (TSA) slants, 1 nutrient broth (TSB), 1 nutrient gelatin deep, 1 motility
The purpose of this project was to identify unknown bacteria species from a mixed culture. The two unknown species were initially plated onto Tryptic Soy Agar (TSA), Eosin Methylene Blue (EMB), Mannitol Salt Agar (MSA), and blood agar plates to distinguish between the two different bacteria using colony size, color, shape, and growth characteristics. By identifying and inoculating the differing types of colonies, the two unknown bacteria were purified and able to be tested
The Effect of pH on the Activity of Catalase Planning Experimental Work Secondary Resources Catalase is a type of enzyme found in different types of foods such as potatoes, apples and livers. It speeds up the disintegration of hydrogen peroxide into water because of the molecule of hydrogen peroxide (H2O2) but it remains unchanged at the end of the reaction.
The Effect of Temperature on the Activity of the Enzyme Catalase Introduction: The catalase is added to hydrogen peroxide (H²0²), a vigorous reaction occurs and oxygen gas is evolved. This experiment investigates the effect of temperature on the rate at which the enzyme works by measuring the amount of oxygen evolved over a period of time. The experiment was carried out varying the temperature and recording the results. It was then repeated but we removed the catalase (potato) and added Lead Nitrate in its place, we again tested this experiment at two different temperatures and recorded the results. Once all the experiments were calculated, comparisons against two other groups were recorded.
Inconsistencies in this lab could have caused variations in data collecting. Collecting data from one petri dish was challenging because something could have been different on other petri dishes if this experiment was tested on several petri dishes. This could have been different because the other petri dishes could have had more micro-organisms in Section 2 instead of Section 1, or no bacteria could have grown at all in every section of the petri dish.- Second, nothing grew in section B even though there were no disinfectants in that section. The reason why the bacteria and mold might have grown in sections 1, 2, and 3 was because in the process of making the experiment, the coffee filter papers were touched with glove free hands and were not clean. If this lab was run again, some changes would be to wear rubber gloves, do not pour the hand sanitizers on the coffee filter paper but just pour one pump straight into the petri dish, have more than one petri dish to collect data off of, and check when the last time someone cleaned the door knob
There were five test solutions used in this experiment, water being the control, which were mixed with a yeast solution to cause fermentation. A 1ml pipetman was used to measure 1 ml of each of the test solutions and placed them in separated test tubes. The 1 ml pipetman was then used to take 1ml of the yeast solution, and placed 1ml of yeast into the five test tubes all containing 1 ml of the test solutions. A 1ml graduated pipette was placed separately in each of the test tubes and extracted 1ml of the solutions into it. Once the mixture was in the pipette, someone from the group placed a piece of parafilm securely on the open end of the pipette and upon completion removed the top part of the graduated pipette.
Leboffe, M. J., & Pierce, B. E. (2010). Microbiology: Laboratory Theory and Application, Third Edition 3rd Edition (3rd Ed.). Morton Publishing
The abnormal presence of bacterial growth can be inspected under a microscope. If the organism inspected is not the bacteria used in the experiment, it means that the growth of the bacterial culture investigated is absent. By using this method, contamination by foreign substances in the surrounding air can be ruled out and the results would be more accurate.