METHODOLOGY
Area of isolate
For the isolate that can produce biosurfactant, we collected the sample from oil spilled area, from automobile workshop because the nature of the place will determine the type of microorganism.
Sampling
Sample of the soil was collected from the automobile workshop. The sample of the soil was taken to the laboratory for analysis via sterile polythene bag. At the time of sample collection, the temperature and pH of the soil sample was 30℃ and 7 respectively.
Collection of bacteria from the sample and its isolation
In 50ml of R2B broth, the soil sample of 5 gram inoculated and incubated at 25℃ for 72 hours. After incubation, serial dilution of the medium taken place from 〖10〗^(-1) to 〖10〗^(-6) in sterile
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After solidification of agar, the fresh colonies from the culture were taken and streaked on the blood agar. Incubated at 37℃ for 48-72 hours. After incubation, we were looking for the clear zone around the microbial colonies. This clear zone are the sign of biosurfactant producing microorganisms.
Biochemical analysis of biosurfactant producing microorganisms
The biosurfactant producing microorganisms then characterized by different types of test includes Motility Test, Gram Staining, Indole Test, Methyl Red Test, Citrate Test, Spore Staining, Catalase Test, Voges-Proskauer Test, Casein Hydrolysis, Starch Hydrolysis, Lipid Hydrolysis, Gelatin Liquifaction Test, Gelatin Hydrolysis, Oxidase Test.
Results after these test, we will able to find the closest match of our sample with known bacterial genus and assign the bacterial signature according to Bergey’s manual.
Biosurfactant
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Incubated at 25℃ for 7 days under the shaking condition. After 7 days, centrifuge it at 5000rpm and at temperature of 4℃ for 20 minutes. Discard the pellet that contains bacterial cells, take supernatant. Make the pH of supernatant of 2 by using "M" "H" _2 "S" "O" _4. Add choloroform and methanol of ratio 2:1 and of equal volume. Mix the mixture and left it for overnight for the process of evaporation. The white coloured sediment appeared that is the biosurfactant.
Drying of biosurfactant
Poured the sediment in the sterile petriplate of known weight. Put the plate in the Hot Air Oven at 100℃ for 30 minutes. After drying, the weight of biosurfactant was calculated with the help of following formula-
Dry wt. of biosurfactant = (wt. of plate after drying – wt. of empty plate)
Preliminary characterization of biosurfactant
It was done by TLC method that is Thin Layer Chromatography method. Silica gel poured on the glass plate. A mark of biosurfactant was placed on the glass plate which is poured by silica gel. The biosurfactant ran on silica plate by a solution which is made up of chlororform, methanol and water. Ninhydrin reagent and Throne reagent was sprayed on the silica plate for detection of lipopeptide biosurfactant as red mark and glycolipid biosurfactant as yellow mark respectively.
Molecular weight of biosurfactant
The sediment of biosurfactant mixed with chloroform and given for GC-MC
Identification of bacteria within Unknown Culture #21 In this experiment, an unknown culture of two different types of bacteria was assigned to each person, a number of tests were performed to isolate and identify these bacterial cells. Based on knowledge from the previous experiments completed in lab, a basic understanding of each type of bacteria was used to create a flow chart that would aid the process of identifying the unknown bacteria within the culture. A gram stain that is performed initially will narrow down the types of tests certain bacteria will and will not respond to. In addition to the gram stain, some of the tests that were used include, a catalase test, an Eosin methylene blue (EMB) agar test, a bile esculin test, and a 6.5% sodium chloride (NaCl) test.
The purpose of this lab report is to employ a myriad of skills, tools and, methods learned throughout this semester to perform the appropriate tests for the identification of the assigned unknown bacteria. Add more background information here!!! The most important tools and techniques used during this identification include aseptic technique, microscopic examination and, the use of selective and differential media. Aseptic technique is an important tool for microbiologists. It is imperative that aseptic technique is maintained throughout the length of any test to avoid any cross-contamination that may lead to inaccurate results.
In this experiment, we cultivated an unknown specimen containing two microorganisms. The purpose of this experiment was to use a variety of biochemical test previously learned in the lab to identify the unknown bacteria. The identification of unknown bacteria is a major part of microbiology. Microbiologist observe samples such as blood and sputum in the laboratory for the presence of microorganisms. Identifying unknown bacteria is extremely important in clinical settings because it helps physicians find treatment for infections.
The Unknown Identification Lab was an experiment that provided the opportunity to apply all the tests that were learned in the semester of lab, to identify the two bacterias that remain unknown. Gram- staining and two other tests will be used to identify the unknowns. This experiment is crucial to the understanding of each test, and can benefit in the ability to identify the characteristics of specific bacteria. Having a clearer understanding of the bacteria can further the research of bacteria for medicine, such as antibiotics. The understanding can also help the development of research in the environment.
Unknown Paper I Introduction This lab is a presentation of lab tests performed to finalize a conclusion based on results to identify the given unknown bacteria. The unknown bacteria was identified based on lab test results in the table provided in class for the possible unknown bacteria. The unknown bacteria identified as #36, and based on the lab tests is Enterobacter II Materials and Methods Catalase Test- this test determines whether bacteria have the enzyme catalase which catalyzes the breaks down hydrogen peroxide.
When given an unknown bacteria there are a multitude of steps one must go through to be able to correctly identify what bacteria was given. It is important to correctly identify the bacteria because some bacteria are more harmful than others. The gram stain is the first test that should be performed because it helps narrow down the possibilities by telling one whether the bacteria is gram positive or gram negative. After this test is performed, one shall place bacteria on/in Mannitol Salt agar, MacConkey agar, Eosin Methylene Blue agar, Urea agar, Simmon’s Citrate, Purple Beef broth with Lactose and finally Purple Beef broth with Sucrose. A streak plate should also be made up, this helps one identify the morphology of the colonies.
The unknown bacteria was then tested on multiple selective and differential media. Growth was present on the MacConkey Agar and the colonies were the same color as the plate, which told me my bacteria was gram negative and did not ferment lactose. There was no growth on the Mannitol Salt Agar, and this told me the unknown was not salt tolerant and did not
What information does this tell you about the bacteria in the sample, and why did the colony change colour? [2 marks] This tells me that the bacteria in the sample contains coliforms such as Escherichia coli (E.coli). The colonies changed colour as the enzymes produced by the bacterial cells reacted with the red galactosidase in the agar medium, this reaction causes the colonies to turn pink making them easily
Another variable of the experiment that was controlled was the time in which the agar cubes spent in the sulphuric acid. The time allowed calculation of the rate of diffusion. The size of the agar cubes was controlled by using a grid and scalpel to, as accurately as possible, cut the agar cubes into the appropriate sizes. The shape of the agar cubes was also controlled. In future, this could be experimented with to investigate how different shaped agar blocks affect surface area to volume ratio and hence the rate of
Exercise 14: Unknown Identification Lab Report The purpose of the study was to identify the unknown bacterium using various biochemical tests in addition to using scientific methods in determining the outcome of the hypothesis. Each biochemical test will help determine the bacteria based on specific characteristics of each organism. I was giving unknown number 232. The first procedure that needed to be done after obtaining unknown bacterial mixture was to isolate the two bacteria in a pure culture using the streak plate method described in Microbiology Laboratory Manual Eight Edition. The material used was trypticase soy agar (TSA) plate, nutrient plate, starch agar, hydrogen peroxide, iodine reagent and microscope.
Prior to the amylase extract being placed into the wells, the wells were prepared with 3 drops of iodine solution which works as an indicator of starch presence. We measured the presence of the starch with a 5 number color system. The lightest color, yellow, being number one and having the lowest concentration of starch, while the darkest color, a blue black,being number 5 and having the highest concentration of starch. The results showed that the optimal temperature for the bacterial amylase, Bacillus licheniformis, was 55℃, while the optimal temperature for the fungal amylase, Aspergillus oryzae, was of 25℃. The bacterial amylase, Bacillus licheniformis, had the
The Another medium used was MAC, it is used to isolate and differentiate gram-negative organisms and it is a pink, dusty rose color. Lastly, the Citrate Slant is a green color and it was used as a differential test to examine enzymes. The media were inoculated at 37°C for 48 hours, then it was observed to determine the
Aseptic technique was initiated at the beginning of this experiment by cleaning the work surface with disinfected wipes. Personal protectives equipment was also worn. The material utilized in this experiment was: S. epidermidis culture broth, sterile cotton swab, streak plate, forceps in 70% alcohol, a lit tea light, and the three antibiotic disks (novobiocin, gentamicin, penicillin). The first step, I divided a plate into three quadrants and labelled them with the different antibiotic names. Using the lit tea light, like a bursen burner, I flamed the mouth of the S. epidermidis culture.
The bacteria, Pseudomonas Aeruginosa, is gram- negative. This means it is does not have a violet stain. It is also considered a bacillus bacteria. Pseudomonas aeruginosa can be strictly found in aerosols, this means it is compressed in pressure and released into a fine spray. Research has been made and found that this bacterium harms a lot of plants, animals, but mainly humans in such case.
Catalase Test, in this test the microbial culture from Nutrient Agar plates were used. This test determines the production of catalase by the microorganisms. Catalase is an enzyme which decomposes hydrogen peroxide to water and oxygen gas thereby, protecting the microorganisms from the lethal effect of hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism. (Bahrami-Hessari et. al.