In order to identify an unknown bacterium a variety of tests can be performed. The unknown bacterium that underwent a few of these tests was determined to be Escherichia coli. A Gram stain, citrate utilization test using Simmons citrate agar, and a urease detection test with phenol red were performed on the assigned bacterium. The unknown bacterium was determined to be E. coli because the tests concluded that the specimen was Gram-negative, bacilli, citrate utilization negative, and urease production negative. It is concluded that E. coli cannot utilize citrate as its sole carbon source and it cannot convert urea into ammonia and carbon dioxide.
Introduction
It is important in microbiology to be able to identify an unknown microorganism. Many tests that characterize functions of bacteria have been developed to accomplish this task. The unknown bacterium that was tested was discovered to be Escherichia coli. E. coli is a Gram-negative, bacillus bacterium. Gram-negative means there is an outer membrane surrounding the thin peptidoglycan layer of the cell. Bacillus means that the bacterium is rod-shaped. The E. coli was identified as Gram-negative and bacilli from performing a Gram stain. A Gram stain uses crystal violet for the primary dye and safranin as a counter stain (1). Gram-positive bacteria have a very thick peptidoglycan layer in their cell wall. This thick cell wall will stay stained purple by the crystal violet-iodine complex, and Gram-negative bacteria will be decolorized and stained pink from the safranin counter stain (1). The peptidoglycan layer’s thickness determines whether it is stained pink or purple.
To test an organism’s ability to use citrate as a source of carbon, a citrate utilization test is. This test is ...
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...n dioxide. The ammonia would cause the broth’s pH to be alkaline, which would turn the broth bright pink (3). Since the broth stayed the same orange/yellow color, we know that the urea in the broth was not degraded to ammonia, meaning that the organism does not produce the enzyme urease.
Literature Cited
1) Bartholomew, J.W., and T. Mittwer. 1952. The Gram stain. Microbiology and molecular biology reviews. 16: 1-29.
2) Krajewska, B. 2009. Ureases I. Functional, catalytic and kinetic properties: A review. Journal of Molecular Catalysis B: Enzymatic. 59: 9-21.
3) Roberts, G.D., C.D. Horstmeier, G.A. Land and J.H. Foxworth. 1978. Rapid urea broth test for yeasts. Journal of Clinical Microbiology. 7: 584-588.
4) Vaughn, R.H., J.T. Osborne, G.T. Wedding and J. Tabachnick. 1950. The utilization of citrate by Escherichia coli. Journal of Bacteriology. 60: 119-127.
Upon receiving the unknown Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed 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. After ten minutes had passed, I collected the ingredients needed to perform a gram stain. I got the primary stain, crystal violet, and flooded my smear for sixty seconds, and then rinsed the color off with water until the water ran clear. I then flooded the smear with the mordant, grams iodine, and let that sit on the slide for sixty seconds as well. I then rinsed the grams iodine off with water and applied alcohol to the smear to decolorize the cells; however I made sure not to over decolorize and only put enough drops on the smear till the purple ran clear. I then rinsed the slide with water and flooded the smear with safranin the counter stain and let it sit for sixty seconds and then rinsed the color off with water. I blo...
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
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
The Gram positive bacteria has been nicknamed Posi. The Gram positive species’ morphology includes having an opaque opacity with a smooth margin. The moisture content of the Gram positive species is shiny and the pigmentation is gold. The Gram positive species grows at an optimal temperature of 37°C. The shape of the Gram positive species is a cocci, with an arrangement of grapelike clusters. The Gram positive species’ size ranges from .5-1.5 µm. Oxygen requirement of the Gram positive species is facultative, and has complete lysis of red blood cells. All results are summarized in Table
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.
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 eighteenth exercise of the laboratory manual titled Unknown Identification and Bergey’s Manual is an experiment to identify an unknown bacterium. In this exercise, a student must randomly choose a numbered bacterium available to the class. The keys in Appendix H, located on the last pages of the book, are the major helpful tools in this exercise because it provides completed steps of tests that needs to be performed in order to distinguish certain bacteria. This means that in this exercise, various types of tests and techniques must be performed to identify the chosen unknown bacterium. The unknown bacterium that I selected was number thirty-nine in which I discovered as the Bacillus megaterium after conducting several tests.
I was given unknown organism #14, in order to find out what organism I had, I had to perform several different biochemical tests to identify it. Starting with the Gram stain test, which is performed to differentiate Gram-positive and Gram-negative cells. After staining, when observed through the microscope Gram-positive cells are a purple color with thick peptidoglycan cell walls. Gram-negative cells are a pinkish/red color with thinner cell walls. (handout G. s.) My organism was observed to be pinkish rod shaped meaning it is Gram-negative bacteria.
The purpose of this investigation was to identify an unknown bacterium. “At any time there are millions of bacteria living around, on, or inside us” (The Plague). Bacterium can’t be identified by merely looking at it. Many bacteria have the similar appearances in growth. “In most cases, detection is based on the reaction of an enzyme with a certain substrate” (Sigma-Aldrich). Identification is usually based on the results of the bacterium’s cells metabolic capacities.
Urea Hydrolysis: This test result was negative. There was no accumulation of sufficient ammonia from the hydrolysis of urea. An alkaline environment was not created by organism
The main purpose of this lab was to identify unknown bacteria, every student got different organisms. By using different types of procedures, we obtain results in order to classify the bacteria as gram positive (+) or gram (-) and from there, continue with the processes ending with the name of the bacteria. All the material including a flow chart was provided from the instructor.
The gram stain was negative. The bacterial cells observed under the microscope were pink in color from the safranin stain, but all traces of the crystal violet were washed away with the alcohol wash. The bacterial cells were observed to be small and bacillus or rod shaped.
Talaro , K., & Chess, B. (2012). Foundations in microbiology. (8th ed., pp. 563-564). New York, NY:
A scientist named Christian Gram invented a technique called gram staining by which is colorized that is separated into two groups Gram positive and Gram negative. In bacteria most have rigid cell walls in which is accountable for the shape of the organism. Within the cell wall of the bacteria it identifies whether the bacteria is gram positive or negative. In the cell wall there a lot of difference that determines the bacteria is gram is positive or negative for example the thickness and the amount of peptidoglycan later presences or absence of outer membrane and teichoic acids. Where gram positive have two later and negative has three layers. Gram positive the cell wall is a single layer, primarily composed of peptidoglycan, does not contain outer membrane and contains negatively charged teichoc acids, this result of a blue color indicator. For gram negative is more complex that the Gram positive, its membrane lies outside of th...
ix. citric acid increases the acidity of foods and makes it harder for bacteria to