1. Title: The Process of Determining the Unknown Bacteria #9 Rachel Judecki July 5, 2011 2. Introduction: Each student was given unknown bacteria and was instructed to perform a variety of experimental tests that would help to identify their bacteria. During the process of identification, the unknown bacteria was added to many different testing medias using aseptic technique.

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They are as follows: lactose fermentation on eosin methylene blue (EMB), TSI (Triple Sugar Iron agar), Phenol red sucrose, the SIM test, H2S by SIM, IMViC (indole, motility, voges-proskauer, and citrate), Urease (urea broth), PDase (Phenylalanine Deaminase), Lysine Decarboxylase, and Ornithine Decarboxylase. Colonial morphology on EMB was used to prove the identity of the bacteria. The unknown bacteria was #9. After performing the tests it is determined that the unknown bacteria is Escherichia coli. 3. Methods: All tests were performed using the aseptic technique to assure the cultures were pure and not contamination was present.

The inoculation loops and straight wire stab needles were placed over the flame of a Bunsen burner and heated until red hot (sterile) then cooled before placing them into the bacteria to prevent the killing of the bacteria. The open end of each tube of bacteria was flamed once it was open (before inoculation) and before it was closed (after inoculation). (Morello, 9th Edition. 2008) EMB (eosin methylene blue) agar was used to determine if the bacteria was a lactose fermenter or a non- lactose fermenter. EMB agar is a selective and differential media.

The dyes eosin Y and methylene blue found in the medium inhibit the growth of gram-positive bacteria but not the growth of gram-negatives. Lactose fermenters metabolize the lactose in the media and produce acid byproducts, causing a color change in the colony which is dark purple, almost black. Strong acid productions by organisms result in a metallic green sheen. Weaker fermentation of lactose results in colonies with a pinkish-purple color. Colonies that are non-lactose fermenters remain colorless, or at least no darker than the color of the media.

The unknown bacteria #9 was streak diluted on to an EMB agar plate using a sterile inoculating loop. After incubation, the unknown bacteria #9 produced dark purple/black colonies that had a green metallic sheen. This is a typical test result from the bacteria Escherichia coli. Phenol Red Sucrose broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate (sucrose). Phenol red is a pH indicator which turns yellow below a pH of 6. 8 and fuchsia above a pH of 7. 4.

If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed. The unknown bacteria #9 was added to the phenol red sucrose broth using a sterile inoculation loop.

Only a loop full was added and mixed into the broth. After incubating, the unknown bacteria #9 caused the broth to change color from red to yellow indicating a positive result for sucrose. There were bubbles inside the Durham tube which is a positive result for gas production. H2S (only from SIM) SIM medium is a combination differential medium that tests three different parameters, which are represented by the three letters in the name: Sulfide-Indole-Motility medium. It is a semi-solid agar that is inoculated with a bacterium to test for hydrogen Sulfide, Indole, and Motility of the organism.

The medium is inoculated by a stab method (stab a straight hole through the medium using a straight wire with the bacteria on it). Incubate the bacteria for about 24 hours and then begin testing. If hydrogen sulfide is present, it will react with the sodium thiosulfate in the medium and the indicator, ferric ammonium citrate, to produce ferrous sulfide which falls out of solution as a blackish precipitate. The presence of hydrogen sulfide typically means that the bacteria produces the enzyme cysteine desulfanase which breaks up the cysteine in the medium into hydrogen sulfide. A positive result will turn the medium black.

If the result is negative then the medium will stay semi-clear. The unknown bacteria #9 produced negative results for H2S in the SIM test tube. The medium stayed semi-clear and did not turn black. IMViC reactions are a set of four reactions that are: Indole test, Methyl Red test, Voges Proskauer test and Citrate utilization test. The letter “i” is only for rhyming purpose. The Indole portion of the test is performed by adding Kovac’s reagent to the inoculated SIM medium. The Kovac’s reagent reacts with the indole (if indole is present) to produce a pinkish-red or reddish-purple ring around the top of the test tube.

If indole isn’t present, there will be no color change. The presence of indole indicates that the bacteria produces tryptophanase, an enzyme which breaks down tryptophan into smaller components, one of which being indole. The results for the indole test for unknown bacteria #9 were positive. When the Kovac’s reagent was added to the SIM tube, the top of the medium turned a ruby red color which indicates a positive test result. Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC tests as the medium in which both the Methyl Red and Voges-Proskauer tests can be performed.

It is a simple broth that contains peptone, buffers, and dextrose or glucose. To perform these tests a loop full of the bacteria is added to the broth and incubated. Methyl red is a dye that is acid-sensitive. It turns yellow at a pH above 4. 5 and when the pH is below 4. 5 it turns red. The Methyl Red test involves adding the pH indicator methyl red to an inoculated tube of MR-VP broth. If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium.

When methyl red is added, if acidic end products are present, the methyl red will stay red. The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result.

If, after the reagents have been added, a copper color is present, the result is negative. Neither of these tests were performed during this experiment. The test results were given to the students by the teacher. When methyl red is added to MR-VP broth that has been inoculated with Escherichia coli (#9), it stays red. This is a positive result for the MR test. When the VP reagents are added to MR-VP broth that has been inoculated with Escherichia coli (#9), the media turns a copper color. This is a negative result for the VP test. Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source.

Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicator bromthymol blue. This test is part of the IMViC tests. Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport the citrate into the cell. These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium. At pH 7. 5 or above, bromthymol blue turns royal blue.

At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media. If the medium’s slant turns sapphire blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. A loop full of the unknown bacteria #9 was inoculated onto the citrate slant and incubated. There was no change in the green color of the agar which means the results for citrate for #9 were negative. Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease that hydrolyzes urea to ammonia and carbon dioxide.

The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red. Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment. If the urea in the broth is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink. Phenylalanine deaminase (PDase) medium tests the ability of an organism to produce the enzyme deaminase. This enzyme removes the amine group from the amino acid phenylalanine and releases the amine group as free ammonia. As a result of this reaction, phenylpyruvic acid is also produced.

After incubation, 10% ferric chloride is added to the media; if phenylpyruvic acid was produced, it will react with the ferric chloride and turn the slant a bluegreen color. If the medium remains a straw color, the organism is negative for phenylalanine deaminase production. A loop full of unknown #9 was added to the slant of the PDase test tube. After incubation, five drops of 10% ferric chloride was added to the slant. There was no reaction so the results are negative. Lysine Decarboxylase (LD) is an amino acid that possibly can be broken down by the decarboxylase enzymes that are in some bacteria.

The carboxyl (COOH) group on the amino acid molecule is removed during this process. As a result, alkaline end products that change the pH indicator color are left. The pH indicators are bromcresol purple and cresol red. Bromcresol purple turns purple at an alkaline pH and turns yellow at an acidic pH. The experiment works best when air is excluded from the test tube, so a layer of mineral oil is added after a loop full of the bacteria is inoculated and before incubation. The results for the unknown bacteria #9 were positive. The medium turned purple indicating a positive result.

A negative result would have been if the medium turned yellow. Colonial morphology was the determining test to prove that unknown bacteria #9 was indeed Escherichia coli. The dark purple colonies with a green metallic sheen of the unknown #9 was the same as Escherichia coli. Enterobacter agglomerans (the only other bacteria left) colonies have a dark purple center surrounded by a wide, light-colored purple, mucoid rim – resulting in a “fish-eye” type of colony. 4. Results: The indole test had an unusual result. The first time the test was performed the Kovac’s reagent was added and it turned an orange color.

After allowing the test tube to sit for a few days, the agar medium turned an orange color. The test was repeated and the results from this second test confirmed that the unknown bacteria #9 was positive for indole. After adding the Kovac’s reagent, it turned a ruby red color indicating a positive result. Another test that had an unusual result was the phenol red sucrose broth. The tube was inoculated and left to incubate. The original color of the broth was red. After incubation the broth had changed to orange, which was a weak reaction.

After letting the test tube incubate for a few more days, the broth had changed to yellow and bubbles were in the Durham tube. This indicated a positive result for both sucrose and gas production. *see attached Bacteria and Tests Performed Chart *see attached Flow Chart 5. Discussion of the Medical or Ecological Significance: The genus Escherichia is named after Theodor Escherich, who isolated the type species of the genus. Escherichia organisms are gram-negative bacilli that exist singly or in pairs. Escherichia coli is a facultative anaerobic with a type of metabolism that is both fermentative and respiratory.

They are either non-motile or motile by peritrichous flagella. Escherichia coli is a major facultative inhabitant of the large intestine. Escherichia coli of many different serotypes, are categorized into four major groups according to virulence mechanisms: enterotoxigenic (ETEC); enteropathogenic (EPEC); enteroinvasive (EIEC); and enteroaggregative (EAgg EC). Other groups (e. g. , diffusely adherent E. coli) are less well established as pathogens. Escherichia coli is a bacterium that is commonly found in the gut of humans and other warm-blooded animals. While most strains are harmless, some can cause severe food borne disease.

Escherichia coli infection is usually transmitted through consumption of contaminated water or food, such as undercooked meat products and raw milk. Symptoms of disease include abdominal cramps and diarrhea, which may be bloody. Fever and vomiting may also occur. Most patients recover within 10 days, although in a few cases the disease may become life-threatening. Escherichia coli is also one of the most frequent causes of many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), and traveler’s diarrhea, and other clinical infections such as neonatal meningitis and pneumonia.

Most Escherichia coli infections can clear up on their own after a few days. The Escherichia coli 0157:H7 infection is well known for causing not only severe diarrhea, but also possible life threatening complications mostly in children and the elderly. Some of the complications are as follows: renal failure, anemia, and dehydration especially for children (termed HUS or Hemolytic-uremic syndrome) and spontaneous bleeding, organ failures, and mental changes in the elderly (termed TTP or thrombotic thrombocytopenic purpura).

Some of these patients develop disabilities or die. This type is transmitted through fecal-oral transmission. Prevention of this infection is done through cooking meats thoroughly, not drinking raw milk, not swallowing water when swimming in pools, rivers, etc. , wash hands after changing a child’s diaper, and eliminate cross-contamination on cutting boards and knives by washing them in between uses. Bibliography Black, Jaquelyn G. , Microbiology: Principles and Explorations, 7th Edition, John Wiley & Sons, Inc, Hoboken, NJ, 2008 Davis, Charles P. Marks, Jay W. , (2011) E. coli 0157:H7 (Escherichia coli 0157:H7 infection). Retrieved from: http://www. medicinenet. com/e_coli__0157h7/article. htm Morello, Josephine A. , Mizer, Helen Eckel, Granato, Paul A. , Laboratory Manual &Workbook in Microbiology: Applications to Patient Care, 9th Edition, McGraw-Hill Higher Education, New York, NY, 2008 Unknown author, Enterotoxigenic Escherichia coli (ETEC) page last updated: 11/25/2009. Retrieved on 7/2/2011 from: http://www. cdc. gov/nczved/divisions/dfbmd/diseases/enterotoxigenic_ecoli/