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Gen Micro Lab

by: Toby Cremin

Gen Micro Lab HSCI 3321

Toby Cremin
GPA 3.92

Eric Mustain

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Eric Mustain
Class Notes
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This 3 page Class Notes was uploaded by Toby Cremin on Sunday October 11, 2015. The Class Notes belongs to HSCI 3321 at East Tennessee State University taught by Eric Mustain in Fall. Since its upload, it has received 35 views. For similar materials see /class/221390/hsci-3321-east-tennessee-state-university in Information System at East Tennessee State University.

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Date Created: 10/11/15
Background Information of Enterobacteriaceae or the Enterics There are over 28 different genera that make up this family and over 80 different species Differentiating between the species can be done by using fermentation and biochemical tests The only downside is that the fermentation and biochemical tests cannot distinguish between the virulent and nonvirulent strains of E coli E coli 0157H7 has the Otype antigen number 157 and the Htype antigen number 7 The Enterics are gram negative bacilli They are facultative anaerobes are able to reduce nitrate are catalase positive most are oxidase negative and all of them ferment glucose In order to recover Enterics from a sample plate on a mildly selective medium such as MacConkey Agar or Eosin Methylene Blue EMB Agar and also plate on a stringently selective agar such as SalmonellaShigella SS Agar or HektoenEnteric HEK Agar Serotyping methods are used to distinguish between virulent and nonvirulent strains of bacteria Serotyping methods use certain antibodies to determine the presence of specific antigens that are only found on virulent bacteria There are three different antigens that are tested for when serotyping the Enterics 1 OAntigen O 39 39 id of LPS quotI 39 39 iu39e 2 HAntigen Flagellin protein of the flagellum 3 KAntigen Capsule polysaccharide MacConkey Agar is selective and differential for gram negative bacteria MAC is selective because of the presence of bile salts in the medium Bile salts are found in the stomach and are able to break down fats The bile salts also inhibit the growth of gram positive bacteria MAC is also selective because of the presence of crystal violet which also has inhibitory effects on gram positive bacteria MAC is differential because it distinguishes between lactose and nonlactose fermentors The medium differentiates because of the presence of the pH indicator neutral red Neutral red is colorless to a weak red in neutral conditions and bright pink in acidic conditions The fermentation of lactose produces acidic products so a positive fermentor will produce bright pink colonies Slower lactose fermentors will produce just pink colonies while faster fermentors will produce lots of acidic products resulting in a pink precipitate EMB is also selective and differential EMB is selective because of the presence of the dyes eosiny and methylene blue Both of these dyes inhibit the growth of gram positive bacteria EMB is differential by detecting the fermentation of lactose The characteristic of this test is that fast lactose fermentors will produce green sheen on the colonies The green sheen is produced because lots of acid product causes both dyes to precipitate on the surface of the colonies SalmonellaShigella SS agar is more selective than the previous two because it contains both bile salts and brilliant green dye The combination of the two makes it more selective inhibiting the growth of gram positive and some gram negative bacteria SS is differential through the detection of lactose fermentation and HzS The pH indicator neutral red detects the fermentation of lactose and HzS is detected by the presence of a black precipitate Neutral red is colorless to a weak red in neutral conditions and bright pink in acidic conditions HZS is produced as a result of the enzyme thiosulfate reductase reducing sodiumthiosulfate to sulfite and HzS Sodiumthiosulfate is present in the medium and ferric citrate it the indicator for the test Ferric citrate reacts with H ZS to form the black precipitate and ferrous sulfide Organisms that reduce sodiumthiosulfate use sulfate as an alternate electron acceptor in respiration anaerobic respiration HektoenEnteric HEK is also selective and differential The presence of bile salts in the medium makes it selective against gram positive bacteria The medium is differential through the detection of HzS production Lactose and sucrose are present but the preferential fermentation is not found on this medium There are two pH indicators acid fuchsin and bromthymol blue Acid fuchsin is clear in neutral conditions and red in acidic conditions Bromthymol blue is green in neutral conditions and orangepink in acidic conditions Triple Sugar ron TSI slants contain three sugars glucose lactose and sucrose There is one part glucose to ten parts lactose and ten parts sucrose The pH indicator is phenol red which is a weak red in neutral conditions and yellow in acidic conditions Sodium thiosulfate and ferric ammonium citrate is also presence in order to detect HzS production The results of the TSI slant can be used to determine the different genera of the Enterics The results are found on the slant and in the butt of the tube Acidic conditions indicate the fermentation of organic acids such as lactic acid and acetic acid Production of hydrogen gas and carbon dioxide can also occur In order for the species to be a true alkaline slantacid butt producer the whole face of the slant must be pink Some pathogens cause the butt to appear pink halfway down indicating slow fermentation Enterobacter is characteristic of this Sometimes HzS production can prevent the butt of the test tube from being observed but it is safely assumed that if the slant is acidic or alkaline the butt is probably the same Motility ndole Ornithine MIO Agar is a semisolid soft medium The purpose of using it is to observe whether or not the organism in question is motile If the organism is motile it should be able to travel throughout the tube causing it to become cloudy all the way through Ornithine detects the decarboxylation of itself by ornithine decarboxylase through detecting the production of carbon dioxide and putresine The pH indicator is bromocresol purple which is a weak purple in neutral conditions a strong purple in alkaline conditions and yellow in acidic conditions Ornithine is not a common amino acid and decarboxylation by the enzyme will only occur under acidic conditions Since all of the Enterics ferment glucose it is present in the medium so that the bacteria can create acidic conditions Activation of the enzyme creates alkaline conditions though The production of indole is done through the catabolism of tryptophan Tryptophan is 39 39 Iand alpha 39 39 39 Iby u 39 into pyruvate indole and ammonia ndole is detected by adding two to three drops of Kovac s reagent to the sample A positive test occurs when the reagent reacts with indole to produce a cherry red color Urea Agar Slants detect the presence of urease Urease is produced by many pathogens to neutralize acidic environments and to produce nitrogen from urea to convert it to biomass for growth The metabolism of urea by urease creates an alkaline environment by producing ammonium ions which in turn react with carbon dioxide forming ammonium carbonate an alkaline product The pH indicator is phenol red which is a weak red in neutral conditions and yellow in acidic conditions Simmons Citrate Agar contains citric acid which is the only carbon source in the medium This medium detects growth of bacteria that are able to utilize citrate Growth is difficult to see but can be detected by the catabolism of citrate which produces alkaline products The pH is detected by bromothymol blue which is green in neutral conditions and blue in alkaline conditions Citric acid is metabolized by citrase into oxaloacetate and acetic acid Oxaloacetate is then decarboxylated into pyruvate The pH change occurs when carbon dioxide reacts with the sodium ions in the medium forming sodium carbonate Phenylalanine Agar detects the deamination of phenylalanine Phenylalanine is deaminated by phenyldeaminase producing phenylpyruvate and ammonia In order to detect the presence three to four drops of ferric chloride which detects the phenylpyruvate must be added to the sample This test is only positive for the Proteus Providencia ancl Morganella genuses This test should only be used to confirm the growth of Proteus species Arginine and Lysine broths are enriched with the appropriate amino acids and contain the pH indicator bromocresol purple Bromocresol purple is purple in alkaline conditions and yellow in acidic conditions This detects the decarboxylation of the appropriate amino acid In order for the enzyme that is responsible for metabolizing arginine and lysine it must be in acidic conditions which activate the enzyme The broth contains glucose and since all of the Enterics ferment glucose it is suitable enough so that the products are acidic enough to get the enzyme going After decarboxylase is activated it will return the conditions of the test tube to alkaline In order for the test to be positive the sample must be purple and turbid The ONPG test is an enzymesubstrate test The test detects the presence 3 galactosidase which is the enzyme required to ferment lactose The direct testing of the enzyme takes less time to get results that streaking on a plate B galactosidase is encoded by the Lac 2 gene of the Lac operon Galactosidase breaks lactose down into glucose and galactose The Lac Y gene encodes for the Lac permease which is responsible for transporting lactose into the cell Lac A gene has an unknown function


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