The Determination of Unknown Bacteria using API 20E System PDF

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The Determination of Unknown Bacteria using API 20E System

Abstract The goal of this experiment is to identify an unknown bacteria using the API 20E System which is a series of tests to determine the positivity or negativity of a compound based on its

reactions. The observations gathered from these reactions aid in the identification of the unknown bacteria obtained through the comparison of each positive and negative test results. The plastic strip of the API 20E System hold twenty mini-test chambers containing dehydrated media which is inoculated with certain substrates to produce specific reactions. With the provided charts, I identified the unknown bacteria given by analyzing the results of the API 20E System and the accepted results of what the unknown bacteria would be. After performing the experiment, I determined that the unknown bacteria I received was Salmonella enteritidis. Introduction The Analytical Profile Index or the API 20E System is a panel for identification and differentiation of members of the family Enterobacteriaceae and other gram-negative Bacteria. The family enterobacteriaceae includes commonly known pathogenic bacterias such Escherichia Coli, Salmonella enteritidis, Citrobacter freundii, Enterobacter aerogenes, and Proteus mirabilis. Therefore, the API 20E System has crucial applications in the identification and prevention of the spread of disease-causing bacteria. The plastic strip of the API 20E System contains approximately twenty chambers consisting of a microtube with dehydrated media and a cupule. The dehydrated mediums include: β-galactosidaseenzyme, Arginine, Citrate Utilization, H2S production, Urease Test, Tryptophan Deamination, Indole Production, Voges Proskauer, Gelatinase, Sugar Fermentation (9), Nitrate Reduction, and Oxidase. To inoculate each compartment, a bacterial saline or broth suspension of the unknown bacteria is created which rehydrates the substrates in the microtube causing a reaction to occur. The cupule receives the suspension and allows it to flow into the tube containing the medium. In the chambers of ADH, LDC, ODC, H2S, and URE, it was instructed to add mineral oil to create an anaerobic condition. The results are determined by examining the color changes produced by the reactions.

· ONPG Test: O-Nitrophenyl-β-D-galactopyranoside (ONPG) is structurally similar to lactose, except that ortho nitrophenol has been substituted for glucose. On hydrolysis (the chemical breakdown of a compound due to reaction with water), through the action of the enzyme βgalactosidase, ONPG cleaves into two residues, galactose and o-nitrophenol. ONPG is colorless

compound: O-nitrophenol is yellow, providing visual evidence of hydrolysis. The yellow color is usually distinct and indicates that the organism has produced o-nitrophenol from the ONPG substrate through the action of β-galactosidase.Non-lactose fermenting bacteria are devoid of βgalactosidase and are incapable of producing the color change (Hardy Diagnostics, 2008). · ADH Test: Test of decarboxylation of the amino acid arginine by arginine dihydrolase. Decarboxylation is the removal of carboxyl group from an organic molecule. The amine products increase the pH of the solution in the tube. Arginine is first converted to citrulline via dihydrolase reaction, in which NH2 group is removed from arginine. Citrulline in next converted to ornithine. This process increases the pH of the solution in the tube. A positive result will a red/red orange color, and a negative result of this reaction shows yellow/yellow orange (Tankeshwar, 2018). · LDC Test: Tests for the decarboxylations (described in ADH test) of the amino acid lysine by lysine decarboxylase. This test has the same concept as the ADH test, with the same color results - positive reaction shows a red/red orange color, and negative is yellow/yellow orange. The test is used to determine if bacteria can use the amino acid, Lysine, as a source of carbon and energy for growth ( Tankeshwar, 2018). · ODC Test: Tests for decarboxylations (described in ADH & LDC tests) of the amino acid ornithine by ornithine decarboxylase. Same concept as ADH and LDC tests - positive reaction red/red orange and negative is yellow/yellow orange. The test is used to determine if bacteria can use the amino acid, Ornithine, as a source of carbon and energy for growth.

· CIT Test: This test is used to determine the ability of a microorganism to use citrate as its sole carbon source. Organisms which can utilize citrate as their 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. The citrate media is green before inoculation, and turns blue if the culture is positive for citrate utilization (Tankeshwar, 2016). · H2S Test: Tests for production of hydrogen sulfide. Some Bacteria that can reduce sulfur containing compounds such as thiosulfate to hydrogen sulfide during metabolism. Once H2S is

produced, it combines with ferrous ammonium sulfate forming black precipitate. The black precipitate shows a positive test for H2S production, and no black precipitate is a negative result. · URE Test:Tests for the enzyme Urease. Some bacteria can use urea as a source of nitrogen. As a result, they are able to produce an enzyme called urease that breaks the nitrogen and carbon bond in urea forming ammonia, water and carbon dioxide.The ammonia produces from the utilization of urea by bacteria, raises the pH of the solution yellow to red. Therefore, a red color shows that there is urease in the bacteria, and a yellow color shows that there is not (Tankeshwar, 2017). · TDA Test: (Tryptophan deaminase): detection of the enzyme tryptophan deaminase: Reagent to put- Ferric Chloride. Deaminase is an enzyme that hydrolyzes amino compounds (such as amino acids) with removal of the amino group.This test determines the ability of bacteria to deaminate amino acid L-tryptophan to produce intermediate product indole-pyruvic acid. Indolepyruvic acid produces a red/brown color in the presence of ferric chloride. If color is yellowish, then the result is negative (Hardy Diagnostics, 2008). · IND (Indole) Test: Production of indole from tryptophan by the enzyme tryptophanase. Reagent- Indole is detected by addition of Kovac’s reagent. It tests for the bacteria species’ ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one. Bacteria is grown in a tryptone broth which contains tryptophan. After the incubation time has passed, Kovac’s Reagent (yellow) is added to culture. Kovac’s reagent reacts with indole and creates a red color at the top part of the test tube. A red ring shows a positive result, and yellow color shows negative result. · VP (Voges-Proskauer) Test: Tests for the detection of acetoin produced by fermentation of glucose by bacteria utilizing the butylene glycol pathway. Also, the Voges Proskauer test uses Barrit’s reagent A (which contains alpha-naphthol) and Barrit’s reagent B ( which contains potassium hydroxide) to test for the presence of acetoin. Acetoin is an intermediate of the 2,3butanediol fermentation pathway. After the reagents are added, the tube is shaken vigorously then allowed to sit for 5-10 minutes. A red color means the culture is positive for acetoin. A negative result would show a colorless mixture. · GLU Test: Tests fermentation of glucose. Ph Indicator used is Bromothymol blue, color is blue at pH 7.8. As pH decreases, its color turns to green (6.8), then yellow (6.0). Acids produced by fermentation lower the pH of the solution. The reagents added to the GLU microtube are

sulfanilic acid and N,N-dimethyl-1-naphthylamine. The Nitrate Reduction test for the GLU test for microbes that produce the enzymes nitrate reductase and Nitrite reductase. The two enzymes catalyze two reactions and converts nitrate to nitrogen gas. The nitrite ions are detected by the addition of sulfanilic acid and N,N-dimethyl-1-naphthylamine. Any nitrite will react with these reagents to produce a pink or red color. If no color change occurs, there are 3 possibilities: bacteria possess nitrate reductase and reduce nitrite to ammonia, other enzymes reduced nitrite to ammonia, or nitrates are not reduced. If nitrates reduced past nitrite, addition of zinc powder can confirm as zinc reduces nitrates to nitrites. No red color produced with addition of zinc powder nitrates reduced past nitrite. Pink red color appears with addition of zinc powder - nitrates not reduced (Tankeshwar, 2016). · MAN (Mannose), INO (inositol), SOR (sorbitol), RHA (rhamnose), SAC (sucrose), MEL (melibiose), AMY (amygdalin), and ARA (arabinose) Tests: Test for fermentation of the compounds listed in parentheses. Fermentation is the chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving bubbles and the giving off of heat. Reagent: hydrogen peroxide. A positive result for fermentation will show a yellow color to the mixture, and a negative result will show a green/blue-green color (Tankeshwar, 2016).

Hypothesis: To determine the unknown bacteria given through the various tests from the API 20E System.

Materials · Broth cultures of: Escherichia Coli, Salmonella enteritidis, Citrobacter freundii, Enterobacter aerogenes, and Proteus mirabilis. · Unknown bacteria: Salmonella enteritidis. · API 20E System with the dehydrated mediums of: ONPG, ADH, LDC, ODC, CIT, H2S, URE, TDA, IND, VP, GLU, MAN, INO, SOR, RHA, SAC, MEL, AMY, AND ARA. · Mineral Oil · Ferric chloride (10%) · Kovac’s Reagent · Potassium hydroxide (40%)

·

α -naphthol (6%)

· Sulfanilic acid (0.8%) · N, N-dimethyl-a-naphthylamine (0.5%) · Zinc powder · 1.5% H2O2

Procedure 1. Take picture of API strip prior to the experiment. 2. Use chart 35.1 from manual to guide through the experiment. 3. ONPG, ADH, LDC, ODC, CIT, H2S, and URE tests in microtubes have already been taken care of. Just follow given chart and note results based on colors of tubes. 4. For TDA tube, add 1 drop of 10% Ferric chloride through cupule and note color change result. 5. For the IND tube, add 1 drop of Kovac’s Reagent and mark color change/results. 6. VP: add 1 drop of 40% Potassium hydroxide, then 1 drop 6% α -naphthol and wait ten minutes before considering reaction. 7. GLU: before adding reagents, look for bubbles as it determines if nitrate reduction occurs or not. 8. GLU: add 2 drops 0.8% Sulfanilic acid and 2 drops 0.5% N,N-dimethyl-anaphthylamine. Add zinc powder to reagent to further determine positive or negative test. Mark down results observed. 9. Add 1 drop 1.5% H2O2 to the microtubes containing the reagents MAN, INO, SOR, RHA, SAC, MEL, AMY, and ARA. 10. Observe which tests were (+) or (-). 11. Use Chart III to see characterization of gram negative.

Results After conducting the experiment and reviewing my results, I was able to firmly identify my unknown bacteria through the different reactions of the API 20E System and the charts provided (bacteria test chart and gram negative rod chart.) I compared and contrasted the color

changes and characteristics of these reactions to the accepted characteristics of the bacterias it could be. By analyzing my results, I concluded that ADH, LDC, ODC (+/-), CIT, H2S, GLU, MAN, SOR, RHA, MEL and ARA exhibited positive results to each of their own individual tests. On the other hand, URE, TDA, VP, INO, and SAC exhibited negative results to their own individual tests. The microtubules of ONGP, IND, and AMY tested positive as it had a yellow, red, and yellow color change, respectively while it should have a colorless, yellow, and blue color change indicating it was negative. I attribute these faulty results to contamination. Discussion After conducting the experiment and reviewing my results, I identified my unknown bacteria to be Salmonella enteritidis through the color changes of the reactions and using the charts given such as the gram negative rod chart. Prior to the experiment, the ONPG through URE tests were conducted, so we were able to easily observe the color changes in these microtubules. In the ONPG test, I observed a yellow color to the media indicating a positive outcome for B-galactosidase enzyme by hydrolysis of the substrate o-nitrophenyl-b-Dgalactopyranoside. This color change is a characteristic of the bacterias Escherichia Coli, Citrobacter freundii, and Enterobacter aerogenes. For the bacteria Salmonella enteritidis, this test should be colorless signifying a negative result. I conclude that this false result is due to contamination inside the microtubule. In the ADH microtubule, there was a red/pink color change which is positive result for the bacteria Salmonella enteritidis. This specific result is only a characteristic of Salmonella enteritidis which greatly aided in the identification of my unknown bacteria. In the LDC microtubule, there was a red color produced which is a positive result for the bacterias Escherichia Coli, Salmonella enteritidis, Enterobacter aerogenes, and Proteus mirabilis. In the ODC microtubule, I observed a red color which is positive. In the CIT microtubule, there was a turquoise color indicating a positive result. In the H2S microtubule, it was positive for production of hydrogen sulfide as it had a black deposit as shown in Figure 2. In the URE microtubule, it showed a yellow color change which is a negative result for the enzyme urease. In the TDA microtubule, I added a drop of Ferric chloride which produced a color reaction of golden orange indicating a negative result for the detection of the enzyme tryptophan deaminase. In the IND microtubule, I added a drop of Kovac’s reagent and waited two minutes for a reaction to occur. After, I observed a red ring color reaction indicating a positive result for the production of indole from tryptophan which is an incorrect result for the bacteria Salmonella

enteritidis. The correct result should be a yellow color change to prove negative for the production of indole from tryptophan. In the VP microtubule, I added a drop of Potassium hydroxide and a drop of

α -naphthol which produced a pale color, evidential of a negative

reaction for the detection of acetoin produced by fermentation of glucose. In the GLU microtubule, I added two drops of Sulfanilic acid and two drops of N,N-dimethyl-anaphthylamine and then checked to see if bubbles were visible which is indicative of reduction of nitrate to nitrogenous state. I observed red bubbles forming and then added zinc powder to further prove a negative or positive result. After doing this procedure, I observed a yellowish grey color in the microtubule from the substrate mixture which is indicative for a positive result for fermentation and nitrates were reduced. In the MAN, SOR, RHA, MEL and ARA microtubules, there were yellow color changes indicating positive results for fermentation. In the INO and SAC microtubules, there was a display of a blue color specifying there was no fermentation of those compounds or a negative result. However, I believe there was contamination in the AMY microtubule because instead of a blue color indicating no fermentation or negative reaction, there was a yellow color change. Although there were contamination factors which lead to some results being faulty, I was able to narrow down the list of bacterias given to Salmonella enteritidis based on key color characteristics and the charts provided. With the Characterization of Gram-Negative Rods chart, I was able to clarify my results for the negativity and positivity of each test for each bacteria. Numbers ranging 0-50 indicate negative for the test and above this range indicate positive. Based on my observations and the knowledge I gained performing this experiment, I accept the hypothesis that the API 20E System can aid in the determination of the given unknown bacteria. Therefore, I conclude that my unknown bacteria is Salmonella enteritidis.

Citations

● ONPG RAPID TEST BROTH. (n.d.). Retrieved from https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/ONPGBroth.htm

● Hardy Diagnostics. “FERRIC CHLORIDE REAGENT, 10%.” Ferric Chloride Reagent, 10% for Deaminase Reactions in Bacteria, 2008, catalog.hardydiagnostics.com/cp_prod/Content/hugo/FerricChlorideRgnt10.htm. ● Citrate utilization test: Principle, Procedure, expected results and positive organisms. (2016, August 10). Retrieved from https://microbeonline.com/citrate-utilization-testprinciple-procedure-expected-results-and-positive-organisms/ ● Tankeshwar. “Urease Test: Principle, Procedure, Interpretation and Urease Positive Organisms.” Microbeonline, 1 Feb. 2017, microbeonline.com/urease-test-principleprocedure-interpretation-and-urease-positive● Tankeshwar. “Decarboxylation Test: Types, Uses, Principles, Procedure and Results.” Microbeonline, 14 Apr. 2018, microbeonline.com/decarboxylation-test-types-usesprinciples-procedure-results/organsims/. ● Tankeshwar. “Citrate Utilization Test: Principle, Procedure, Expected Results and Positive Organisms.” Microbeonline, 10 Aug. 2016, microbeonline.com/citrateutilization-test-principle-procedure-expected-results-and-positive-organisms/....