1 / 23

Media Biochemical Tests

nani
Download Presentation

Media Biochemical Tests

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

    1. Media & Biochemical Tests Laboratory Objectives

    2. Tests To Know Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production Triple Sugar Iron Nitrate Reduction Test Oxidase Thioglycollate

    3. Indole Test How to Perform Test: Inoculate Tryptone broth with inoculating loop. Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. 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. Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color. Reading Results: Kovac’s reagent reacts with indole and creates a red color at the top part of the test tube.

    4. Indole

    5. Methyl Red/Voges Proskauer (MR/VP) How to Perform Tests: Inoculate 2 glucose broths with inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube. Properties they test for: Both tests are used to help differentiate species of the family Enterobacteriaceae. MR—tests for acid end products from glucose fermentation. VP—tests for acetoin production from glucose fermentation. Media and Reagents Used: Glucose Broth Methyl Red indicator for acid Voges Proskauer reagents—A: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water.

    6. MR/VP continued Reading Results: MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production) VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change.

    7. Citrate How to Perform Test: Inoculate slant with inoculating loop. Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source. Media and Reagents Used: Simmon’s Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator—bromthymol blue. Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a – result remains green

    8. Citrate

    9. H2S Production in SIM How to Perform Test: Stab SIM media with inoculating needle. Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. This test is used to determine the ability to reduce sulfur into H2S. Media and Reagents Used: SIM media contains the sulfur containing amino acid, cysteine, sodium thiosulfate, & peptonized iron or ferrous sulfate. Reading Results: H2S will react with the iron or ferrous sulfate and produce a black precipitate. A positive result has a black precipitate present and a negative result has no black precipitate.

    10. Urea Hydrolysis How to Perform Test: Inoculate Urea broth with inoculating loop. Property it tests for: This test is done to determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease. Media and Reagents Used: Urea broth contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator. Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.

    11. Motility Test How to Perform Test: Stab motility media with inoculating needle. Property it tests for: This test is done to help differentiate species of bacteria that are motile. Media and Reagents Used: Motility media contains tryptose, sodium chloride, agar, and a color indicator. Reading Results: If bacteria is motile, there will be growth going out away from the stab line, and test is positive. If bacteria is not motile, there will only be growth along the stab line. A colored indicator can be used to make the results easier to see.

    12. Motility

    13. Lactose Fermentation How to Perform Test: Inoculate lactose broth with inoculating loop. Property it tests for: This tests for the bacteria’s ability to ferment lactose. Media and Reagents Used: Lactose broth contains beef extract, gelatin peptone, and lactose. A phenol red indicator is added to indicate acid production from fermentation. Results A positive result is yellow after several drops of phenol red is added (indicating lactose fermentation) A negative result is reddish in color.

    14. Sucrose Fermentation How to Perform Test: Inoculate sucrose broth with inoculating loop. Property it tests for: This test is done to help differentiate species of the family Enterobacteriaceae. This tests for the bacteria’s ability to ferment sucrose and production of acid end-product Media and Reagents Used: Sucrose broth contains beef extract, gelatin peptone, and sucrose. Phenol red indicator is added to indicate an acid end-product. Results A positive result is yellow after several drops of indicator is added (indicating sucrose fermentation) A negative result is reddish in color.

    15. Glucose Fermentation & Gas Production How to Perform Test: Inoculate broth with inoculating loop. Property it tests for: This test is done to help differentiate species of the family Enterobacteriaceae. This tests for the bacteria’s ability to ferment glucose and produce gas and/or an acid end-product.. Media and Reagents Used: Glucose broth contains beef extract, gelatin, peptone, and glucose. A phenol red indicator is added to indicate an acid end product. A Durham tube is added to indicate gas production. Results A positive result for acid is yellow after indicator is added (indicating glucose fermentation) A positive result for gas is a bubble in the Durham tube. A completely negative result is reddish in color.

    16. Sugar Fermentation Tests

    17. Nitrate Test How to Perform Test: Inoculate Nitrate broth using an inoculating loop and incubate 48 hours. Property it tests for: This test is used to determine if an organism can reduce nitrate to either nitrite or ammonium Media and Reagents: Media contains beef extract peptone and potassium nitrate. Three reagents are added: sulfanilic acid, dimethyl-alpha-napthylamine and zinc.

    18. Nitrate Reduction Three possible test results: Nitrate reduction to nitrite: This is detected after sulfanilic acid (SA) and dimethyl-alpha-napthylamine (DAN) are added and the media turns red. Nitrate reduction to ammonium: If the media did not turn red after the first two reagents were added then zinc is added. Zinc reacts with nitrate to produce a red color. If the media remains yellowish after zinc is added then this indicates a positive result for nitrate reduction to ammonium. Negative result for nitrate reduction: This occurs if the tube did not turn red after SA and DAN, but did turn red after zinc. Zinc detects nitrate and if nitrate is still present that means it was not reduced.

    19. A: is the media before the reagents are added. B and C: after addition of SA and DAN only B indicates a positive result for nitrate reduction to nitrite C indicates a possible negative result for nitrate reduction; must add zinc to confirm. After addition of SA, DAN and zinc. Left tube indicates a confirmed negative result for nitrate reduction. Means that nitrate is still in media. Zinc reacted with nitrate to turn media red. Right inicates a positive result for complete nitrate reduction to ammonium. Media does not turn red; therefore nitrate is completely reduced.

    20. Triple Sugar Iron Test (TSI) How to Perform Test: Inoculate slant with inoculating needle, streak slant, then stab it. Property it tests for: This tests for the bacteria’s ability to ferment lactose, sucrose, and/or glucose, H2S and CO2 production. This is also a confirmatory test for the other lactose, sucrose, and/or glucose fermentation tests, gas production from fermentation, and H2S production tests. Media and Reagents: TSI media contains beef extract, yeast extract, peptone, proteosepeptone, dextrose, lactose, sucrose, ferrous sulfate, sodium chloride, sodium thiosulfate, agar, and phenol red indicator.

    21. TSI continued Reading Results: If acid is produced because of glucose fermentation, the bottom of the slant turns yellow for a positive test result (K/A). If acid is produced because of lactose, and/or sucrose, as well as glucose, the whole slant will turn yellow for a positive test result (A/A). If H2S is produced, because of the sulfur components of the media, a black precipitate will form for a positive result for H2S production. If CO2 (gas) production occurs, this can be observed by cracks or bubbles seen in the media for a positive test result. If there is no color change from reddish to yellow, then the test is negative for fermentation. If no black precipitate forms, then the test is negative for H2S production. If there are no cracks or bubbles, the test results are negative for gas production.

    22. TSI Slant From left to right Tube 1: un-inoculated media Tube 2: A/A ?fermentation of glucose and another sugar (lactose or sucrose) Tube 3: A/A and H2S+ ?same as 2 but also positive for H2S production Tube 4: K/A ?fermentation of glucose, but no fermentation of another sugar (lac or suc) Tube 5: K/A and H2S+

    23. Oxidase How to Perform Test: Inoculate Nutrient agar slant with organism. Grow for 24-48 hours at optimal temperature. Put a smear of culture onto a filter paper disk. Add 1-2 drops of oxidase reagent to smear. Property it tests for: It tests to see if the organism uses the enzyme cytochrome oxidase. Cytochrome oxidase is an enzyme found in some bacteria that transfers electrons to oxygen, the final electron acceptor in some electron transport chains. Thus, the enzyme oxidizes reduced cytochrome c to make this transfer of energy. Media and Reagents: Oxidase reagent Results: Reagent turns purple when exposed to bacteria= + result for the enzyme oxidase Reagent stays the same= - result for oxidase

    24. Oxidase Results

More Related