1 / 14

BIOCHEMICAL DIFFERENTIAL TESTS Ex. 41-43

Lab 12. BIOCHEMICAL DIFFERENTIAL TESTS Ex. 41-43. Metabolism (all biochemical reactions in a living cell) is divided in:. 1) Catabolism: Breakdown of molecules and production of Energy 2) Anabolism: Synthesis of new cellular material, using the Energy produced in Catabolism. O/F Test.

taryn
Download Presentation

BIOCHEMICAL DIFFERENTIAL TESTS Ex. 41-43

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. Lab 12 BIOCHEMICAL DIFFERENTIAL TESTSEx. 41-43

  2. Metabolism (all biochemical reactions in a living cell) is divided in: • 1) Catabolism: Breakdown of molecules and production of Energy • 2) Anabolism: Synthesis of new cellular material, using the Energy produced in Catabolism

  3. O/F Test • Results • Fermentative and oxidative • Oxidative • Glucose not metabolized

  4. BIOCHEMICAL DIFFERENTIAL TESTS • CARBOHYDRATE FERMENTATION • pH indicator: Phenol Red • Aerogenic fermenters • Anaerogenic fermenters Yellow: Acid produced

  5. BIOCHEMICAL DIFFERENTIAL TESTS • METHYL RED TEST • Culture medium: Glucose peptone broth • Differentiates between mixed acid fermenters and 2,3-butanediol fermenters • Differentiate among the coliforms • E. coli vs. Enterobacter-Klebsiella group • Indicator: Methyl red • Interpretation • Addition of methyl red indicator and development of red color = positive (mixed acid fermentation) • Orange or orange-red = negative

  6. BIOCHEMICAL DIFFERENTIAL TESTS • CITRATE UTILIZATION TEST • Basis: Utilization of citrate as sole source of carbon • Medium: Citrate Broth/Simmon’s Citrate Agar slant • Sodium citrate Acetate + Oxaloacetate • Citrate permease also required for entry • Indicator: Bromthymol blue • Accumulation of alkaline products lead to indicator color change • Positive: growth & change in color indicator to blue • Negative: no growth nor change in indicator color (remains green-acidic) Mg2+ Citratase Na+ Pyruvate + CO2

  7. BIOCHEMICAL DIFFERENTIAL TESTS • NITRATE REDUCTION • Nitrate Reductase • NO3- + 2 e- + 2 H+ —> NO2- + H2O • Nitrite Reductase • 2 NO2- + 10 e- + 12 H+ —> N2 + 6 H2O • Evidence for a multistep process • NO3- NO2- NO • NO N2O N2 Nitrate Reductase Nitrite Reductase Nitric Oxide Reductase Nitrous Oxide Reductase

  8. BIOCHEMICAL DIFFERENTIAL TESTS NITRATE REDUCTION Reagents: sulfanilic acid/-naphtylamine: detect presence of No2 No color change Red color Bacterial Reduction NO3- NO2- Add powdered Zn: react with NO3 product No color change NO3- NO2- NH2/N2 Nitrate Reductase/Nitrite Reductase both present Red color no Bacterial Reduction

  9. BIOCHEMICAL DIFFERENTIAL TESTS O O CH3—C—C—OH • INDOLE TEST • Detects intracellular enzyme complex Tryptophanase Medium: Tryptone Broth Tryptophanase NH3 Indole Pyruvic Acid Ammonia • Kovac’s Reagent (p-dimethylaminobenzaldehyde) • Added after 24 hr. incubation

  10. BIOCHEMICAL DIFFERENTIAL TESTS • INDOLE TEST RESULTS

  11. Ex. 42: Hydrolytic & Degradative reactions • STARCH HYDROLYSIS • Urea Hydrolysis • Fat Hydrolysis

  12. Tributyrin agar test (Fat Hydrolysis) Tributyrin agar is a differential medium that tests the ability to produce an exoenzyme, lipase, that hydrolyzes tributyrin oil.  Tributyrin oil forms an opaque suspension in the agar. When an organism produces lipase a clear halo surrounds the areas where the lipase-producing organism has grown. Pseudomonas aeruginosa positive for lipase production (clearing surrounding the growth.). Salmonella negative for lipase production.

  13. II) Eosin Methylene Blue Agar • Selective-differential medium for the isolation of gram-negative bacteria. It is especially used in the isolation of coliforms • EMB agar is selective because the aniline dyes in this purple media inhibit growth of Gram-positive organisms. Lactose fermenters metabolize the lactose in the media and produce acid byproducts, causing a color change in the colony. Thus, EMB is also a differential medium. • Strong acid production by organisms such as E. coli results in a metallic green sheen. • Weaker fermentation of lactose results in colonies with a pinkish-purple color. • Colonies of nonlactose fermenters remain colorless, or at least are no darker than the color of the media. • III) Nutrient Agar (Rich medium): Detection of colony types

  14. Ex. : You will inoculate all biochemical test following the handout instructions (for work distribution) and your Lab Manual (for procedures) WORK Physiological Unknown • Check if you have well isolated colonies for the two types • Consider results analyzed last week • Make a gram stain of each if you have not done it • Show your dichotomous key to your instructor and start doing the biochemical tests accordingly.

More Related