Lab 12 BIOCHEMICAL DIFFERENTIAL TESTSEx. 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 • Results • Fermentative and oxidative • Oxidative • Glucose not metabolized
BIOCHEMICAL DIFFERENTIAL TESTS • CARBOHYDRATE FERMENTATION • pH indicator: Phenol Red • Aerogenic fermenters • Anaerogenic fermenters Yellow: Acid produced
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
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
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
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
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
BIOCHEMICAL DIFFERENTIAL TESTS • INDOLE TEST RESULTS
Ex. 42: Hydrolytic & Degradative reactions • STARCH HYDROLYSIS • Urea Hydrolysis • Fat Hydrolysis
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.
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
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.