2420 Lab Week 11 Experiments 15,16,17

2420 Lab Week 11 Experiments 15,16,17 Nutritional Types Carbon Energy Heterotroph Autotroph Phototrophs Chemotroph must obtain carbon an organism that gain energy gain energy from in an organic form uses CO2, an through chemical inorganic gas as photosynthesis compounds nutritionally its carbon source dependent on other life forms not nutritionally dependent on proteins, other life forms carbohydrates, lipids, nucleic acids Metabolic Strategies Processing nutrients in many cases is based on three catabolic pathways that convert glucose to CO2 and gives off energy Aerobic respiration Anaerobic respiration Fermentation glycolysis glycolysis Kreb’s cycle Kreb’s cycle glycolysis respiratory chain respiratory chain organic Final electron Not Oxygen but 2- compounds acceptor: Oxygen sulfate (SO4 ), - nitrate (NO3 ) Fermentation • Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen • Uses organic compounds as terminal electron acceptors • Yields a small amount of ATP • Production of ethyl alcohol by yeasts acting on glucose • Formation of acid, gas, and other products by the action of various bacteria on pyruvic acid Comparing Aerobic Respiration, Fermentation and Anaerobic Respiration Interpreting carbohydrate fermentation tests Negative control Acid only Acid and gas TRIPLE SUGAR IRON INGREDIENTS FUNCTION RESULTS INTERPRETATION Phenol red turns yellow in an acid Indicates whether the acids of a pH indicator: environment. fermentation have been produced. below 6.8 it is Phenol red yellow; Failure to turn the butt yellow Indicates that no fermentation has above 8.2 it is red occurred, and that the bacterium is an obligate aerobe. if only glucose is 0.1 % fermented, only a Butt yellow. Slant red. Only glucose is fermented glucose small amount of acid is produced if the culture can ferment either Indicates the ability of the 1.0 % lactose lactose (lac+) culture to ferment either 1.0% sucrose and/or sucrose Butt yellow. Slant yellow. (suc+), a large lactose or sucrose amount of acid is produced A few bacteria are capable of reducing the SO4= to H2S (hydrogen FeSO4 A source of iron sulfide). (ferrous and Butt black. The iron combines with the H2S to sulfate) sulfur form FeS (ferrous sulfide) a black compound. gas formed Standard notation SR/BR SR/BY SY/BY SY/BY/+ G IMViC tests IMViC tests are used to differentiate the enterics (Enterobacteriaceae). Indole test (tryptone broth) Methyl Red and Voges-Proskauer tests (MRVP broth) Citrate test (Citrate agar slants) Significance: When testing drinking water for the presence of the sewage indicator E. coli, one must be able to rule out Enterobacter aerogenes E. aerogenes is not always associated with sewage, and its presence in water would not necessarily indicate sewage contamination. Indole test Detects Tryptophan hydrolysis Some bacteria split tryptophan into indole and pyruvic acid Indole can be detected with Kovac's reagent Test is very important in differentiating E. coli (indole positive) from some closely related enteric bacteria. It also differentiates Proteus mirabilis (indole negative) from all other Proteus species (indole positive). Tryptone broth is used for this test as it contains a large amount of tryptophan. Indole test Interpretation After incubation: The broth must be turbid A clear broth indicates that your organism did not grow and cannot be tested Add 5 drops of Indole reagent (Kovac’s reagent) to the tryptone broth culture + - DO NOT SHAKE THE TUBE A positive result has a red layer at the top A negative result has a yellow or brown layer Methyl red test Mixed acid fermentation Many Gram-negative intestinal bacteria can be differentiated based on the products produced when they ferment the glucose in MR-VP medium. Escherichia, Salmonella, and Proteus ferment glucose to produce lactic, acetic, succinic, and formic acids and CO2, H2, and ethanol. The large amounts of acids produced lowers the pH of the medium Methyl red (a pH indicator) will turn red when added to the medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas. Methyl red test interpretation The MR-VP broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Add 5-6 drops of methyl red to the original broth. + - SHAKE THE TUBE. Positive result: Medium turns red. Negative result: Medium remains yellow. Voges-Proskauer (VP) test Organisms that are negative in the methyl red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products do not lower the pH as much as acids do. Enterobacter, Serratia and some species of Bacillus produce these substances. There is no satisfactory test for determining production of 2, 3 butanediol. A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent. Voges-Proskauer test interpretation Barritt's solution A contains naphthol. Barritt's solution B contains KOH. 1) Add 15 drops of Barritt's solution A. GENTLY SHAKE THE TUBE. 2) Add 10 drops of Barritt's solution B. GENTLY SHAKE THE TUBE. Leave tube on rack for 30-45 minutes. + - Shake tubes every 10 minutes Positive result: Medium turns red. Negative result: Medium remains yellow. Citrate test Simmon's citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH. Citrate test interpretation + - A positive reaction is indicated by growth and a slant with a Prussian blue color. A negative slant will have no growth of bacteria and will remain green..

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