Metabolism part

Lab 10 Microbial metabolism part I Biochemical characteristics of bacteria  Many of the detectable differences between bacteria are biochemical  We take advantage of these principals by performing biochemical test on bacteria  These test give us the ability to distinguish between different bacteria  Over these days you will perform various biochemical test that are used to distinguish between different bacteria Flow chart for bacteria identification (example) http://www.kcom.edu/faculty/chamberlain/Website/lab/idlab/4gram-.jpg Objective Carbohydrate fermentation  Carbohydrate are big molecules – Require enzymes to be broken down in smaller molecules – If the bacteria does not have the enzyme it can not break down/use the carbohydrate  Fermentation is noted by acid production Objective Carbohydrate fermentation A Lactose broth    Escherichia coli Proteus vulgaris Positive result: yellow color change with or without gas production  Negative result: no color change  Gas production  bubble in the durham tube http://www2.austin.cc.tx.us/microbugz/images/26suc.jpg http://biosci.usc.edu/courses/2002-fall/documents/bisc300-lab_Carbohydrate_Fermentation_Sucrose.jpg Objective Carbohydrate fermentation B OF test (oxidation-fermentation)    Contains a Glucose pH indicator: bromothymol blue  turn yellow under acidic condition Determine whether a bacterium has the enzymes necessary for the aerobic breakdown of glucose (oxidation ) and/or for the fermentation of glucose (anaerobic) OF glucose test Use the needle oil 1: no oxidation, no fermentation 3: Fermentation 2: oxidation but no fermentation http://www.jlindquist.net/generalmicro/DMimages/newglucof2.jpg Escherichia coli Pseudomonas aeruginosa Objective Starch hydrolysis  Starch is a big sugar molecule  Is broken into smaller molecules by the enzyme amylase  Area of starch hydrolysis becomes apparent on the media following the addition of iodine  Positive result: clear halo around the colonies  Negative result: areas of dark blue Bacillus subtilis Escherichia coli Starch hydrolysis - Add Gram Iodine + Objective 3: MR-VP  Methyl red  Identify bacteria that produce stable acids as end-products when fermenting glucose  pH indicator added after incubation  After 24 hours,Add Methyl Red – drops  Positive: Red  Negative: yellow E Coli E Aerogenes http://www.marietta.edu/~spilatrs/biol202/labresults/methyl%20red.jpg Objective 3: MR-VP  Voges-Proskauer  Identify bacteria that produce butanediol as end-products of fermentation  Add 15 drops of reagent  Add drops of reagent  Color change can take up to 30 60 minutes  Positive: red color  Negative: yellow/translucent color Objective Catalase test Streptococcus faecalis  Aerobic respiration results in the formation of hydrogen ions (H+) which are converted into H2O2.n (hydrogen peroxide)  The Catalase enzyme breaks down the peroxide into non toxic water Staphylococcus aureus TSA Catalase test 24 hours later add hydrogen peroxide on the colony Bubble: positive result http://biology.ucok.edu/Microbiology/images/catalase.jpg Objective Oxidase  Cytochrome oxidase is an enzyme involved in the reduction of oxygen at the end of the electron transport chain  React with oxygen and the reagent to produce a purple (dark almost black) color change – Positive result: purple color change – Negative result: no color change Oxidase test P aeruginosa  Aerobic respiration results in the formation of hydrogen ions (H+) which are converted into H2O2.n (toxic)  The Catalase enzyme breaks down the peroxide into non toxic water E coli TSA Oxidase 2-3 drops oxidase reagent Purple color change Positive result http://biology.ucok.edu/Microbiology/images/oxidase2.jpg Objective Citrate utilization  Test the ability of the bacteria to use citrate as their sole carbon source  Test whether they possess the citrase enzyme or not  It test for the presence of alkaline (basic) products as a result of citrate utilization  Bromothymol blue  detect pH change Citrate utilization    Inoculate slant E coli Enterobacter aerogenes  Incubate  Positive: media change from green to blue (citrate was used)  Negative: no color change http://www.marietta.edu/~spilatrs/biol202/labresults/citrate.jpg Objective Nitrate reduction test  It test the ability of some bacteria to reduce Nitrate to nitrite  Media contains peptone and beef extracts to support growth as well as potassium nitrate   if the organism can reduce nitrate it will react with the reagents – 1) sulfanillic acid – 2) naphthalamine acetate – 3) Addition of zinc dust might be necessary if the nitrate has been reduced all the way to elemental nitrogen Nitrate reduction test  positive In slant :Staphylococcus aureus Streptococcus faecalis Incubate at 37 + +Z n du s t http://www.cdc.gov/std/Gonorrhea/lab/images/NO3pos.GIF Objective TSI    Triple Sugar iron Used for identification of Gram-negative enteric rods measures a bacterium's ability to utilize three sugars – – – Glucose 0.1% Sucrose 1% Lactose 1%  pH indicator in the media detect acid production  if acid turn yellow  Also measure the reduction of sodium thiosulfate ** tests in one tube** TSI  Inoculation is a two step procedure – First, a loopful of bacteria is spread across the surface of the agar using a needle – Second, the needle is stabbed into the bottom (butt) of the tube  Acid production only in the butt  indicative of glucose fermentation only – the small amount of glucose in the medium is fermented within the first hours of incubation – After, the bacteria get their energy by oxidizing peptone  media on the slant turn red (alkaline conditions) – Peptone can not be used in the butt because there is no O2  butt remains yellow TSI  Acid production in the slant and butt  sucrose and/or lactose fermentation – high concentrations of these sugars  extensive acid production  Reduction of sodium thiosulfate to hydrogen sulfide – Hydrogen sulfide production will turn parts of the agar black  Production of gas – cracks in the agar – air bubbles trapped at the bottom of the tube TSI K: alkaline Escherichia coli Pseudomonas aeruginosa Proteus vulgaris http://users.stlcc.edu/kkiser/TSIreact.jpg A: acid [...]... butt  sucrose and/or lactose fermentation – high concentrations of these sugars  extensive acid production  Reduction of sodium thiosulfate to hydrogen sulfide – Hydrogen sulfide production will turn parts of the agar black  Production of gas – cracks in the agar – air bubbles trapped at the bottom of the tube TSI K: alkaline Escherichia coli Pseudomonas aeruginosa Proteus vulgaris http://users.stlcc.edu/kkiser/TSIreact.jpg