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  1. 1. Lab # 9: IMVIC Test <ul><li>Identification of enteric bacilli is important in controlling intestinal infections by preventing the contamination of food and H2O supplies. </li></ul><ul><li>Bacteria that is found in the intestinal tract of humans are members of the family Enterobacteriaceae. </li></ul><ul><li>Enterobacteriaceae are aerobic or facultative anaerobes, gram neg., nonendospore forming, rod shaped bacteria. </li></ul><ul><ul><li>Coliforms are Enterobacteriaceae that ferment lactose with acid and gas formation within 48 hrs at 35 deg. C </li></ul></ul>
  2. 2. Lab # 9: IMVIC Test pg. 141, 221 <ul><li>IMViC, TSIA, Sims are done individually </li></ul><ul><li>You will inoculate all your tubes with one of your controls </li></ul><ul><li>Make sure all the controls are covered </li></ul><ul><li>If you have more than 4 in your group, make your additional inoculations with E. coli and Enterobacter aerogenes </li></ul>
  3. 3. Lab # 9: IMVIC Test pg. 141, 221 <ul><li>The IMViC uses a standard combination of 4 tests with each capital letter in IMViC representing a test: the i is added to make pronunciation easier </li></ul><ul><li>The tests are as follows: </li></ul><ul><ul><li>I = indole test – indole production from tryptophan </li></ul></ul><ul><ul><li>M = Methyl red test – for acid production from glucose </li></ul></ul>
  4. 4. Lab # 9: IMVIC Test <ul><li>IMViC test </li></ul><ul><ul><li>V = Voges - Proskauer test – for production of acetoin from glucose </li></ul></ul><ul><ul><li>C = Citrate test – checks to see if the organism can use citrate as it’s sole source of carbon </li></ul></ul><ul><ul><ul><li>Citrate is inoculated into 2 tubes </li></ul></ul></ul><ul><ul><ul><ul><li>Koser’s citrate – broth to broth transfer </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Simmon’s citrate – broth to slant transfer </li></ul></ul></ul></ul>
  5. 5. Lab # 9: IMVIC Test <ul><li>Inoculate one loopful of bacteria into one tube, then the tubes are incubated for 24 – 48 hours for the bacteria to grow </li></ul><ul><li>Controls for IMViC test, TSIA, Sims are: </li></ul><ul><ul><li>Enterobacter aerogenes </li></ul></ul><ul><ul><li>E. coli </li></ul></ul><ul><ul><li>Citrobacter freundii </li></ul></ul><ul><ul><li>Staphylococcus aureus </li></ul></ul>
  6. 6. Lab 9: Triple Sugar Iron Agar pg. 149, 227 <ul><li>TSI agar contains: </li></ul><ul><ul><li>0.1 % glucose </li></ul></ul><ul><ul><li>1.0 % lactose </li></ul></ul><ul><ul><li>1.0 % sucrose </li></ul></ul><ul><ul><li>0.02% ferrous sulfate </li></ul></ul><ul><ul><li>Phenol red </li></ul></ul><ul><ul><li>Nutrient agar </li></ul></ul>
  7. 7. Lab 9: Triple Sugar Iron Agar <ul><li>Checks for fermentation – acid and gas production, indicates if hydrogen sulfide (H2S) has been produced – black ppt </li></ul><ul><li>Procedure: </li></ul><ul><ul><li>Stab a sterile loop all the way to the bottom of the butt of the tube and pull it straight out </li></ul></ul><ul><ul><li>When you get to the top of the agar make your normal streak up the slant of the tube </li></ul></ul>
  8. 8. Sulfide Indole Motility Agar pg 155, 233 <ul><li>Sims agar contains: </li></ul><ul><ul><li>Peptone and sodium thiosulfate as the sulfur substrates </li></ul></ul><ul><ul><li>Ferrous sulfate (FeSO4) which behaves as a H2S indicator </li></ul></ul><ul><ul><li>Sufficient agar to make the media semisolid and enhance anaerobic respiration </li></ul></ul>
  9. 9. Sulfide Indole Motility Agar <ul><li>Checks for motility, indole production, and H2S production of organism </li></ul><ul><li>Procedure: </li></ul><ul><ul><li>Stab a loopful of your organism straight down, in the middle, about ½ way down </li></ul></ul><ul><ul><li>Don’t wiggle your loop </li></ul></ul><ul><ul><li>You want to be able to see if your organism is motile </li></ul></ul>
  10. 10. Lab 8 Results <ul><li>Rubber band all your gelatin stabs together and put them in a beaker to be refrigerated </li></ul><ul><li>When the extracellular gelatinase hydrolyzes the protein gelatin it will remain liquid after refrigeration </li></ul><ul><li>If the media becomes solid your organism does not have the enzyme gelatinase </li></ul>
  11. 11. Lab 8 Results <ul><li>Extracellular Metabolism with extracellular enzymes </li></ul><ul><li>1. Skim milk (Casein agar) – checks to see if organism produces enzyme caseinase </li></ul><ul><ul><li>If it does , there will be a clearing around your colonies where the protein was broken down to amino acids </li></ul></ul>
  12. 12. Lab 8 Results <ul><li>2. Starch agar – checks to see if your organism produces the enzyme amylase which breaks down starch to simple sugars </li></ul><ul><li>a. procedure: Flood plate with gram iodine </li></ul><ul><li>b. positive rx = yellow zone around the colonies </li></ul><ul><li>c. negative rx = purple around colonies </li></ul>
  13. 13. Lab 8 Results <ul><li>CH2O Metabolism </li></ul><ul><li>1. Durham tube fermentation method </li></ul><ul><li>a) sugar (lactose) fermented </li></ul><ul><li>b) acid production = yellow media </li></ul><ul><li>c) gas production = bubble in durham tube </li></ul><ul><li>d) red tube means an alkaline broth – no acid produced, no fermentation </li></ul>
  14. 14. Lab 8 Results <ul><li>Check your agar plates and record which organisms grew </li></ul><ul><ul><li>MSA </li></ul></ul><ul><ul><li>EMB </li></ul></ul><ul><ul><li>MacConkey’s </li></ul></ul><ul><ul><li>Blood – check for alpha, beta, or gamma hemolysis </li></ul></ul>