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- 1. Lab #9: IMVIC Test Identification of enteric bacilli is important in controlling intestinal infections by preventing the contamination of food and H2O supplies. Bacteria that is found in the intestinal tract of humans are members of the family Enterobacteriaceae. Enterobacteriaceae are aerobic or facultative anaerobes, gram neg., nonendospore forming, rod shaped bacteria. Coliforms are Enterobacteriaceae that ferment lactose with acid and gas formation within 48 hrs at 35 deg. C
- 2. Lab #9: IMVIC Test pg. 141, 221 IMViC, TSIA, Sims are done individually You will inoculate all your tubes with one of your controls Make sure all the controls are covered If you have more than 4 in your group, make your additional inoculations with E. coli and Enterobacter aerogenes
- 3. Lab #9: IMVIC Test pg. 141, 221 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 The tests are as follows: I = indole test – indole production from tryptophan M = Methyl red test – for acid production from glucose
- 4. Lab #9: IMVIC Test IMViC test V = Voges - Proskauer test – for production of acetoin from glucose C = Citrate test – checks to see if the organism can use citrate as it’s sole source of carbon Citrate is inoculated into 2 tubes Koser’s citrate – broth to broth transfer Simmon’s citrate – broth to slant transfer
- 5. Lab #9: IMVIC Test Inoculate one loopful of bacteria into one tube, then the tubes are incubated for 24 – 48 hours for the bacteria to grow Controls for IMViC test, TSIA, Sims are: Enterobacter aerogenes E. coli Citrobacter freundii Staphylococcus aureus
- 6. Lab 9: TripleSugar Iron Agar pg. 149, 227 TSI agar contains: 0.1 % glucose 1.0 % lactose 1.0 % sucrose 0.02% ferrous sulfate Phenol red Nutrient agar
- 7. Lab 9: TripleSugar Iron Agar Checks for fermentation – acid and gas production, indicates if hydrogen sulfide (H2S) has been produced – black ppt Procedure: Stab a sterile loop all the way to the bottom of the butt of the tube and pull it straight out When you get to the top of the agar make your normal streak up the slant of the tube
- 8. Sulfide Indole MotilityAgar pg 155, 233 Sims agar contains: Peptone and sodium thiosulfate as the sulfur substrates Ferrous sulfate (FeSO4) which behaves as a H2S indicator Sufficient agar to make the media semisolid and enhance anaerobic respiration
- 9. Sulfide Indole MotilityAgar Checks for motility, indole production, and H2S production of organism Procedure: Stab a loopful of your organism straight down, in the middle, about ½ way down Don’t wiggle your loop You want to be able to see if your organism is motile
- 10. Lab 8 ResultsRubber band all your gelatin stabs together and put them in a beaker to be refrigerated When the extracellular gelatinase hydrolyzes the protein gelatin it will remain liquid after refrigeration If the media becomes solid your organism does not have the enzyme gelatinase
- 11. Lab 8 ResultsExtracellular Metabolism with extracellular enzymes 1. Skim milk (Casein agar) – checks to see if organism produces enzyme caseinase If it does , there will be a clearing around your colonies where the protein was broken down to amino acids
- 12. Lab 8 Results2. Starch agar – checks to see if your organism produces the enzyme amylase which breaks down starch to simple sugars a. procedure: Flood plate with gram iodine b. positive rx = yellow zone around the colonies c. negative rx = purple around colonies
- 13. Lab 8 ResultsCH2O Metabolism 1. Durham tube fermentation method a) sugar (lactose) fermented b) acid production = yellow media c) gas production = bubble in durham tube d) red tube means an alkaline broth – no acid produced, no fermentation
- 14. Lab 8 ResultsCheck your agar plates and record which organisms grew MSA EMB MacConkey’s Blood – check for alpha, beta, or gamma hemolysis
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