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

Lab # 9: IMVIC Testpg. 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

Lab # 9: IMVIC Testpg. 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

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

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

Lab 9: Triple Sugar Iron Agarpg. 149, 227

TSI agar contains:– 0.1 % glucose– 1.0 % lactose– 1.0 % sucrose– 0.02% ferrous sulfate– Phenol red– Nutrient agar

Lab 9: Triple Sugar 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

Sulfide Indole Motility Agarpg 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

Sulfide Indole Motility Agar

• 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

Lab 8 Results

• Rubber 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

Lab 8 Results

• Extracellular 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

Lab 8 Results

2. Starch agar – checks to see if your organism produces the enzyme amylase which breaks down starch to simple sugarsa. procedure: Flood plate with gram iodine

b. positive rx = yellow zone around the coloniesc. negative rx = purple around colonies

Lab 8 Results

• CH2O 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

Lab 8 Results

• Check your agar plates and record which organisms grew– MSA– EMB– MacConkey’s– Blood – check for alpha, beta, or gamma

hemolysis