Ch 14 Imvic Test - Micro Lab

Front 1

human intestinal microbiome

Back 1

• trillions of bacteria; 500 - 1000 different species
• primarily Gram-negative anaerobes of the genus Bacteriodes
• also, facultative anaerobic enterobacteria which are short Gram-negative rods

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intestinal microbiota

Back 2

• serve to protect the human body by outcompeting pathogens
• ex: Escherichia and Enterobacter

(members of the genera Salmonella and Shigella are pathogenic and can cause serious diseases of the digestive system)

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Why is identification of enteric bacteria important and what test is used?

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• enteric bacteria are similar morphologically and physiologically
• important to know that food and water sources are not contaminated by these bacteria
• IMViC test is used

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IMViC Test

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used to test for enteric bacteria

Indole

Methyl Red

Voges-Prokauer

Citrate test

(i is added for pronunciation)

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tryptophan

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• an amino acid found in almost all proteins
• bacteria synthesize tryptophanase to break down tryptophan into its metabolic products: indole, pyruvic acid, and ammonia
• pyruvic acid & ammonia are used by the cell
• indole accumulates in the medium

Front 6

Kovac's reagent

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• tests for the presence of indole
• p-dimethylaminobenzaldehyde reacts w/ indole to produce the red dye rosindole
• indole positive will have a visible red layer on the surface of the medium

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Indole Test

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• tests whether the bacteria can metabolize tryptophan by detecting accumulation of indole
• uses a SIM deep tube with stab inoculation
• after incubation Kovac's reagent is added to the media
• if indole is present a positive result will have a red layer
• a negative result will have a light yellow/orange layer

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mixed-acid fermentation

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• byproducts are mixtures of large amounts of stable acids
• ex: E. coli
• uses the Methyl Red Test
• positive red result would indicate an acidic pH below 4.4

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MR-VP broth media

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contains:

• glucose - fermentable CHO
• peptone - protein
• phosphate buffer - buffers pH changes

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Methyl Red Test

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• used to test for mixed-acid fermentation in MR-VP media
• organisms that produce enough acid to overcome the buffering capacity of the broth
• after incubation the pH indicator Methyl Red is added
• red = positive = pH below 4.4
• orange = negative = intermediate pH
• yellow = negative = pH above 6.0

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Barritt's reagent

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• used in the Voges-Proskauer Test
• contains alcoholic α-naphthol (VP-A) and 40% potassium hydroxide solution (VP-B) that will react with acetoin and oxygen to produce a red wine color
• reaction may take up to 30 min for color to develop

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Voges-Proskauer Test

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• test for organisms able to ferment glucose, but quickly convert their acidic products to neutral acetoin and 2,3 butanediol
• inoculation of MV-VP media; incubation for 72 hours; production of acetoin is dependent on incubation time and pH
• after incubation Barritt's reagent is added
• positive result will give a red color at the top of the tube

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Which tests are used to determine if the organisms can ferment glucose?

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Methyl Red - tests for mixed-acid production

Voges-Proskauer - tests for acetoin or 2,3 butanediol products

(difference is in the products)

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Citrate Utilization Test

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• tests if the organism can use citrate as a sole carbon source; bacteria that synthesize the enzyme citrate permease
• uses Simmon's Citrate agar in a slant tube
• negative result = green
• positive result = blue / can use citrate
• differential and selective

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Simmon's citrate agar

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differential and selective medium that contains:

• citrate - carbon source
• ammonium salts - nitrogen source
• bromthymol blue - pH indicator

only bacteria that synthesize the enzyme citrate permease will grow on this medium

green = negative blue = positive

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Which bacteria were used in this experiment?

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E. coli

Enterobacter aerogenes

Klebsiella pneumoniae

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Examples of differential media

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PRB, SIM Agar, MRVP

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Examples of selective media

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Simmons Citrate, PE Agar