Microbiology Lab: Exercise 24-Biochemical Tests, SIM

What are the three biochemical properties the SIM test reveals?

1) Sulfide production from the amino acid cysteine 2) Indole production from the amino acid tryptophan 3) Motility

Procedure for the three SIM tests

1. Label three SIM tubes "E. coli", "K. pneumoniae", and "P. vulgaris". 2. Inoculate each SIM tube with the apropriate bacteria. Remember to stab each tube ONCE right in the middle for each bacteria. 3. Incubate for 48 hourse at 37 degrees C. 4. First, determine if bacteria were motile. Examine tubes. 5. Next, determine if bacteria produced cysteine desulfurase. (Look for lack precipitate). 6. Last, determine if bacteria produced tryptophanas. Add 10 drops of Kovac's Reagent to each of the tubes. Use a pipette for this. Look for a dark red color change.

SIM

A biochemical test. Stands for Sulfide, Indole, and Motility. It is a medium used for these three tests.

How to determine if an indole is present.

Add Kovac's Reagent.

Cystein desulfurase

An enzyme produced by bacteria that can catabolize the amino acid cysteine, which contains sulfur. The SIM medium contains this amino acid.

What is a positive outcome for the sulfide test?

Blackening of the medium.

What does the cysteine desulfurase do?

Breaks down the cysteine to produce the end products of pyruvate (used for energy) and hydrogen sulfude and ammonia.

What does tryptophanase do?

Catabolizes the amino acid tryptophanase

Motility test

Determines if the bacteria inoculated are motile.

What does the second test determine? (The indole test)

Determines if the bacterium produces the enzyme cysteine desulfurase.

What is the indicator called that combines with hydrogen sulfide to produce the black color?

Ferrous iron (Fe2+)

What does the appearance of the red dye indicate?

Indicates that the bacterium is positive for the production of tryptophanase. Absence of the redo color indicates the bacterium is negative for tryptophanase production.

What reagent is added to determine if indole was present in the media? What is the name of the red dye that forms when this reagent and the indole react?

Kovac's reagent is added. The red dye called rosindole forms if indole is present.

What is a negative outcome for the sulfide test?

No blackening of the medium.

What three products does this enzyme break tryptophane down into?

Produces Pyruvate, Ammonia, and indole (a by product)

What are the three products of the breakdown of cysteine?

Pyruvate, Ammonia (NH3), and hydrogen sulfide (H2S)

What does Kovac's Reagent do?

Reacts with indole to produce a red dye called rosindole.

What does the Acronym SIM stand for?

Sulfide, Indole, Motility.

A cloud of bacteria along the stab line indicates what?

That the bacteria are motile.

How to detect this reaction (the breakdown of cysteine by systeine desulfurase).

The SIM medium contains the indicator ferrous iron, which combines with hydrogen sulfide to produce a black precipitate. If the culture blackens from the precipitate, then the bacterium is positive for the production of cysteine desulfurase.

How is the motility test determined?

The motile bacteria will migrate by means of flagella away from the stab line. This will produce turbidity, or a cloud of growth throughout the tube. Non-motile bacteria will not move away from the stab line, and will only grow right around the stab line.

What is the SIM test designed for?

To detect three biochemical properties of a bacterium from the inoculation of one SIM tube.

What is the Tryptophanase test used for (the indole test)?

To determine if a bacterium produces the enzyme tryptophanase.