Exercise # 18 (Protein Catabolism) Sulfide, Indole and Motility (SIM) Test

Sulfide, Indole and Motility (SIM) Test

• The (S) or sulfur component of this test identifies the production of hydrogen sulfide (H2S). If a bacterial species produces hydrogen sulfide (H2S), the H2S will react with the iron ions in the ferrous sulfate (an ingredient in the medium) to produce a black color in the bottom of the tube

Sulfide, Indole and Motility (SIM) Test

Bacteria produce H2S using the enzymes cysteine desulfhydrase and/or thiosulfate reductase. Cysteine and thiosulfate are differential ingredients in this medium. SIM media does not allow us to distinguish between these types of sulfide production

black

indicates that the organism is a potential pathogen

indole (I) component of this test

analyzes bacteria for the presence of the enzyme tryptophanase.

Tryptophanase

an enzyme that both deaminates the amino acid tryptophan (the differential ingredient for this test) and also works on the R-group of tryptophan

Tryptophanase

breaks down proteins that contain the amino acid tryptophan (an ingredient in the media) to produce pyruvate, indole and ammonia

Haemophilus influenzae

ability to produce tryptophanase and break down tryptophan to produce indole is positively correlated with higher pathogenicity

(M) test for motility

indicates the presence of flagella

• Beef extract

• Peptones (source of cysteine, differential ingredient)

• Thiosulfate, differential ingredient

• Ferrous sulfate as a source of iron to indicate H2S production

Components of T-soy Semisolid Agar Plus

Sulfide Production

black precipitate in the butt of the tube is positive

• The bacteria digest cysteine to H2S using the enzyme cysteine desulfhydrase or reduces thiosulfate using the enzyme thiosulfate reductase to produce H2S. H2S reacts with ferrous sulfate to produce the black precipitate.

• If bacteria produce a black precipitate, it also has flagella. Flagella allow motility and contribute to pathogenicity.

Indole Production

• Presence of a red ring at the top of the media after Kovac’s reagent is added is positive

• The bacteria digest tryptophan to indole, indicating the presence of tryptophanase (a deaminase that also works on the R-group)

• Tryptophan is digested to indole, which reacts with Kovac’s reagent for form a red ring. Indole can contribute to pathogenicity.

Motility

• Cloudiness away from the stab line or BLACK is positive

• The bacteria produces one or more flagella