SIM Test: Principle, Procedure, Result Interpretation and Limitations
Introduction of SIM test
SIM (Sulfide, Indole, Motility ) medium is useful for for the differentiation of gram-negative enteric bacilli. SIM test helps to isolate the organisms on the basis of sulfide production, indole formation, and motility.
Principle of SIM test
The medium having the constituents ferrous ammonium sulfate and sodium thiosulfate, that together serve as indicators for the production of hydrogen sulfide (H2S). Hydrogen sulfide production detects when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with hydrogen sulfide gas. Casein peptone of this medium is rich in tryptophan. Organisms having the enzyme tryptophanase degrade tryptophan to indole. Indole detection is achieved after the addition of Kovac’s reagent following incubation of the inoculated medium. Indole combines with p-dimethylaminobenzaldehyde and produces a red band at the top of the medium. A negative indole test produces no color change after the addition of Kovac’s reagent i.e. Yellow color of Kovac’s reagent. Lower concentration of agar added to the medium provides a semi-solid structure allowing for the detection of bacterial motility. Motile organisms diffuse from the stab line and produce turbidity or cloudiness throughout the medium. Growth of non-motile bacteria is restricted along the stab line and leave the surrounding medium clear. Another constituent, animal tissue of this medium which provides amino acids and nutrients necessary for bacterial growth.
Composition of the medium
Ingredients for 100 ml of distilled water-
Pancreatic Digest of Casein: 2.0gm
Peptic Digest of Animal Tissue: 0.61gm
Ferrous Ammonium Sulfate: 0.02gm
Sodium Thiosulfate: 0.02gm
Final pH 7.3 +/- 0.2 at 25°C.
Requirements for SIM test
Inoculating wire and
Quality control strains
Escherichia coli ATCC 25922
Salmonella enterica ATCC 14028
Procedure of SIM test
Take pure colonies from an 18-24-hour old culture on solid medium.
Inoculate the SIM Medium by stabbing the center of the medium to a depth of half inch.
Incubate the inoculated medium aerobically at 37°C for 18-24 hours.
Observe for hydrogen sulfide production and motility of test organism.
Only apply Kovac’s reagent (three drops ) after reading the result of H2S and motility reaction to the surface of the medium.
Observe for the development of a pink to red color.
Result interpretation of SIM test
Positive H2Stest :blackening of the medium
A negative H2S test:absence of blackening
Positive motility test :a diffuse zone of growth flaring from the line of inoculation
Negative motility test: restricted growth along the stab line
Indole positive test: a pink to red color ring is formed at the top of the medium after addition of Kovac’s reagent
Indole negative test:A yellow color denotes a negative indole test after addition of Kovac’s reagent
Escherichia coli ATCC 25922: Growth; Motility: positive, H2S: negative and Indole: positive (It turns pink after addition of Kovac’s reagent)
Caps should be loose during incubation otherwise erroneous results may occur
The inoculum should take from a solid medium because from a liquid or broth suspension will delay the initiation of growth and may cause erroneous results.
When inoculating semi-solid media, it is important that the inoculating needle be removed along the exact same line used to inoculate the medium. A fanning motion may result in growth along the stab line that may result in false-positive interpretation.
Take motility and hydrogen sulfide (H2S) reaction results prior to addition of Kovac’s reagent.
Weakly motile organisms or organisms that possess damaged flagella (due to heating, shaking, or other trauma) often result in false-negative motility tests and therefore, motility results should be confirmed by performing a hanging drop motility test.
Some microorganisms like Yersinia enterocolitica that demonstrates motility best at 25°C.
Obligate aerobes , such as Pseudomonas aeruginosa , will produce a spreading film on the surface of the medium and will not extend from the line of inoculation where oxygen is depleted.
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