SIM Test: Introduction, Principle, Test Requirements, Procedure, Result Interpretation, and Limitations

Introduction

SIM stands for sulfide, Indole, and Motility and SIM medium is applied for the differentiation of gram-negative enteric bacilli. SIM test assists to isolate the bacteria on the basis of sulfide production, indole formation, and motility and thus SIM test is a combination of three tests in a single test tube.

Principle of SIM Test

The medium has the constituents ferrous ammonium sulfate and sodium thiosulfate, which together serve as indicators for the production of hydrogen sulfide (H₂S). H₂S production detects when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with hydrogen sulfide gas. The Casein peptone of this medium is rich in tryptophan. Bacteria having the enzyme tryptophanase degrade tryptophan to indole. Indole detection is achieved after the addition of Kovac’s reagent following the 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 (yellow color of Kovac’s reagent). A lower concentration of agar (0.2-0.4%) added to the medium provides a semi-solid structure allowing for the detection of bacterial motility. Motile bacteria diffuse from the stab line and produce turbidity or cloudiness throughout the medium.  The growth of non-motile bacteria is restricted along the stab line and leaves the surrounding medium clear.  Another constituent, animal tissue of this medium provides amino acids and nutrients necessary for bacterial growth.

Composition of the SIM Medium

• Ingredients for  100 ml of distilled water-
• Pancreatic Digest of Casein: 2.0gm
• Peptic Digest of Animal Tissue (PDAT): 0.61gm
• Ferrous Ammonium Sulfate: 0.02gm
• Sodium Thiosulfate: 0.02gm
• Agar: 0.35gm
• Final pH 7.3 +/- 0.2 at 25°C.

Requirements for SIM Test

• Test organisms
• SIM medium
• Inoculating wire
• Bunsen burner
• Incubator

Quality control strains

1. Escherichia coli ATCC 25922
2. Salmonella enterica ATCC 14028

Procedure of SIM Test

1. Pick up pure colonies from an 18-24-hour old culture on a solid medium.
2. Inoculate the SIM medium by stabbing the center of the medium to a depth of half an inch.
3. Incubate the inoculated medium aerobically at 37°C for 18-24 hours.
4. Observe for H₂S production and motility of the test organism.
5. Only use Kovac’s reagent (three drops ) after reading the result of H₂S and motility reaction to the surface of the medium.
6. Observe the development of a pink-to-red color.
7. Follow the same above steps for control strains too for validation of the test procedure.

Result interpretation of SIM Test

• H₂S test Positive: Blackening of the test medium
• H₂S test Negative: Absence of blackening in the medium
• Motility test Positive: A diffuse zone of growth flaring from the line of inoculation
• Motility test Negative: Restricted growth along the stab line
• Indole positive test: A pink to red color ring is formed at the top of the medium after the addition of Kovac’s reagent.
• Indole negative test: A yellow color denotes a negative indole test after the addition of Kovac’s reagent
• Escherichia coli ATCC 25922: Growth; Motility: positive, H₂S: negative, and Indole: positive (It turns pink after the addition of Kovac’s reagent)
• Salmonella enterica ATCC 14028: Growth; Motility: positive, H₂S: positive, and Indole: negative

Limitations of SIM Test

1. Caps or cotton plugs should be loose during incubation otherwise erroneous results may occur
2. The inoculum should take from a solid medium since a liquid or broth suspension will delay the initiation of growth and may cause erroneous results.
3. 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 a false-positive interpretation.
4. Take motility and hydrogen sulfide (H₂S) reaction results prior to the addition of Kovac’s reagent.
5. 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.
6. Some bacteria like Yersinia enterocolitica demonstrate motility best at 25°C.
7. Obligate aerobes like 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.

Further Readings

1. Cowan and Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
2. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher El
3. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
5. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
6. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
7. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.