SIM Test for Gram Negative Bacteria: Introduction, Principle, Procedure, Result Interpretation

SIM test for hydrogen sulphide , indole production and motility test for gram negative bacteria

Introduction of SIM Test 

SIM test stands for sulphide, indole, motility for hydrogen sulphide , indole production and motility test for gram negative bacteria as shown above picture.

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 Kovacs 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.

Requirements for SIM Test 

Test organisms

SIM medium

Inoculating wire and

Bunsen burner

Incubator

Quality Control Strains

Escherichia coli ATCC 25922

Salmonella enterica  ATCC 14028

Procedure of SIM Test

  1. Take pure colonies from an 18-24-hour old culture on solid medium.
  2. Inoculate the SIM Medium by stabbing the center of the medium to a depth of half inch.
  3. Incubate the inoculated medium aerobically at 37°C for 18-24 hours.
  4. Observe for hydrogen sulfide production and motility of test organism.
  5. Only apply Kovac’s reagent (three drops ) after reading the result of H2S and motility reaction to the surface of the medium.
  6. Observe for the development of a pink to red color.

Result Interpretation of SIM Test

Positive H2S test : 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)

Salmonella enterica ATCC 14028: Growth; Motility: positive, H2S: positive and Indole: negative

Further Readings

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. Clinical Microbiology Procedure Hand book Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  5. 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.
  6. 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.
  7.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
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