10.9: SIM Deep Tests

HGTC BIO 225: Microbiology Lab Manual · LibreTexts

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    Learning Objectives
    • Demonstrate proficiency in aseptic techniques while handling microbial cultures and performing the SIM test to prevent contamination and ensure accurate results.
    • Demonstrate an understanding of the principles behind the SIM test, including its purpose, components, and the biochemical reactions involved.
    • Perform the SIM test procedures, including inoculating the SIM medium, interpreting results, and identifying any observed reactions.
    • Interpret the results of the SIM test, distinguishing between positive and negative reactions for sulfide production, indole production, and motility.

     

    SIM Medium

    SIM (sulfur reduction, indole, motility) medium is an example of a combination differential medium, meaning that one can determine several bacterial activities/characteristics through the use of one medium. SIM medium tests for sulfur reduction, indole production, and motility. The form of medium used for this test is an agar deep. The composition of SIM medium includes pancreatic digest of casein, peptic digest of animal tissue, ferrous ammonium sulfate (Fe(NH₄)₂(SO₄)), sodium thiosulfate (Na₂S₂O₃), agar (3.5 g/L), and distilled or deionized water.

     

    SIM deep media can be used for three different tests of bacterial characteristics

    Figure 1: SIM medium is for deep cultures and an inoculation needle is used to inoculate it. After incubation, a black color indicates that the bacteria has produced hydrogen sulfide (H2S). (Left) This culture turned black and therefore this bacterial species is positive for hydrogen sulfide production. (Middle & Right) These cultures did not turn black and are therefore negative for hydrogen sulfide production. If bacteria are motile, the growth will fan away from the stab (left). In this image (right), the bacteria are localized to the stab and therefore no motility occurred and is negative for motility. After incubation of a SIM deep, Kovac's reagent may be added to the top of the SIM medium. When Kovac's reagent turns red, this indicates that the bacterial species produced indole and is an indole-positive species (middle).

     

    SIM - Sulfur Reduction

    Sulfur can be reduced, producing hydrogen sulfide (H2S), by bacteria in two unrelated ways:

    1. One process occurs during putrefaction. When proteins putrefy, the resulting foul “rotten egg” smell is due to the production of hydrogen sulfide gas (H2S). Hydrogen sulfide is a byproduct of the conversion of the amino acid cysteine to pyruvate by the enzyme cysteine desulfurase.
    2. The second mode of H2S generation involves anaerobic respiration. In some prokaryotes, thiosulfate (S2O32-) is the terminal electron acceptor in anaerobic respiration. When thiosulfate is reduced (picks up electrons) the result is H2S gas. In either case, invisible H2S gas is produced.

    Because hydrogen sulfide gas is colorless, SIM medium uses an indicator reaction. Iron (in the form of ferrous ammonium sulfate) in the medium combines with H2S gas to form iron sulfide, FeS, a black precipitate. Any black color in the medium indicates the bacterial species is positive for sulfur reduction. If there is no black color in the medium, the bacterial species is negative for sulfur reduction.

    Unfortunately, this test does not distinguish between the hydrogen sulfide produced as a result of putrefaction and hydrogen sulfide produced at the end of anaerobic respiration.

     

    H2S and motility results and interpretations

    Figure 2: Results of SIM deep cultures (no Kovac's reagent). (Left) The medium is black throughout indicating hydrogen sulfide production and motility. (Middle) The medium is black along the stab line only indicating hydrogen sulfide production and no motility. (Right) The medium is not black and there is no growth away from the stab line indicating this bacterial species is hydrogen sulfide negative and motility negative.

     

    SIM - Indole

    Tryptophan is an amino acid found in most proteins. Some bacteria produce tryptophanase, an enzyme that breaks tryptophan down into indole, ammonia, and pyruvate (see below). Not all bacterial species produce tryptophanase. Whether a bacterial species produces tryptophanase is dependent on its genes. Testing for the activity of tryptophanase using the indole test is an effective way to differentiate one bacterial species from another and to characterize bacteria.

    Chemical reaction catalyzed by tryptophanase

    Figure 3: Chemical reaction catalyzed by tryptophanase. Bacteria that have the tryptophan gene can break down tryptophan to produce indole. Indole is not used by the bacteria and therefore will accumulate in the medium. Kovac's reagent can be used to detect the accumulated indole and therefore is an effective way of determining if the bacterial species produces the enzyme tryptophanase. Pyruvate is utilized by bacteria in either the cellular respiration metabolic pathway or in a fermentation pathway.

     

    The presence of indole indicates that an organism produces the enzyme tryptophanase. Indole can be detected using a chemical known as Kovac’s reagent. Indole forms a red ring with the addition of Kovac’s reagent indicating the bacterial species is indole positive and that it produces tryptophanase. When a bacterial species is indole negative (indicating no tryptophanase activity), Kovac's reagent will produce a dark yellow ring.

     

    indole test results labeled as indole positive an indole negative

    Figure 4: Results from indole test. Kovac's reagent is on top of the SIM agar deep inside these test tubes. The reagent appears yellow when the bacterial species is indole negative (left) and the reagent appears red when the bacterial species is indole positive (right).

    Kovacs reagent chemical reaction

    Figure 5: Reaction that occurs between indole and Kovac's reagent. A reaction between indole and Kovac's reagent produces a dye that is pink-red when indole is present.

     

    SIM - Motility

    Motility is the ability of a microbe to “swim” using flagella. The reduced agar content of this medium, 3.5 g/L compared to 12-15 g/L in most solid media, creates a semi-liquid environment allowing motile cells to spread from their original placement. The stab technique deposits cells in a straight line down the center of the deep using an inoculation needle rather than an inoculation loop. If growth is observed beyond the stab line into the periphery of the tube, the test is positive for motility. Avoid confusing growth produced by the lateral movement of the needle during an imperfect stab inoculation with actual motility. Rotating the tube for a side view will help you determine if growth is confined to the original inoculation line or has truly spread into the periphery of the tube.

     

    how to inoculate a deep medium with an inoculation needle

    Figure 6: How to inoculate a deep with an inoculation needle. Hold the inoculation needle vertically and stab straight down the center of the deep. Then remove the inoculation needle along the original stab line.

     

    Motility is indicated by the ability of the organism to ‘fan’ away from the streak. Or, the entire tube may appear cloudy when compared to an un-inoculated control. If the organism is non-motile, the growth will only appear along the stab line.

     

    motility_results.png

    Figure 7: SIM deep results shown with Kovac's reagent present on top of the medium. (Left) Bacterial growth has moved away from the stab and therefore motility is positive. The Kovac's reagent is not red and therefore is indole negative. The medium did not produce any black coloration and is therefore hydrogen sulfide reduction negative. (Right) Bacterial growth stayed close to the initial stab and there is motility negative. The Kovac's reagent is not red and therefore is indole negative. The medium did not produce any black coloration and is therefore hydrogen sulfide reduction negative.

     
    Real-World Connections

    Salmonella

    Salmonella is a bacterial pathogen responsible for a variety of gastrointestinal (GI) infections, including food poisoning and typhoid fever, both of which can pose serious health risks, especially in vulnerable populations like young children, the elderly, and immunocompromised individuals. Infection typically occurs through ingesting contaminated food (e.g., undercooked poultry, eggs, and produce) or water.  Symptoms include diarrhea, abdominal cramps, fever, nausea, and vomiting. Severe cases can lead to dehydration, septicemia, or even death. 

    One of the most infamous Salmonella outbreaks occurred in 1985 when contaminated milk from Hillfarm Dairy in Illinois led to over 16,000 reported cases of salmonellosis. This outbreak was caused by Salmonella typhimurium, a strain commonly associated with foodborne illness. The contaminated milk, which bypassed proper pasteurization, was distributed across multiple states, leading to widespread disease.

    The SIM test is critical in diagnosing GI infections, particularly those caused by Salmonella species.  A key feature of Salmonella is its ability to produce hydrogen sulfide (H₂S). This production occurs when the bacterium breaks down sulfur-containing compounds, producing a black precipitate in the SIM medium. By detecting H₂S production in stool samples from infected individuals, the SIM test confirmed the presence of Salmonella; ultimately curbing the spread of this deadly pathogen.

    Laboratory Instructions

    1. Obtain three (3) SIM medium deep tests.
    2. Using labeling tape, label the test tubes with your group name, the medium name (SIM), the bacterial species name, the course section, and the date.
    3. Using an inoculating needle, aseptically stab the medium about 2/3 of the way down and out the same pathway as quickly as possible with the bacterial species provided by your instructor.
    4. Repeat steps 1-3 in additional SIM deeps for other bacterial species.
    5. Incubate the tube for at least 48 hours at 37 °C.
    6. After the incubation period, examine your tube.

     

    Indole Test

    1. After the SIM deep has incubated for at least 48 hours, examine results for motility and hydrogen sulfide production and record results.
    2. Add 3 drops of Kovac’s reagent to the top of the SIM medium tube.
      • if Kovac's reagent produces a dark yellow ring indicates the species is indole negative
      • if Kovac's reagent produces a red ring indicates the species is indole positive
     

    Attributions

    10.9: SIM Deep Tests is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by LibreTexts.