9.9: SIM Deep Tests
- 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 s ulfur reduction, i ndole production, and m otility. 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.
S IM - Sulfur Reduction
Sulfur can be reduced, producing hydrogen sulfide (H 2 S), by bacteria in two unrelated ways:
- One process occurs during putrefaction. When proteins putrefy, the resulting foul “rotten egg” smell is due to the production of hydrogen sulfide gas (H 2 S). Hydrogen sulfide is a byproduct of the conversion of the amino acid cysteine to pyruvate by the enzyme cysteine desulfurase .
- The second mode of H 2 S generation involves anaerobic respiration. In some prokaryotes, thiosulfate (S 2 O 3 2 - ) is the terminal electron acceptor in anaerobic respiration. When thiosulfate is reduced (picks up electrons) the result is H 2 S gas. In either case, invisible H 2 S 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 H 2 S 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.
S I M - 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.
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.
SI M - 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.
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.
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
- Obtain three (3) SIM medium deep tests.
- 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.
- 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.
- Repeat steps 1-3 in additional SIM deeps for other bacterial species.
- Incubate the tube for at least 48 hours at 37 °C.
- After the incubation period, examine your tube.
Indole Test
- After the SIM deep has incubated for at least 48 hours, examine results for motility and hydrogen sulfide production and record results.
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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
- Centers for Disease Control and Prevention. (1985). Milk-borne salmonellosis—Illinois . Morbidity and Mortality Weekly Report, 34(19), 265-266. https://www.cdc.gov/mmwr/preview/mmwrhtml/00000520.htm
- Centers for Disease Control and Prevention. (2024, September 6). Salmonella . https://www.cdc.gov/salmonella/index.html
- "Microbiology Laboratory Manual: Labs, 1.23 SIM Deep Tests" by Dr. Rosanna Hartline , West Hills College Lemoore, LibreTexts: Biology is licensed under CC BY-NC 4.0
- General Microbiology Lab Manual (Pakpour & Horgan) by Nazzy Pakpour & Sharon Horgan is licensed under CC BY-SA 4.0
- Klamm’s Microbiology Laboratory Manual by Loretta Sanderson Klamm is licensed under CC BY-NC-SA 4.0
- Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation by Susan McLaughlin and Joan Petersen is licensed under CC BY-NC
- Microbiology for Allied Health Students: Lab Manual by Molly Smith and Sara Selby ( GALILEO Open Learning Materials ) is licensed under CC BY 4.0
- Red Mountain Microbiology by Jill Raymond Ph.D.; Graham Boorse, Ph.D.; Anne Mason M.S. is licensed under CC BY-NC 4.0
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