# 1.23: SIM Deep Tests

$$\newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} }$$ $$\newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}}$$$$\newcommand{\id}{\mathrm{id}}$$ $$\newcommand{\Span}{\mathrm{span}}$$ $$\newcommand{\kernel}{\mathrm{null}\,}$$ $$\newcommand{\range}{\mathrm{range}\,}$$ $$\newcommand{\RealPart}{\mathrm{Re}}$$ $$\newcommand{\ImaginaryPart}{\mathrm{Im}}$$ $$\newcommand{\Argument}{\mathrm{Arg}}$$ $$\newcommand{\norm}[1]{\| #1 \|}$$ $$\newcommand{\inner}[2]{\langle #1, #2 \rangle}$$ $$\newcommand{\Span}{\mathrm{span}}$$ $$\newcommand{\id}{\mathrm{id}}$$ $$\newcommand{\Span}{\mathrm{span}}$$ $$\newcommand{\kernel}{\mathrm{null}\,}$$ $$\newcommand{\range}{\mathrm{range}\,}$$ $$\newcommand{\RealPart}{\mathrm{Re}}$$ $$\newcommand{\ImaginaryPart}{\mathrm{Im}}$$ $$\newcommand{\Argument}{\mathrm{Arg}}$$ $$\newcommand{\norm}[1]{\| #1 \|}$$ $$\newcommand{\inner}[2]{\langle #1, #2 \rangle}$$ $$\newcommand{\Span}{\mathrm{span}}$$$$\newcommand{\AA}{\unicode[.8,0]{x212B}}$$

##### Learning Objectives
• Explain what a SIM deep is and tell the tests that can be conducted in a SIM deep.
• List possible metabolic reasons why a species of bacteria could be producing hydrogen sulfide.
• Tell what the indole test examines and what enzyme it tests for.
• Explain what the motility is and that is indicates whether or not bacterial species produce flagella for motility.
• Successfully conduct SIM tests and Interpore results of these tests.

## SIM Medium

SIM (sulfur reduction, indole, motility) medium is an example combination 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. SIM is an example of a The form of medium used for this test is an agar deep. SIM Medium contains the following: 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 - 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 an 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 (though not odorless!), 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 an anaerobic respiration.

### 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.

The pyruvate and ammonia (NH3) are converted into other molecules, but the indole accumulates, and thus can be detected in the media.

The presence of indole therefore 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), the Kovac's reagent will produce a dark yellow ring.

### 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.

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.

## Laboratory Instructions

1. Obtain a deep of SIM medium.
2. Label the test tube with your group name, the medium name, and the bacterial species name.
3. Using an inoculating needle, 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 if testing multiple bacterial species.
5. Incubate the tube for at least 48 hours.
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 10 drops of Kovac’s reagent to the top of the SIM meidum 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

## Results & Questions

bacterial species medium color hydrogen sulfide production (+/-) Kovac's reagent color indole (+/-) location of growth (along stab / fanned out) motility (+/-)
Escherichia coli
Proteus vulgaris
Staphylococcus aureus
1. Complete the table above with results from the SIM deep tests.
2. A bacterial species that is positive for hydrogen sulfide production could be producing H2S in one of two ways. What are these?