Sulfur, Indole, and Motility (SIM) test (combined differential medium)

What does SIM media test for?

• Sulfur reduction

• Indole Production 

• Motility

How to determine the production of Indole after incubation?

Addition of Kovac’s reagent

What are components of SIM tube?

• Pancreatic digest of casein, peptic digest of animal tissue

• Ferrus Ammonium Sulfate

• Sodium Thiosulfate

• Agar

What are the indicators of SIM test? How do they work?

• Ferrus Ammonium Sulfate — Sulfur reduction

bind with H₂S→ black precipitation

• Kovac’s reagent — Indole production

react with Indole → red color

After incubation, the SIM tube has black color and bubble ring on surface

Sulfur reduction (+)

Bubble of H₂S gas

How does Indole production take place?

If organism possess tryptophanase enzyme that breaks down tryptophan AA in the media → indole, pyruvate and ammonia

After incubation and addition of Kovac’s reagent, the SIM tube has a yellow butt and red slant or red ring on surface

What does this indicate?

Production of Indole (+)

After incubation, the SIM tube appears to be turbid compared to the control tube

What does this mean?

Motility (+)

Organisms have flagella that help them move in the small section of agar in the tube

After incubation and addition of Kovac’s reagent, the SIM appear pink ring, turbid black growth

This means

Indole (+)

Motility (+)

Sulfur (+)

After incubation and addition of Kovac’s reagent, the SIM appear clear, growth around stab mark, no black color change nor ring pink

This mean

Sulfur (-)

Indole(-)

Motility (-)

Brief protocol of SIM test

Day 1:

1. Obtain 5 SIM tubes, label each (P.mirabilis, M.luteus, E.coli, C.freundii)

2. Obtain 4 both culture, vortex. Have 1 control tubes

3. Aseptically stab into ½ agar, then pull straight up. No squiggle

4. Incubate for 24-48 hours at 37 degrees

Day 2:

• Add 5 drops of Kovac’s reagent

• Wait for 3 minutes to see change

• Record results