Trio Sugar Iron Agar (TSI) (Energy metabolism, AA metabolism)

What are components of TSI?

• Pancreatic Digest of Casein, yeast extract, beef extract

• Sodium Chloride

• 1% Lactose, 1% Sucrose, 0.1% Glucose

• Ferric Ammonium Citrate

• Sodium Thiosulfate

• Phenol Red

What 3 fermentable sugar substrates in TSI

• Glucose

• Sucrose

• Lactose

Why does Glucose have the smallest concentration out of all 3 fermentable sugars?

Because Glucose is the most preferred sugar

What is the source of reducible sulfur in TSI?

Sodium Thiosulfate

What are the indicators of TSI? How do they work?

• Phenol Red — pH indicator

Fermentation lowers pH → Phenol Red change color to yellow

• Ferric Ammonium Citrate — sulfur reduction indicator

Sulfur reduction produce H₂S that binds to Ferric Ammonium Citrate → black precipitation 

What does TSI test for?

• Sugar fermentation of either Glucose or Lactose and/ or Sucrose

• Sulfur reduction 

• Gas production

• Maybe peptone degradation

Is sulfur reduction aerobic or anaerobic process?

Strictly anaerobic

Can we conclude that sulfur reduction happens independently of sugar fermentation?

No, it requires at least one sugar fermentation

Can we conclude that gas production happens independently of sugar fermentation?

Yes, it may or may not partner with sugar fermentation

T/F: Slant with a deep butt creates both anaerobic and anaerobic environments

True

What does the result of peptone degradation (protein breakdown) look like in TSI tubes?

The basic products will raise the pH

Phenol Red turn pink/ red 

After incubation, the TSI tube has yellow butt and yellow slant

This indicates …

Glucose / Lactose /+ Sucrose fermentation

The appearance of cracking and lifting of agar butt after incubation indicates …

Production of gas

Within the first 12 hours, which nutrient source will be metabolized?

Glucose

Then organisms can switch to other sugar fermentation, AA catabolism

After incubation, the TSI tube has red slant, yellow butt and no cracking in agar

What does this indicate?

Glucose fermentation only → lower pH, color change to yellow

Then the organism switched to catabolized AA, resulting in reversion → increase pH around surface, color change to red

No gas production

After incubation, the TSI tube has yellow/ red slant, black butt

What does this indicate?

After only glucose or/ with lactose and/ or sucrose fermentation, the organism switched to reduce sodium thiosulfate

Product H₂S react with ferric ammonium citrate → black precipitate

After incubation, the TSI tube has the same color as the control tube

Pink or red slant, no change in butt

What does this mean?

No sugar fermentation occurred

The organism is an obligate aerobe

After incubation, the TSI tube has the same color as the control tube

Pink or red slant and red/ pink butt

What does this mean?

No sugar fermentation occurred

The organism degrade peptone anaerobically and aerobically

Brief protocol of TSI

1. Obtain 5 TSI slant tubes, label (E.coli, P.aureus, P.stuartii, P.mirabilis) and one control tube

2. Aseptically stab into ½ of the slant and squiggle on surface for 4 organisms and 1 control

3. Incubate for 24-47 hours at 37 degrees