3.2.2: Triple Sugar Iron Agar Test Activity

In this activity you will test five bacterial samples.

1. Turn on the Bunsen burner and sterilize the inoculating needle by passing it through the flame of the burner.
2. Gently raise the lid to the agar plate containing the bacterial culture to be sampled and touch the inoculating needle to a bacterial colony to obtain a bacterial sample.
3. Pick up the triple sugar iron agar tube to which the sample is to be transferred. Remove the cap and pass the mouth of the tube through the flame of the Bunsen burner.
4. Insert the inoculating needle by stabbing straight into the butt of the slant and then streak up the surface of the agar.
5. Pass the mouth of the triple sugar iron agar tube through the flame and recap it, and return it to the test tube rack.
6. Sterilize the inoculating needle by flaming the loop in the flame of the Bunsen burner.

Repeat steps 1 - 6 for the other four bacterial species and triple sugar iron agar tubes to be inoculated.

7. Incubate the inoculated nitrate broth tubes at 37°C for 24 hours.
8. Observe the triple sugar iron agar tubes for color changes, precipitation formation, and cracks in the media. Use the chart as a guide to interpret your results.

Triple Sugar Iron (TSI) Agar Reactions and Interpretations

Results		Abbreviations	Interpretation
Slant	Butt
No change	No change	-/-	No fermentation
Red	Red	K/K	Protein catabolism yielded alkaline products
Red	Yellow	K/A	Glucose fermentation
Red	Yellow with bubbles	K/A, G	Glucose fermentation and gas production
Red	Yellow with black precipitate	K/A, H2S	Glucose fermentation and hydrogen sulfide gas production
Yellow	Yellow	A/A	Glucose and lactose or sucrose fermentation
Yellow	Yellow with bubbles	A/A, G	Glucose and lactose or sucrose fermentation; gas production
Yellow	Yellow with black precipitate	A/A, H2S	Glucose and lactose or sucrose fermentation; hydrogen sulfide gas production

In the table below write the name of the species tested and the results for each of the tests performed.

Experimental Results

Bacterial Species	Results	Abbreviations	Interpretation

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