TRIPLE SUGAR IRONAGAR AND ITS USE ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103.
Prep of TSI slants	This page has been accessed  times since 3 August 2001.	Variety of TSI reactions (reactions analyzed)

INTRODUCTION:

Triple Sugar Iron medium is a differential medium that can distinguish between a number of Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to:

a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H₂S.

The medium contains 1.0% each of sucrose and lactose and 0.1% glucose. If only glucose is fermented, acid produced in the butt will turn it yellow, but insufficient acid products are formed to affect the methyl red in the slant. However, if either sucrose or lactose are fermented, sufficient fermentation products will be formed to turn both the butt and the slant yellow. If gas is formed during the fermentation, it will show in the butt either as bubbles or as cracking of the agar. If no fermentation occurs (as for an obligate aerobe), the slant and butt will remain red.The medium also contains ferrous sulfate. If the bacterium forms H₂S, this chemical will react with the iron to form ferrous sulfide, which is seen as a black precipitate in the butt (a black butt).  The following table summarizes these reactions:

PREPARATION OF MEDIA: (for 40 tubes)(NOTE: prepare at beginning of class.)

1. Weigh out medium, dissolve: 17.8 g dry medium, add to 300 mL water, heat to boiling to dissolve.
2. Deliver aliquots to tubes: 6 mL to16 x 150 mm tubes with a repipet. Cover with closures.
3. Autoclave capped aliquoted tubes 13 lb, 15 min.
4. Creating the slant:  Using a ringstand and clamp, clamp the rack so that the tubes (with liquid medium in them) have a 3 cm slant with a 2-3 cm butt. Let cool until solid. Incubate 48 hr at37ºC to assure sterility.

INOCULATION:
 

1. Inoculate the slants with a pure culture by streaking over the entire surface of the slant (zig-zag to cover surface)and then stabbing deep into the butt.
2. Incubate at 37ºC for 24 hours (48 hr may be necessary to show all H₂S reactions).

TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS

SCORING THE SLANTS: Examine the slant and butt, and record data using the following criteria:
 

SLANT COLOR:	Code letter:	Interpretation	Example
RED	R	does not ferment either lactose or sucrose	Example red slant
YELLOW	Y	ferments lactose and/or sucrose	Example yellow slant

SCORING THE BUTT COLOR AND CONDITION:
 

BUTT COLOR/CONDITION	Code Letter	Interpretation	Example
RED	R	no fermentation, the bacterium is an obligate aerobe	Example red butt
YELLOW	Y	some fermentation has occurred, acid has been produced, it is a facultative anaerobe.	Example yellow butt
GAS FORMED	YG	Seen as cracks in the agar, bubbles, or the entire slant may be pushed out of the tube.  (Caution: these gassy fermenters may have bacteria close to the opening.)	Example yellow butt with gas
BLACK	"+"	H2S has been produced	Example black butt

 

Here are six different reactions for bacteria inoculated into triple sugar iron slants. Look at the left image first and record what you think the reactions are in each slant.  Then check your answers with the image on the right.

After you have scored your TSI Agar slants, you should suggest a species of bacterium which matches those metabolic traits discovered. While by no means definitive, the following are TSI Agar reactions typical of a number of prominent species which can be distinguished with this medium:
 

TYPICAL REACTIONS OF SELECTED ENTERICBACTERIA: