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 differentialmedium that can distinguish between a number of Gram-negative enteric bacteriabased 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 also contains ferrous sulfate.If the bacterium forms H₂S, this chemical will react with theiron to form ferrous sulfide, which is seen as a black precipitate in thebutt (a black butt).
 

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

1. Weigh out medium, dissolve: 17.8 gdry medium, add to 300 mL water, heat to boiling to dissolve.
2. Deliver aliquots to tubes: 6 mL to16 x 150 mm tubes witha repipet. Cover with closures.
3. Autoclave capped aliquoted tubes 13lb, 15 min.
4. Creating the slant:  Using a ringstandand clamp, clampthe rack so that the the tubes (with liquid medium in them) have a3cm 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 bystreaking 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 hrmay be necessary to show all H₂S reactions).

TRIPLESUGAR 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 redslant
YELLOW	Y	ferments lactose and/or sucrose	Example yellowslant

SCORING THE BUTT COLOR AND CONDITION:
 

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

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

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

TYPICAL REACTIONS OF SELECTED ENTERICBACTERIA:
 
 

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