Triple Sugar Iron Agar and Its Use and Interpretation

Triple Sugar Iron Agar and Its Use and Interpretation


see Difco Manual, 9th Ed, (1953) pp. 166-168 (Table on p. 161.)


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:

phenol red a pH indicator:

below 6.8 it is yellow

above 82., it is red

Phenol red turns yellow in an acid environment.  It thus indicates whether the acids of fermentation have been produced.  Failure to turn the butt yellow indicates that no fermentation has occured, and that the bacterium is an obligate aerobe.
0.1 % glucose if only glucose is fermented, only  a small amount of acid is produced If only glucose is fermented, only enough acid is produced to turn the butt yellow.  The slant will remain red.
1.0 % lactose

1.0% sucrose

if the culture can ferment either lactose (lac+) and/or sucrose (suc+), a large amount of  acid is produced a large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose

(ferrous sulfate)

A source of iron and sulfur A few bacteria are capable of reducing the SO4= to H2S (hydrogen sulfide).
The iron combines with the H2S to form FeS (ferrous sulfide) a black compound.  This will turn the butt black.  Thus, a black butt indicates H2S production.

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.


  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).


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


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:


Shigella dysenteriae R Y Causes food infection dysentery
Salmonella typhimurium R YG + Causes food poisoning
Salmonella typhi R Y + Causes typhoid fever
Aerobacter aerogenes Y YG Similar to Klebsiella,but nonrespiratory
Escherichia coli Y YG  Most common of GIflora
Citrobacter freundii Y YG + one of “paracolon” group (non-pathogenic)
Proteus vulgaris Y YG + Causes GU infections, highly motile
Klebsiella pneumoniae Y R or YG Pneumonia in debilitated patients (nosocomial)
Pseudomonas aeruginosa R R GI inhabitant, causes wound, GU infections
Alcaligenes faecalis R R GI inhabitant, opportunistic pathogen of GU