TRIPLE SUGAR IRON AGAR

Code: CM0277

a composite medium for the differentiation of Enterobacteriaceae by three sugar fermentations and hydrogen sulphide production

* Adjusted as required to meet performance standards

Directions
Suspend 65g in 1 litre of distilled water. Bring to the boil to dissolve completely. Mix well and distribute. Sterilise by autoclaving at 121°C for 15 minutes. Allow the medium to set in sloped form with a butt about 1 in.deep.

Description
A composite medium for the differentiation of Enterobacteriaceae according to their ability to ferment lactose, sucrose and glucose, and to produce hydrogen sulphide.

Not only does this medium perform most of the functions of Kligler Iron Agar but, in addition, its sucrose content permits the recognition and exclusion of sucrose-fermenting species. These organisms may ferment lactose slowly or not at all during the incubation period, but they attack sucrose readily. Some Proteus and other species may give similar reactions to salmonellae and shigellae and it is necessary to distinguish them by their ability to hydrolyse urea. For this reason Triple Sugar Iron Agar should be used in parallel with Urea Broth or Urea Agar.

This medium was formerly considered to be interchangeable with Kligler medium for the detection of hydrogen sulphide producing Enterobacteriaceae. It is now thought that Triple Sugar Iron Agar is not suitable for the detection of hydrogen sulphide production by sucrose-fermenting organisms, such as some Citrobacter and Proteus species, in which the sucrose fermentation masks the hydrogen sulphide indicator in the medium ^†1.

Triple Sugar Iron Agar is recommended for the presumptive identification of colonies or sub-cultures from plating media such as Salmonella Shigella Agar (Modified) (CM0533), Bismuth Sulphite Agar (CM0201), Brilliant Green Agar (CM0263), MacConkey Agar No.3 (CM0115), or Desoxycholate Citrate Agar (Hynes) (CM0227).

Technique
The USA techniques are described elsewhere^2,3 but the following is suggested as a simple method:
1. Pick a single colony from the surface of a selective plating medium and smear a MacConkey Agar (CM0007) plate. Incubate for 18 hours at 37°C and inoculate two separate tubes of media from one single isolated colony:
(i) Triple Sugar Iron Agar - smear the slope and stab the butt.
(ii) Urea Broth Base (CM0071) with added Urea Solution (SR0020).
2. Incubate at 35°C.
3. Examine the Urea Broth tube after 5 hours and again after 18 hours incubation. Discard tubes showing a red or pink coloration, which is due to urea hydrolysis by Proteus or other organisms.
4. Where there is no urea hydrolysis, examine the Triple Sugar Iron Agar tubes after 18 hours and 48 hours. The following are typical reactions:

AG = acid (yellow) and gas formation
A = acid (yellow)
NC = no change
ALK = alkaline (red)
+ = hydrogen sulphide (black) ^†
- = no hydrogen sulphide (no black) ^†
† See note in text.
The presumptive evidence so obtained may be confirmed serologically after sub-culturing the organism from the Triple Sugar Iron Agar slope in Nutrient Broth No.2 (CM0067).

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Quality control
Typical reactions of organisms in Triple Sugar Iron Agar:

* This organism is available as a Culti-Loop®

References
1. Bulmash J. M. and Fulton M. (1966) J. Bact. 88. 1813.
2. American Public Health Association (1976) Compendium of Methods for the Microbiological Examination of Foods. APHA Inc. Washington DC.
3. Edwards P. R. and Ewing W. H. (1972) Identification of Enterobacteriacea. 3rd Edn. Burgess Publishing Co. Minneapolis. USA.