Triple sugar iron (TSI) agar is a useful laboratory medium for identifying clinically important Gram negative bacteria in a microbiology laboratory (principally the Enterobacteraceae). Commercial preparations are available from Difco and Oxoid.
It has properties that are very similar to Kligler iron agar.
The principal components in approximate proportions are
The agar is poured into a test tube around 20mm in diameter and set at an angle so it forms a slope (the sloping part of the agar which is exposed to the air) and a butt (the agar collecting at the bottom of the test tube that is not exposed to air).
A pure inoculum of the organism to be identified taken from a single colony is spread on the slope and then stabbed straight down into the butt. The culture is incubated at 37 degC for 24 hours.
The pH is 7.4 at the start, and the agar therefore starts off pink.
The possible outcomes are:
- Slope & butt: pink
- Slope & butt: yellow
- Slope: pink. Butt: yellow
- Gas formation: bubbles form in the agar or the agar cracks
- Slope & butt: black
Non fermentors
Bacteria that do not ferment lactose, glucose or sucrose (e.g. Pseudomonas species) will grow without producing acid: the slope and butt therefore both remain pink.
Lactose non-fermentors
Slope & butt: yellow. Organisms that ferment glucose or sucrose will produce sufficient acid to colour the entire agar yellow. This is because of the large amount of sucrose and glucose in the agar. Examples include Salmonella and Shigella.
Lactose fermentors
Organisms that ferment lactose but not glucose or sucrose will colour the agar yellow. The amount of acid produced, however, is small compared with the amount produced by glucose and sucrose fermentors, because of the relative amounts of the sugars present. Spontaneous decarboxylation of the amino acids present in the slope (being the only part of the agar exposed to air) results in the production of alkali. Because the relative amount of acid produced is small, the means that the slope remains pink, whereas the butt alone turns yellow.
Lactose fermentors include Escherichia, Enterobacter and Klebsiella.
Gas producers
Gas accumulates in bubbles and if produced in sufficient quantities, will crack the agar in the butt. Examples of organisms that do this include Enterobacter, Escherichia, Proteus and Salmonella.
Hydrogen sulphide producers
Bacteria that are able to utilise sulphur in the agar (in the form of thiosulphate) will generate hydrogen sulphide gas. The forms bubbles and cracks in the agar, but will also react with the ferric ions in the agar to form ferrous sulphide which colours the agar black. Examples include Salmonella and Proteus. | 
