25 Triple Sugar Iron Medium
Emilie Miller, Ph.D
Triple Sugar Iron is a slant medium with two growth environments: aerobic (on the slant) and anaerobic (in the “butt”). The medium contains three sugars in varying concentrations and a pH indicator that turns yellow at pH measurements below 6.8, and a deeper red at pH measurements above 8.2. Bacteria that ferment typically produce one or more types of acid as a byproduct, therefore, fermentation (both aerobic on the slant and anaerobic in the butt) is noted as a change in the color of the media. The medium also identifies strict aerobes that only grow on the slant surface, and also bacteria that produce H2S, either as a way to produce ATP anaerobically using sulfur or sulfate as a final electron acceptor, or as a result of the breakdown of proteins that contain high numbers of sulfur-containing amino acids (cysteine or methionine).
The results of this test are reported as appearance of the slant/appearance of the butt, using A to indicate acid reaction (yellow color), K to indicate an alkaline reaction, and NC to indicate no change in the medium. H2S (detected as a blackening in the media) and the production of gas (CO2) as a byproduct of fermentation are also reported if observed.
Incubations longer than 24 hours may result in a false result known as a reversion. If the bacteria is a vigorous fermenter, it will use up all the sugars and start metabolizing proteins which alkalizes the medium. The media may then revert to its original red color.
As an example, and for practice, the interpretation and outcomes for the 4 TSI tests shown are provided in the table below. Note that many other possible reactions may also occur so proper interpretation of this test is important.
| Table 1. TSI reactions shown in the cultures in Figure 4, from left to right. | |
| Outcome | Interpretation |
| Uninoculated control | For color comparison with inoculated samples |
| K/NC | Aerobic respiration (dark red on the slant) only. Bacteria are strict aerobes. |
| A/A; gas | Fermentation of all three sugars with CO2 produced. Bacteria are facultative anaerobes. |
| K/A; H2S | Aerobic respiration (dark red on slant), fermentation of glucose (acid only in butt), anaerobic respiration (black in butt). Bacteria are facultative anaerobes. |
| K/A | Aerobic respiration (dark red on the slant); fermentation of glucose (acid only in butt). Bacteria are facultative anaerobes. |
Procedure: Inoculate 4 tubes with 4 different microbes by inserting the inoculating needle straight down to 0.5 cm above the bottom. Pull the needle out and create a fishtail smear on the top of the slant.