Triple Sugar Iron Agar
Triple sugar iron agar and Kliger’s iron agar (KIA)are very useful in the presumptive identification of gramnegative enteric bacteria, particularly in screening for intestinal pathogens. The formulas for TSI and KIAare identical except that TSI contains sucrose in addition to glucose and lactose. Lactose is present in a concentration 10 times that of glucose (l %lactose and 0.1% glucose). In TSI, sucrose is also present in a 1% concentration. Ferrous sulfate and sodium thiosulfate are added to detect the production of hydrogen sulfide gas CH2S).Phenol red is used as the pH indicator, which is yellow below the pH of 6.8. Uninoculated medium is red because the pH is buffered at 7.4. Both TSI and KIA are useful in detecting the ability of the microorganism to ferment carbohydrates, glucose, and lactose in KIA and glucose, lactose, or sucrose in TSI;to produce gas from the fermentation of sugars; and to detect the production of H2S.
Both TSI and KIA agars are poured on a slant (Figure 9-3). The slant portion is aerobic; the butt, or deep portion, is anaerobic. To inoculate TSI or KIA agars, the laboratory scientist should pick a wellisolated colony with an inoculating needle and stab the butt almost all the way to the bottom of the tube.
Then the laboratory scientist moves the needle back and forth (known as fish tailing), which puts more organisms on the surface of the slant. The cap is replaced loosely to allow oxygen to enter the tube, and the medium is incubated in a non-C02 incubator for 18 to 24 hours. The reaction patterns are written with the slant results first, followed by the butt reaction, separated by a backslash (slant reaction/butt reaction), and it is important that the reactions be read within an 18- to 24-hour incubation period; otherwise, erroneous results are possible.
Reactions on TSI or KIA Agars
1. No fermentation: Alkaline slant/alkaline butt (ALK/ ALK or K/K) or alkaline slant/no change (ALK/no change or K/NC) These reactions are typical of organisms that are not members of the Enterobacteriaceae. Although nonenteric bacilli are unable to ferment either lactose or glucose, these organisms can degrade the peptones present in the medium aerobically or anaerobically, resulting in the production of alkaline byproducts in the slant or deep respectively, changing the indicator to a deep red color.
2. Glucose fermentation only, no lactose (or sucrose in TSI) fermentation: Alkaline slant/acid butt (K/ A)
TSI and KIA agar contain glucose in a 0.1% concentration. The acid produced from this concentration of glucose is enough to change the indicator to yellow initially throughout the medium. After about 12 hours, however, the glucose will be consumed, and bacteria on the slant will utilize the peptones aerobically, producing an alkaline reaction, which changes the indicator to a deep red color. Fermentation of glucose (anaerobic) in the butt produces larger amounts of acid, overcoming the alkaline effects of peptone degradation; therefore the butt remains acid (yellow).
Reading the results after fewer than 12 hours of incubation gives the false appearance of an organism capable of fermenting glucose and lactose (or sucrose in the case of TSI). For this reason, TSI or KIA agar must be incubated for 18 to 24 hours.
3. Lactose (and/or sucrose) fermentation: Acid/acid (A/A)
Glucose fermenters will attack the simple sugar glucose first and then the lactose or sucrose. The acid production from the fermentation of the additional sugar(s) is sufficient to keep both the slant and the butt acid (yellow) when examined at the end of 18 to 24 hours. If the medium is incubated beyond 24 hours, however, it is possible that the lactose or sucrose could be consumed and an alkaline slant be formed. It is important that the TSI and KIA tests are not read after 24 hours of incubation.
4. Hydrogen sulfide production: Alkaline slant/acid butt, HzS in butt (K/A. HzS) or acid slant/acid butt, HzS in butt (A/A HzS)
Two indicators are present in the medium to detect HzS formation: sodium thiosulfate and ferrous sulfate. The HzS production is a two-step process. In the first step, HzSis formed from sodium thiosulfate.
Because HzS is a colorless gas, the second indicator, ferrous sulfate, is necessary to detect its production visually. In some cases, the butt of the tube will be completely black, obscuring the yellow color from carbohydrate fermentation. Because HzSproduction requires an acid environment, even if the yellow
color cannot be seen, it is safe to assume glucose fermentation. a. Bacterium (acid environment) + Sodium
thiosulfate –i> H2S gas
b. HzS + Ferric ions –i> Ferrous sulfide (black precipitate)
5. Gas production (aerogenic) or no gas production ( nonaerogenic)
The production of gas results in the formation of bubbles or splitting of the medium in the butt or complete displacement of the medium from the bottom of the tube. Figure 9-3 illustrates the reactions on TSI agar.