Triple Sugar Iron Agar

Triple Sugar Iron (TSI) Agar and iron Agar (KIA) from Kligler are useful in the presumptive identification of gram-negative enteric bacteria, especially in the screening for intestinal pathogens. The formulae for TSI agar and KIA are identical except that in addition to glucose and lactose, TSI agar contains sucrose. TSI forms hydrogen sulfide (H2S) and is used to determine whether a gram-negative rod ferments glucose and lactose or sucrose. The test is primarily used to distinguish members of the Enterobacteriaceae family from other gram-negative rods.

PRINCIPLE

TSI is composed of 10 parts of lactose: 10 parts of sucrose: 1-part glucose and peptone. Phenol red and ferrous sulfate act as indicators of acidification and H₂S formation, respectively. An organism that ferments glucose turns the entire medium acid (yellow) in 8 to 12 hours. After the recommended 18- to 24-hour incubation period, the butt remains acidic due to the presence of organic acids resulting from the fermentation of glucose in the tip of the tube under anaerobic conditions. However, the slant returns to the alkaline (red) state under aerobic conditions due to oxidation of fermentation products on the slant. This change is the result of CO₂ and H₂O formation and peptone oxidation in alkaline amines in the medium. When fermented, in addition to glucose, lactose and/or sucrose, the large quantity of fermentation products formed on the slant neutralizes the alkaline amines and makes the slant acid (yellow), provided that the reaction is read in 18 to 24 hours. TSI reactions should not be read beyond 24 hours of incubation, as aerobic oxidation of lactose and/or sucrose fermentation products proceeds and the slant eventually returns to alkaline state. CO₂ and hydrogen gas (H₂) formation is indicated by the presence of bubbles or cracks in the agar, or by separation of the agar from the sides or bottom of the tube.

The action of sodium thiosulfate from the bacteria produces hydrogen sulfide. H2S is formed out of sodium thiosulfate in the first step. Since H2S is a colorless gas, it is important to visually detect its development through an indicator, ferrous sulfate. For certain situations, the ass of the tube becomes fully dark, which obscures the yellow light from the fermentation of carbohydrates. Since H2S development needs an acidic environment, it is safe to assume the fermentation of glucose even though the yellow color is not evident.

PROCEDURE

• Warm medium to room temperature and examine for cracks. Do not use if cracks appear.
• Using a sterile inoculating needle, touch the center of a well-isolated colony.
• Stab to within 3 to 5 mm from the bottom of the tube.
• Withdraw the needle, and streak the entire surface of the agar slant.
• Optional for fastidious organisms: add a strip of lead acetate paper to top of tube and hold in place with the cap of the tube.
• Place cap loosely on tube. Do not tighten the cap.
• Incubate aerobically at 35 to 37C for 18 to 24 h.
• Examine the reaction in the slant and the butt. Observe for gas and hydrogen sulfide production.
• Do not attempt to interpret sugar fermentation reactions after 24 h. Refrigerate tubes if readings will be delayed.
• If desired, extend incubation only to detect H2S production. Campylobacters may take 3 days for production of H2S.

INTERPRETATION

• No fermentation: Alkaline slant/alkaline butt (ALK/ALK or K/K) or alkaline slant/no change (ALK/no change or K/NC). Although 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 by-products in the slant or deep, respectively, changing the indicator to a deep red color. These reactions are typical of organisms that are not members of the family Enterobacteriaceae. Example: Acinetobacter, Pseudomonas
• Glucose fermentation only, no lactose (or sucrose in TSI) fermentation: Alkaline slant/acid butt (K/A). TSI agar and KIA 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. However, after about 12 hours, the glucose is consumed, and bacteria on the slant 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 acidic (yellow). Reading the results after less than 12 hours of incubation (acid/acid) gives the false appearance of an organism capable of fermenting glucose and lactose (or sucrose in the case of TSI agar). For this reason, TSI agar or KIA must be incubated for 18 to 24 hours. Examples: Proteus, Salmonella, Morganella
• Lactose (or sucrose or both) fermentation: Acid/acid (A/A). Glucose fermenters attack the simple sugar glucose first and then lactose or sucrose. The acid production from the fermentation of the additional sugar is sufficient to keep both the slant and the butt acidic (yellow) when examined at the end of 18 to 24 hours of incubation. If the medium is incubated beyond 24 hours, however, it is possible that the lactose or sucrose could be consumed, and an alkaline slant could be formed. It is important that the TSI agar and KIA tests are not read after 24 hours of incubation. Examples: Escherichia, Klebsiella, Enterobacter

LIMITATIONS:

• Do not read the test before 18 h, since false readings of acid in the slant may result.
• Copious amounts of H₂S may mask the glucose reaction. If this exists, glucose has been fermented and should be recorded as such. Check for gas production.
• When using KIA for screening fecal cultures, it is not able to separate some uncommon non-lactose fermenters (e.g., Providencia) that ferment sucrose from fecal pathogens which are lactose and sucrose negative.
• If TSI is used to screen fecal cultures, the sugar reactions of acid/acid may be obtained with Yersinia enterocolitica and Edwardsiella tarda, since both can ferment sucrose but not lactose.
• Gas production is better in TSI than in KIA. However, H₂S production may be inhibited on TSI for organisms that utilize sucrose and suppress the enzyme mechanism that results in production of H2S.
• Sulfide-indole-motility (SIM) agar is more sensitive in the detection of H₂S than either TSI or KIA.

References:

• Clinical Microhandbook Isenberg
• Bailey Scott’s Diagnostics Microbiology, 13^th Edition
• Textbook of Diagnostic Microbiology