Each bacterial genus possesses distinctive biochemical activities and enzyme systems, forming the basis for the differentiation and identification of bacteria through their biochemical properties.
Although carbohydrates like glucose, lactose, and sucrose are simple sugars, diverse bacteria employ them in various ways, leading to the production of distinct end products. Furthermore, some bacteria may not utilize one or all of these sugars, with variations in their ability to ferment these sugars and produce acid and gas as byproducts.
The triple sugar iron agar (TSIA) test serves as a biochemical assay designed to differentiate bacteria based on their capacity to ferment these three sugars and generate acid and hydrogen sulfide gas.
In 1944, S. Edward Sulkin and Joseph C. Willett first introduced the TSIA medium as a selective and differential medium for distinguishing bacteria, particularly those of the Enterobacteriaceae family. In 1945, Hajna A.A. introduced modifications to this medium, and it continues to be an essential tool in microbiology laboratories, primarily for the differentiation and tentative identification of Gram-negative enteric pathogens.
The TSIA test closely resembles Kligler's Iron Agar (KIA) test, with the key distinction being the inclusion of '1% sucrose' as an additional sugar in the TSIA medium, alongside glucose and lactose, which aids in the differentiation process.
Table of Contents
- Objectives of TSIA Test
- Principle of TSIA Test
- Requirements for TSIA Test
- Procedure of TSIA Test
- Result and Interpretation of TSIA Test
- Quality Control
- Precautions
- Applications of TSIA Test
- Limitations of TSIA Test
Objectives of TSIA Test
- To distinguish Gram-negative enteric bacilli from one another.
- To assess whether the bacteria can metabolize glucose, lactose, and/or sucrose while generating hydrogen sulfide gas.
- To discern between bacteria that ferment lactose and those that do not.
Principle of TSIA Test
The TSIA test relies on the unique metabolic behaviors of different bacterial genera when metabolizing glucose, lactose, sucrose, and sodium thiosulfate, a sulfur compound. Fermentation of these sugars leads to the production of acid, resulting in a decrease in the medium's pH and a shift to a red color. Additionally, the metabolic activity involving sodium thiosulfate is reflected in the medium turning black.
Initially, bacteria capable of fermenting glucose produce metabolic acids, causing the medium's pH to drop and both the slant and butt regions to turn yellow. However, as glucose becomes depleted, this acid production diminishes.
In the case of bacteria unable to ferment lactose and/or sucrose, they are incapable of utilizing these sugars as carbon sources. Consequently, once glucose is consumed, the oxidative breakdown of peptone in the slant occurs. This oxidative process elevates the medium's pH to an alkaline level, turning the yellow slant to red. However, due to limited oxygen in the butt region, oxidative degradation of peptone doesn't occur there, and the butt remains unchanged in color. Therefore, bacteria that don't ferment lactose and/or sucrose but ferment glucose will exhibit a red slant and a yellow butt (designated as Red/Yellow or K/A).
In contrast, when bacteria are capable of fermenting lactose and/or sucrose, they initiate the fermentation process, resulting in acid production that lowers the medium's pH. This acid production causes both the slant and butt regions to turn yellow, given the abundant presence of carbohydrates (sucrose and lactose). Since there is no triggering of the oxidative metabolism of peptone in this scenario, bacteria that ferment lactose and/or sucrose (or all three sugars) will display a yellow slant and yellow butt (designated as Yellow/Yellow or A/A).
If bacteria are unable to ferment any of the sugars, the medium remains red (labeled as Red/Red or K/K).
Bacteria capable of producing hydrogen sulfide (H2S) can break down the sodium thiosulfate in the culture medium, leading to the release of H2S gas. This H2S gas subsequently reacts with ferric ions, forming water-insoluble ferrous sulfide, which appears black. The presence of H2S is indicated by the development of this insoluble, black-colored substance in the culture medium.
Additionally, the emergence of gas bubbles above the medium or any disturbance or displacement of the medium serves as indicators that gas is being produced during sugar fermentation.
Requirements for TSIA Test
1. Culture Media
Triple sugar iron agar medium is used for the TSIA test.
Composition of TSIA Medium per 1000 mL
- Peptone: 20.00 grams
- HM Extract (Meat Extract): 3.00 grams
- Yeast Extract: 3.00 grams
- Dextrose (Glucose): 1.00 grams
- Lactose: 10.00 grams
- Sucrose: 10.00 grams
- Sodium Chloride: 5.00 grams
- Ferric Citrate: 0.300 grams
- Sodium Thiosulfate: 0.300 grams
- Phenol Red: 0.024 grams
- Agar: 12.00 grams
Preparation of TSIA Medium Slant
- Weigh out the appropriate quantity of TSIA media powder, which should be 64.62 grams per 1000 mL, and dissolve it in the required volume of water in a conical flask or glass bottle.
- Ensure thorough dissolution by stirring the mixture vigorously using a magnetic stirrer or by manual agitation. Heat the mixture to the boiling point to ensure complete dissolution of all components, including the agar, in the water.
- Dispense approximately 5 to 7 mL of the prepared medium into each test tube and lightly cover the opening with a cap or a cotton plug.
- Subject the test tubes to autoclaving at 121°C and 15 lbs of pressure for a duration of 15 minutes.
- After autoclaving, allow the tubes to cool, and position them at a slant of approximately 30° inclination to facilitate the formation of an agar slant with a butt that is about 2.5 to 5 cm in depth.
2. Reagents
There is no need for any extra reagents.
3. Equipment
- Test tubes
- Incubator
- Weighing Machine
- Autoclave
- Bunsen Burner
- Inoculating Loop
- PPE and other general laboratory materials
4. Sample Organism (Test Bacteria)
5. Control Organism
- Salmonella Paratyphi A ATCC 9150
- Proteus vulgaris ATCC 6380
- Salmonella Typhimurium ATCC 14028
- Citrobacter freundii ATCC 8090
- Shigella flexneri ATCC 12022
- Escherichia coli ATCC 25922
- Pseudomonas aeruginosa ATCC 27853
Procedure of TSIA Test
- With a sterile inoculating wire, pick a well-isolated colony from a fresh culture of the test bacterium that has been incubated for 18 to 24 hours.
- Insert the inoculating wire into the butt of the test tube, piercing it to a depth of 3 to 5 mm above the base. While withdrawing the wire, streak the slant of the medium.
- Place the tube in an aerobic environment with a loosely secured cap and incubate it at a temperature of 35±2°C for approximately 24 hours.
- Examine the tube for any color changes in both the slant and the butt regions, and report these observed colors within 24 hours of the incubation period. (If you wish to assess hydrogen sulfide (H2S) production, continue incubating for an additional 24 to 48 hours, but focus on evaluating sugar fermentation and color changes within the first 24 hours of inoculation and incubation.)
Result and Interpretation of TSIA Test
- When you observe a red slant and a yellow butt (referred to as Red/Yellow or Alkaline (K)/Acidic (A)), it indicates that only glucose has undergone fermentation.
- If both the slant and butt appear yellow (termed Yellow/Yellow or Acidic (A)/Acidic (A)), this suggests the fermentation of lactose and/or sucrose, or possibly the fermentation of all three sugars.
- A scenario where both the slant and butt are red (designated as Red/Red or Alkaline (K)/Alkaline (K)) signifies that none of the three sugars have undergone fermentation.
- The development of a black coloration in the medium or the formation of black spots indicates the production of hydrogen sulfide (H2S).
- The presence of cracks in the medium, the formation of gas bubbles within the medium, or the creation of gaps in the medium implies the generation of gas.
Quality Control
The control organisms yield the following outcomes:
- Escherichia coli ATCC 25922: Displays a Yellow/Yellow result, exhibits gas production (+ve), and does not produce hydrogen sulfide (H2S) (-ve).
- Citrobacter freundii ATCC 8090: Shows a Yellow/Yellow result, indicates gas production (+ve), and produces hydrogen sulfide (H2S) (+ve).
- Proteus vulgaris ATCC 6380: Presents a Red/Yellow result, does not produce gas (-ve), and produces hydrogen sulfide (H2S) (+ve).
- Salmonella Paratyphi A ATCC 9150: Exhibits a Red/Yellow result, demonstrates gas production (+ve), and does not produce hydrogen sulfide (H2S) (-ve).
- Salmonella Typhimurium ATCC 14028: Displays a Red/Yellow result, indicates gas production (+ve), and produces hydrogen sulfide (H2S) (+ve).
- Shigella flexneri ATCC 12022: Shows a Red/Yellow result, does not produce gas (-ve), and does not produce hydrogen sulfide (H2S) (-ve).
- Pseudomonas aeruginosa ATCC 27853: Presents a Red/Red result, does not produce gas (-ve), and does not produce hydrogen sulfide (H2S) (-ve).
Results of TSIA Test
Precautions
- If the medium is damaged or exhibits gaps before inoculation, it is advisable not to use it as it could potentially yield inaccurate results regarding gas production.
- Avoid using an inoculating loop for stabbing, as it may lead to cracking in the medium, potentially causing confusion when assessing gas production.
- It's important to refrain from interpreting the sugar utilization results before the incubation period reaches 18 hours. This is because glucose may not have been entirely utilized at this stage, and as a result, the oxidative metabolism of the peptones might not have commenced.
- Likewise, it's not recommended to delay the result reading long after the 24-hour mark, as the entire tube may become completely black, hindering the observation of any color changes in the medium.
- If there is a need to assess the results after 24 hours, remove the tubes from the incubator and store them in a freezer at a temperature of 4°C.
Applications of TSIA Test
- Preliminary recognition of enteric pathogens, especially those belonging to the Gram-negative bacilli group.
- Distinguishing and categorizing members of the Enterobacteriaceae family.
- Identifying lactose-fermenting organisms, non-lactose fermenters, and those capable of producing hydrogen sulfide (H2S).
- A vital instrument for the biochemical identification of bacteria in both research and diagnostic laboratory settings.
Limitations of TSIA Test
- This test serves as a preliminary and differential assay, necessitating supplementary tests for the final confirmation of bacterial identification.
- It is recommended to interpret the results within the specific time frame of 18 to 24 hours, avoiding both premature and delayed readings. This strict time limit is crucial to prevent the occurrence of misleading outcomes.
- It's worth noting that this test is not universally applicable to all fecal bacteria. For instance, in cases of mixed bacterial populations, accurate results may not be achievable, leading to similar outcomes for different bacteria like E. coli and Klebsiella spp.
- The production of hydrogen sulfide (H2S) may be inhibited in the presence of sucrose fermenting bacteria.
- This test does not enable the distinction between the fermentation of lactose or sucrose individually or both sugars together.
