KIA Test- Principle, Media, Procedure, Results, Uses

Kligler’s Iron Agar (KIA) test is a differential medium that is used to identify and differentiate Gram-negative enteric bacilli based on their ability to ferment glucose and lactose and produce hydrogen sulfide (H2S) gas. It is one of the most commonly used tests in clinical microbiology laboratories for screening fecal cultures and isolating pathogens such as Salmonella spp. and Shigella spp.

The KIA test is a useful tool for the identification and differentiation of Gram-negative enteric bacilli, which are bacteria that inhabit the intestinal tract of humans and animals. These bacteria can cause various diseases, such as typhoid fever, dysentery, gastroenteritis, and urinary tract infections. Therefore, it is important to be able to distinguish them from each other based on their biochemical characteristics.

The main objectives of the KIA test are:

• To test the bacteria`s ability to produce hydrogen sulfide (H2S), a gas that has a rotten egg smell and can be toxic in high concentrations. H2S production indicates the presence of certain enzymes that can reduce sulfur compounds, such as sodium thiosulfate, in the culture medium. Some bacteria that can produce H2S are Salmonella, Proteus, and Citrobacter.
• To test the bacteria`s ability to produce gas, such as carbon dioxide or hydrogen, by fermenting either glucose or lactose or both. Gas production indicates the presence of certain enzymes that can break down sugars and release gas as a by-product. Some bacteria that can produce gas are Escherichia coli, Klebsiella, and Enterobacter.
• To test the bacteria`s ability to produce acid by fermenting either glucose or lactose or both. Acid production indicates the presence of certain enzymes that can convert sugars into acidic end products, such as lactic acid or acetic acid. Acid production lowers the pH of the culture medium and changes its color from red to yellow due to the phenol red indicator. Some bacteria that can produce acid are Escherichia coli, Klebsiella, and Enterobacter.
• To differentiate lactose fermenters from non-lactose fermenters. Lactose fermenters are bacteria that can use lactose as a source of energy and produce acid and gas from it. Non-lactose fermenters are bacteria that cannot use lactose and rely on other sources of energy, such as glucose or peptone. Lactose fermentation is a useful criterion for distinguishing between different groups of enteric bacilli, such as Salmonella and Shigella (non-lactose fermenters) and Escherichia coli and Klebsiella (lactose fermenters).

By combining these four objectives, the KIA test can provide a comprehensive profile of the metabolic capabilities of a given bacterium and help narrow down its identification among the enteric bacilli.

The principle of KIA test is based on the ability of different bacteria to use glucose, lactose, and sulfur compounds as sources of energy and produce metabolic byproducts that change the color and appearance of the medium.

The KIA medium contains two carbohydrates: glucose (0.1%) and lactose (1%). Glucose is present in a much lower concentration than lactose. The medium also contains sodium thiosulfate as a source of sulfur and ferrous sulfate as an indicator of hydrogen sulfide (H2S) production. Phenol red is used as a pH indicator that turns yellow below pH 6.8 and red above pH 8.4.

When the KIA medium is inoculated with a bacterial culture, there are four possible outcomes depending on the fermentation abilities of the bacteria:

• If the bacteria can ferment both glucose and lactose, they will produce acid from both sugars, lowering the pH of the medium throughout. The phenol red indicator will turn yellow in both the slant and the butt of the tube. This indicates a positive result for acid production from glucose and lactose (A/A).
• If the bacteria can ferment only glucose, they will produce acid from glucose initially, lowering the pH of the medium in both the slant and the butt. However, as glucose is depleted, the bacteria will switch to using peptone (a protein source) in the slant, which produces alkaline byproducts that raise the pH of the medium. The phenol red indicator will turn red in the slant and remain yellow in the butt. This indicates a positive result for acid production from glucose and a negative result for acid production from lactose (K/A).
• If the bacteria cannot ferment either glucose or lactose, they will use peptone as their sole source of energy, producing alkaline byproducts that raise the pH of the medium throughout. The phenol red indicator will turn red in both the slant and the butt. This indicates a negative result for acid production from both glucose and lactose (K/K).
• If the bacteria can produce H2S, they will reduce sodium thiosulfate to H2S gas, which reacts with ferrous sulfate to form a black precipitate of ferrous sulfide. This will appear as a blackening of the medium, especially in the butt. This indicates a positive result for H2S production (+).

In addition to these outcomes, some bacteria can also produce gas (CO2 or H2) from carbohydrate fermentation, which can be detected by cracks or bubbles in the agar or lifting of the agar from the bottom of the tube. This indicates a positive result for gas production (+).

The KIA test can help differentiate various members of Enterobacteriaceae, a family of Gram-negative bacilli that includes many pathogens and commensals of the gastrointestinal tract. For example, Escherichia coli produces A/A + gas results, Shigella spp. produce K/A results, Salmonella spp. produce K/A + H2S results, and Pseudomonas aeruginosa produces K/K results.

To perform the KIA test, you will need the following items:

• Culture Media: Kligler’s Iron Agar (KIA) medium is used during the test. It is a differential medium that contains two sugars (glucose and lactose), two indicators (phenol red and ferrous sulfate), and a sulfur source (sodium thiosulfate). The medium can detect the fermentation of sugars, the production of gas, the production of hydrogen sulfide (H2S), and the pH changes in the slant and butt of the tube.
• Reagents: No reagents are required for the KIA test. The medium itself acts as an indicator of the biochemical reactions of the bacteria.
• Equipment: You will need some basic laboratory equipment such as PPE (personal protective equipment), inoculating wires, test tubes, caps or cotton plugs, conical flasks or glass bottles, magnetic stirrer or manual stirrer, autoclave, incubator, and a rack to hold the tubes.
• Test Organism: You will need a pure culture of the bacterium that you want to test. The culture should be 18 to 24 hours old and grown on a suitable medium such as nutrient agar or blood agar. You should pick a well-isolated colony from the culture for inoculation.
• Control Organisms: You will also need some control organisms to compare your results with. These are bacteria that have known reactions on the KIA medium and can serve as positive or negative controls. Some examples of control organisms are Escherichia coli, Citrobacter freundii, Proteus vulgaris, Salmonella Paratyphi A, Salmonella Enteritidis, Shigella flexneri, and Pseudomonas aeruginosa. You should use ATCC (American Type Culture Collection) strains of these bacteria if possible.

1. Measure the appropriate amount of KIA media powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company (57.52 grams per 1000 mL).
2. Stir well using a magnetic stirrer or manually and heat to boiling so that all the components and agar dissolve completely in water.
3. Dispense about 5 to 7 mL of the medium in each test tube and loosely put on the cap or cotton plug the opening.
4. Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes.
5. Let it cool and solidify at a slanting position (around 30° inclinations) to form an agar slant with a deep butt.

The KIA medium is now ready to use for inoculation. Make sure to label the tubes with the name of the medium, date of preparation, and batch number. Store the unused tubes in a refrigerator at 2-8°C and use them within one month. Do not use if the medium is cracked or if there is a gap in the medium prior to inoculation.

To perform the KIA test, you will need the following steps:

1. Using a sterile inoculating needle, pick a well-isolated colony from a fresh culture (18 to 24 hours old) of the test bacterium.
2. Inoculate the KIA medium tube by stabbing the butt up to 3 to 5 mm above the base of the tube and while withdrawing, streak the slant portion.
3. Loosen the cap or cotton plug of the tube and incubate it aerobically at 35±2°C for about 24 hours.
4. Examine the tube for color change of the slant and butt and report the color within 24 hours of incubation. If you want to read the H2S production, incubate it for another 24 to 48 hours, but read sugar fermentation and color change within the first 24 hours of inoculation and incubation.

The image below shows how to inoculate the KIA medium tube:

The result of the KIA test is based on the color change of the slant and butt of the medium, as well as the presence or absence of gas and H2S production. The color change is due to the pH indicator phenol red, which turns yellow below pH 6.8 and red above pH 7.4. The following table summarizes the possible outcomes and interpretations of the KIA test:

Some examples of bacteria that show different reactions on KIA are:

• Escherichia coli: Yellow/Yellow, Gas +ve, H2S -ve
• Citrobacter freundii: Yellow/Yellow, Gas +ve, H2S +ve
• Proteus vulgaris: Red/Yellow, Gas -ve, H2S +ve
• Salmonella Paratyphi A: Red/Yellow, Gas +ve, H2S -ve
• Salmonella Enteritidis: Red/Yellow, Gas +ve, H2S +ve
• Shigella flexneri: Red/Yellow, Gas -ve, H2S -ve
• Pseudomonas aeruginosa: Red/Red, Gas -ve, H2S -ve

It is important to read and interpret the KIA results within 18 to 24 hours of incubation, as longer incubation may cause false or misleading results. For example, the whole tube may turn black due to excessive H2S production, or the slant may revert to red due to peptone utilization. If the result needs to be read after 24 hours, the tubes should be stored in a refrigerator at 4°C.

Some additional points are:

• The KIA test can be used to differentiate Salmonella spp. and Shigella spp., as Salmonella spp. are usually H2S positive and Shigella spp. are usually H2S negative.
• The KIA test can also be used to differentiate lactose fermenters and non-lactose fermenters among Enterobacteriaceae.
• The KIA test is not a confirmatory test for identification of bacteria; it should be used along with other biochemical tests and serological tests.

To ensure the accuracy and reliability of the KIA test, it is important to use quality control organisms that have known reactions on the medium. These organisms can be used to check the performance of the medium, the inoculation technique, and the interpretation of the results. Some examples of quality control organisms are:

• Escherichia coli ATCC 25922: Yellow/Yellow, Gas +ve, H2S -ve
• Citrobacter freundii ATCC 8090: Yellow/Yellow, Gas +ve, H2S +ve
• Proteus vulgaris ATCC 6380: Red/Yellow, Gas -ve, H2S +ve
• Salmonella Paratyphi A ATCC 9150: Red/Yellow, Gas +ve, H2S -ve
• Salmonella Enteritidis ATCC 13076: Red/Yellow, Gas +ve, H2S +ve
• Shigella flexneri ATCC 12022: Red/Yellow, Gas -ve, H2S -ve
• Pseudomonas aeruginosa ATCC 27853: Red/Red, Gas -ve, H2S -ve

The quality control organisms should be inoculated and incubated following the same procedure as the test organism. The results should be read and recorded within 18 to 24 hours of incubation. If the results do not match the expected reactions, the test should be repeated or the source of error should be identified and corrected.

• Do not use the medium if it is cracked or if there is a gap in the medium prior to inoculation.
• Do not use an inoculating loop for stabbing because it can create cracking which will confuse during reporting the gas production.
• Do not read the result for sugar utilization prior to 18 hours of incubation because the glucose may not be depleted and the oxidative metabolism of peptones may not be started yet.
• Do not read the result long after 24 hours because the whole tube may turn black and fails to show the color change of the medium.
• If you have to read the result after 24 hours, remove the tubes from the incubator and store them in a freeze at 4°C.
• Loosen closure on the tube before incubating to allow aerobic conditions.
• Use pure cultures and well-isolated colonies for inoculation.
• Avoid contamination of the medium with other bacteria or chemicals.

KIA test is a useful tool for the identification and differentiation of Gram-negative enteric bacilli, especially the members of the family Enterobacteriaceae. Some of the applications of KIA test are:

• To distinguish lactose fermenters from non-lactose fermenters based on the color change of the slant and butt. For example, Escherichia coli and Klebsiella pneumoniae are lactose fermenters that produce yellow/yellow reactions, while Salmonella spp. and Shigella spp. are non-lactose fermenters that produce red/yellow or red/red reactions.
• To detect the ability of bacteria to produce hydrogen sulfide (H2S) by reducing sodium thiosulfate. H2S production is indicated by the blackening of the medium or part of it. For example, Proteus vulgaris and Citrobacter freundii are H2S producers that produce black precipitates, while Pseudomonas aeruginosa and Shigella flexneri are H2S non-producers that do not produce any blackening.
• To detect the ability of bacteria to produce gas by fermenting glucose or lactose. Gas production is indicated by the formation of bubbles, cracks, or gaps in the medium. For example, Escherichia coli and Citrobacter freundii are gas producers that produce visible gas pockets, while Shigella flexneri and Pseudomonas aeruginosa are gas non-producers that do not produce any gas.
• To screen fecal cultures for the presence of enteric pathogens such as Salmonella spp. and Shigella spp., which are usually non-lactose fermenters and H2S producers.
• To presumptively identify some bacterial species based on their characteristic KIA reactions. For example, Salmonella typhi produces a red slant with a black butt (K/A + H2S), while Yersinia enterocolitica produces a red slant with a yellow butt (K/A).

• KIA test is not a confirmatory test for the identification of enteric bacteria. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
• KIA test has a strict time frame for reading the results. It is advised to read the results within 18 to 24 hours of incubation, not too early and not too late. Reading the results too early may result in false-negative reactions for lactose fermentation and H2S production. Reading the results too late may result in false-positive reactions for H2S production due to the diffusion of H2S from other tubes or the whole tube turning black and obscuring the color change of the medium.
• KIA test cannot differentiate between lactose-only fermenters and lactose-and-glucose fermenters. Both types of bacteria will produce a yellow slant and yellow butt (A/A) reaction on KIA. To distinguish between them, other tests such as indole or methyl red tests are required.
• KIA test may not detect some strains of H2S-producing bacteria that require cysteine for H2S production. Such strains may give a false-negative reaction for H2S production on KIA. To detect them, other media such as SIM or TSI that contain cysteine are required.
• KIA test may not detect some strains of non-lactose fermenting bacteria that produce acid from peptone degradation. Such strains may give a false-positive reaction for lactose fermentation on KIA. To confirm them, other tests such as citrate or urease tests are required.

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