The Procedure, Uses, And Interpretation Of Kligler’s Iron Agar Test

Kligler’s Iron Agar test is used for the detection of carbohydrate fermentation.

If you want to learn more about Kligler’s Iron Agar test but don’t know where to start, you’ve come to the right place.

The Procedure, Uses, And Interpretation Of Kligler’s Iron Agar Test

In this article, we will cover some key information about Kligler’s Iron Agar test, including the procedure, uses, and interpretation of this useful test.

Let’s get started. 

What Is Kligler’s Iron Agar Test?

Kligler Iron Agar was modified from Kligler’s original formula, and was designed to identify pure cultures of colonies picked from primary plating media.

Kligler Iron Agar is used in a laboratory setting for the identification of Enterobacteriaceae on the basis of dextrose and lactose fermentation and hydrogen sulfide production.

The use of two sugars helps to distinguish the organism on the basis of only dextrose fermenting from those where lactose has also fermented.

The Principle Of Kligler’s Iron Test 

  • Kligler Iron Agar contains lactose and glucose, enabling the differentiation of species of enteric bacilli. Phenol red is the pH indicator, which displays a color change as a result of acid that is produced during the fermentation process.
  • Fermentation of dextrose results in production of acid, which turns the indicator from red to yellow. Due to the small level of sugar, acid production is limited and rexoxidation is produced on the surface, causing the indicator to remain red. When lactose is fermented, the larger amount of acid leads to the bypass of reoxidation, meaning that the medium turns yellow.
  • Sulfate as well as sodium thiosulfate enables hydrogen sulfide production to be detected, shown through black coloring through the butt or in a ring.
  • Lactose non-fermenters (Shigella and Salmonella) produce a yellow slant thanks to acid produced by the glucose. When the dextrose supply is used up in the aerobic environment, the reaction reverts to alkaline due to oxidation of the acids produced. Reversion doesn’t occur in the anaerobic environment of the butt, meaning it remains acidic.
  • Lactose fermenters produce yellow butts and slants due to lactose fermentation. The high level of acids maintains an acidic pH under aerobic conditions. The tube showing the medium’s original color indicates that neither glucose or lactose has fermented.
  • The production of gas is detected as bubbles or through splitting of the agar in the butt of the medium.

Media Used – Kligler’s Iron Agar Test

  • Peptone – 15.0gm
  • Proteose peptone – 5.0gm
  • Agar – 12.0gm
  • Lactose – 10.0gm
  • Dextrose – 1.0gm
  • Beef extract – 3.0gm
  • Yeast extract – 3.0gm
  • Sodium chloride – 5.0gm
  • Sodium thiosulfate – 0.3gm
  • Ferrous sulfate – 0.2gm
  • Phenol red – 0.024gm

Final pH ( at 25°C) 7.4±0.2

The Method Of Kligler’s Iron Agar Test

  • Step One – To begin the Kligler’s Iron Agar test, you need to pick the center of well-isolated colonies obtained from solid culture media with an inoculating needle. The reason the medium is recommended is for the identification of colonies from plating media like Bismuth Sulphite Agar, Desoxycholate Citrate Agar, and MacConkey Agar.
  • Step Two – Following this, you will need to stab the middle of the medium in the tube to within 3 to 5mm from the bottom.
  • Step Three – Withdraw the inoculating needle and streak the surface of the slant.
  • Step Four – Loosen the closure on the tube before you incubate it. The cap must be loose during the incubation process, because if it was too tight, an acid reaction caused by dextrose fermentation alone will also involve the slant.
  • Step Five – Now you will need to incubate it aerobically for 18 to 48 hours at 95°F.
  • Step Six – Once this time has passed, you will need to read the tube for hydrogen sulfide reactions, gas production, and acid production of the slant/butt.

What Are The Uses Of Kligler’s Iron Agar Test?

What Are The Uses Of Kligler’s Iron Agar Test?

There are a variety of uses of Kligler’s Iron Agar test that you should be aware of. The Kligler’s Iron test is used:

  • To differentiate Salmonella typhi from other Salmonellae and also Salmonella paratyphi A from Salmonella enteritidis and Salmonella scottmuelleri.
  • As a differentiation medium for typhoid, allied bacilli, and dysentery.
  • To differentiate lactose fermenters from the non-fermenters.

Which pH Indicator Is Used In Kligler’s Iron Agar Test?

As we have mentioned above, phenol red is the indicator for Kligler’s Iron Agar test.

This will then show a change in color as a direct response to the acid produced during the fermentation of lactose and glucose.

Expected Results For Kligler’s Iron Agar Test

Once Kligler’s Iron Agar test has been performed, there are expected results you should be aware of.

Below are some expected results for this test.

Hydrogen Sulfide Production

  • Positive Test – Black color developed throughout medium, a black precipitate in the butt, or a black ring at the juncture of the slant and butt
  • Negative Test – No black color to note

Carbohydrate Fermentation

  • Positive Test for Slant Reaction – Yellow (acid)
  • Negative Test for Slant Reaction – Red (alkaline)
  • Positive Test for Butt Reaction – Yellow (acid)
  • Negative Test for Butt Reaction – Red (alkaline)

Gas Production

  • Positive Test – Bubbles in the medium, separation of the medium from the side and bottom of the tube, or cracking and displacement of the medium 
  • Negative Test – No bubbles and no separation or displacement of the medium

KIA Color Reactions

  • Red slant/ yellow butt – dextrose (+), lactose (-)
  • Yellow slant/ yellow butt – dextrose (+), lactose (+)
  • Red slant/ red butt – dextrose (-), lactose (-)

Limitations Of Kligler’s Iron Agar Test 

There are a variety of limitations of Kligler’s Iron Agar test that you should be aware of. These limitations include but are not limited to:

  • Failure to stab the butt will invalidate the test.
  • The results for Kligler’s Iron Agar test should be recorded after 18 to 24 hours. Failure to do so could result in erroneous results. For instance, a reaction read before 18 hours can be falsely interpreted due to the fact that the carbohydrate that has fermented might not have produced enough acid, and therefore the phenol red indicator hasn’t changed as a result. A reaction read after 24 hours, on the other hand, might be interpreted incorrectly thanks to peptone utilization, resulting in an alkaline pH shift.
  • Biochemical, immunological, molecular, or mass spectrometry testing should always be performed on pure culture colonies to ensure complete identification.
  • Hydrogen sulfide determinations using this test should be limited to members of Enterobacteriaceae. 
  • Hydrogen sulfide producing organisms may produce so much black precipitate that the reaction in the butt is completely masked as a result. If hydrogen sulfide is produced, then you should be aware that dextrose is fermented even if you can’t see it.
  • The tube should only be loosely capped in this test, because if it is capped too tightly, an acid reaction caused by dextrose fermentation alone will involve the slant. 

In Summary 

So, there you have Kligler’s Iron Agar test.

We hope this article has been useful and has given you a better understanding of Kligler’s Iron Agar test as a whole and why it is used.

You should now be aware of the procedure, uses, and interpretation of Kligler’s Iron Agar test.

If you’re struggling with anything to do with the test, be sure to save this article for future reference and information.

Jennifer Dawkins

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