Bacteriology 102:
Custom Formulation of Media
Based on Kligler Iron Agar

How to "program" such a medium to screen for certain physiological types of bacteria.
(A "KIA Tutorial.")

Recall from our discussions about the triple differentiating feature of Kligler Iron Agar (KIA) in that it tests whether or not an organism can (1) ferment glucose, (2) ferment lactose, and (3) produce hydrogen sulfide (H₂S) from the reduction of thiosulfate. (This medium is also explained on this page of our Differential Media Site, along with its variant, TSI Agar.) Whether or not there is enough acid produced from the fermentation of one or more sugars to permeate the medium and over-neutralize the alkaline reaction of the slant (which is caused by the aerobic deamination of amino acids) is the key differential feature in distinguishing between those organisms able to ferment just the "minor" sugar (present in 0.1% concentration – i.e., glucose in KIA) and those able to ferment both the "minor" and the "major" sugars (the latter present in 1.0% concentration – i.e., lactose in KIA). The additional "major" sugar in TSI Agar is sucrose.

Conceivably this medium could be altered to test for fermentation with other sugar combinations, and one could also think about adding a specific amino acid to see if the inoculated organism can decarboxylate it. Such amino acids (often lysine, ornithine and arginine) are usually present in decarboxylation test media in a 0.5 to 1.0% concentration, and an anaerobic, alkaline reaction is indicative of decarboxylation.

Consider the diagram on the right which applies to KIA and related media such as TSI Agar. The influence of decarboxylation of an optionally -added amino acid is also indicated.

Decarboxylation of the added amino acid will result in an alkaline "butt" if it is not overneutralized by excess acid formation by fermentation of the major sugar(s). Gas produced during fermentation will show up as cracks in the medium. Also, H₂S production will be evident by the black color of ferrous sulfide (FeS) in the butt of the tube – if the pH of the medium is not too low from excess fermentation, as the FeS can dissolve and disappear under such conditions.

Here is a question from a recent (Spring, 2000) Bacteriology 102 take-home quiz: (Looks like another one of our thought questions doesn't it?)

We would like to be able to differentiate the enteric genera Sorgobacter, Splammobacter and Wiscobacter in a KIA-based medium – not only from each other but also from all other enterics and also Pseudomonas. Consider the reactions in the following table while answering the questions below.

a.  As you know, KIA contains glucose and lactose as the only fermentable sugars, and it has a H₂S indicator system, and it does not contain any extra added ornithine or lysine. What would you add to KIA (in relatively large amounts) to differentiate Sorgobacter, Splammobacter and Wiscobacter from each other and also other enterics and Pseudomonas?

Choose two:  FRUCTOSE  GALACTOSE  MANNITOL  ORNITHINE  LYSINE

b.  Show how these organisms would appear in this modified KIA medium (i.e., color or pH of slant region, color or pH of butt region, and whether or not H₂S is indicated in the butt region).

Here is the solution:

Choosing fructose and ornithine will give you a "unique" pattern of reactions for each of the five groups of organisms. Fermentation of fructose will cause the production of much acid which will produce an acid (yellow) slant for all enterics other than the three genera specified. (Gas production – which may become evident in the real situation but is unnecessary in our discussion of pH indication – is not shown for any of these tubes.)

corresponding tube no. above	1	2	3	4	5
organisms	Sorgobacter	Splammobacter	Wiscobacter	other enterics	Pseudomonas
deamination of amino acids (aerobic alkaline rx.)	+	+	+	+	+
glucose fermentation (minor acid rx.)	+	+	+	+	–
fructose &/or lactose fermentation (major acid rx.)	–	–	–	+	–
ornithine decarboxylation (anaerobic alkaline rx.)	–	+	–	+ or –	–
H2S production (black color)	–	–	+	+ or –	–

The five tubes give unique reactions as required by the question:

• Sorgobacter: Alkaline (red) slant over acid (yellow) butt. Acid from fermentation of glucose is not enough to over-neutralize the aerobic alkaline reaction from deamination of the various amino acids in the medium (mainly in the peptone). Note from the table that deamination will occur in this and the following tubes as is the rule for enterics and similar organisms.
  
• Splammobacter: Alkaline slant over alkaline butt. Alkaline product from ornithine decarboxylation overneutralizes the small amount of acid from glucose fermentation. Otherwise, the tube would appear as indicated for Sorgobacter.
  
• Wiscobacter: Same reactions as for Sorgobacter with the addition of H₂S production showing up (black color) in the butt.
  
• Other enterics: All ferment glucose and fructose; some also ferment lactose. Therefore much acid from fermentation permeates the medium and overneutralizes the alkaline deamination reaction in the slant – thus the entire tube is yellow.
  
  • Quite probably the tubes of those "other enterics" that decarboxylate ornithine will not show that reaction, as the large amount of acid from fermentation will most likely be enough to over-neutralize it.
    Thus this medium can serve to detect those enterics with the following rare combination of reactions: glucose+, lactose–, fructose–, ornithine+; that would be Splammobacter in this set-up. For those organisms fermenting lactose and/or fructose, the relatively large amount of acid thus produced would probably over-neutralize the alkaline reaction from any ornithine decarboxylation.
    
  • Likewise the H₂S producers in the "other enterics" category may not show up as such, as the high amount of acid may dissolve the black FeS precipitate.
    Thus this medium can serve to detect those enterics with this rare combination of reactions: glucose+, lactose–, fructose–, H₂S+; that would be Wiscobacter. The relatively large amount of acid from lactose and/or fructose fermentation would probably wipe out any indication of H₂S formation.
• Pseudomonas: No fermentation of any sugar, nor any growth at all in the butt. Therefore butt stays neutral (orange), and the slant is alkaline (red) due to deamination.

Page last modified on 4/9/01 at 2:45 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison