Differential Media:
Multipurpose Enteric Screening Media

Kligler Iron Agar (KIA), Triple Sugar Iron (TSI) Agar, Lysine Iron Agar (LIA) and Motility Indole Ornithine (MIO) Medium

As an introduction or review: Many of our pH-based differential media have one or more of the features shown in the following table. This table should be helpful in analyzing the reactions in the various media discussed on this page, and more discussion is given on the introductory page of the Differential Media Site which addresses the concept of "programming" differential media to detect certain physiological types. An example where a KIA-like medium is so formulated is explained here.

Overview of Differential Features of KIA, TSI, LIA and MIO

In the isolation of gram-negative fermenting rods from colonies on the appropriate selective-differential plating media, the media discussed on this page aid in a preliminary characterization of the isolates. They may confirm certain reactions seen on the plating media, and some will detect non-glucose fermenters (and therefore non-enterics). So rather than perform many tests on many colonies – some of which may turn out to be a waste of time and money if fully tested – one or more of these "screening" media can be utilized to help in obtaining desired types of enterics.

Note the relative amounts of sugars in KIA according to the table seen above. By the degree of acid produced from fermentation, differentiation can be made between non-fermenters, glucose-fermenters (which produce a relatively small amount of acid) and those which ferment both glucose and lactose (producing a relatively large amount of acid which diffuses througout the medium and easily overneutralizes the aerobic deamination reaction in the slant). Organisms which produce hydrogen sulfide from the reduction of thiosulfate are easily detected; the H₂S reacts with the iron in the medium to produce ferrous sulfide, a black precipitate. The medium is inoculated with the needle, first stabbing down the center to the bottom of the tube and then streaking up the slant. Incubation is for one day at 37°C. The various combinations of reactions are explained and illustrated below. (Tube "C" is the uninoculated control tube which shows an orange (neutral) reaction throughout.)

corresponding tube no. above	1	2	3	4*	5**
deamination of amino acids (aerobic alkaline rx.)	+	+	+	+	+
glucose fermentation (minor acid rx.)	–	+	+	+	+
lactose fermentation (major acid rx.)	–	–	–	+	+
H2S production (black color)	–	–	+	–	+**
typical examples	Pseudomonas (a non-enteric)	Morganella, Providencia, Shigella	Citrobacter, Salmonella, Proteus, Edwardsiella	E. coli, Enterobacter, Klebsiella	coliform strains of Citrobacter that are H2S+, H2S+ E. coli, lactose+ Salmonella
* Tube 4: Much gas is often seen for this tube, evidenced by cracks in the medium. Also, lactose fermenters which are methyl red-negative may show a "reversion" toward an alkaline reaction as neutral products are formed from some of the acid. This appears as shown in Tube 4A where a slight reddening of the slant occurs as the alkaline deamination reaction becomes no longer over-neutralized by acid from fermentation. How might such a tube appear after two or more days of incubation? (Regarding the methyl red test, recall the activities of enterics in MR-VP Broth, illustrated here.) ** Tube 5: Enough acid from lactose fermentation can be produced to cause the black iron sulfide precipitate to break down and not be seen. In this case, the tube will look like no. 4.

This medium is identical to KIA except that sucrose is added. Thus, those which ferment lactose and/or sucrose (in addition to glucose) will produce a large amount of acid which will diffuse throughout the entire medium, overneutralizing the aerobic deamination reaction occuring in the slant. The medium is inoculated with the needle, first stabbing down the center to the bottom of the tube and then streaking up the slant. Incubation is for one day at 37°C. The various combinations of reactions are explained and illustrated below. (Tube "C" is the uninoculated control tube which shows an orange (neutral) reaction throughout.)

corresponding tube no. above	1	2	3	4*	5**
deamination of amino acids (aerobic alkaline rx.)	+	+	+	+	+
glucose fermentation (minor acid rx.)	–	+	+	+	+
lactose and/or sucrose fermentation (major acid rx.)	–	–	–	+	+
H2S production (black color)	–	–	+	–	+**
typical examples	Pseudomonas (a non-enteric)	Morganella, Providencia, Shigella	Citrobacter, Salmonella, Edwardsiella	E. coli, Enterobacter, Klebsiella	sucrose+ Proteus, coliform strains of Citrobacter that are H2S+, H2S+ E. coli, lactose+ Salmonella, Edwardsiella tarda Biogroup 1
* Tube 4: Much gas is often seen for this tube, evidenced by cracks in the medium. Also, methyl red-negative organisms which ferment lactose and/or sucrose may show a "reversion" toward an alkaline reaction as neutral products are formed from some of the acid. Note the slight orange to red color at the tip of the slant in tube 4A. How might such a tube look at two or more days of incubation? (Regarding the methyl red test, recall the activities of enterics in MR-VP Broth which are illustrated here.) ** Tube 5: Enough acid from lactose and/or sucrose fermentation can be produced to cause the black iron sulfide precipitate to break down and not be seen. In this case, the tube will look like tube 4.

Here is another multipurpose medium in one tube. Reactions for lysine decarboxylation (anaerobic alkaline reaction – overneutralizing the acid formed from glucose fermentation), lysine deamination (formation of red-colored products at the top of the medium) and H₂S production (black precipitate) can be made as seen and explained below. (Tube "C" is an uninoculated "control" tube showing the neutral, purple color.)

corresponding tube no. above	1	2	3	4*
deamination of amino acids (aerobic alkaline rx.)	+	+	+	+
deamination of lysine (dark red slant)	–	+	–	–
decarboxylation of lysine (anaerobic alkaline rx.)	–	–	+	+
glucose fermentation (acid rx.)	+	+	+	+
typical examples	Citrobacter	Proteus Providencia Morganella	E. coli Enterobacter	Salmonella Edwardsiella
* H2S-positive reaction usually seen for Salmonella and Edwardsiella is shown for Tube 4. LIA is not as reliable an indicator of H2S as are KIA and TSI.

The reactions in this medium are observed as follows:

• Motility. Observe for cloudiness in the medium (growth away from the stab line). For a non-motile organism, growth may be seen along cracks in the medium caused by gas production, but there will be clear pockets of no growth.
  
  
• Ornithine Decarboxylation. Observe the lower three-quarters (anaerobic region) of the medium for change in color of the pH indicator; growth must be present in this part of the tube for correct analysis of result:
  
  • Gray, blue or purple color: Positive reaction for ornithine decarboxylation – formation of a highly alkaline product, over-neutralizing the acid produced from glucose fermentation.
    
  • Yellow color: Negative reaction. Yellow color is due to the "default" acid production from glucose fermentation.
    
    
• Indole Production. About one-half dropperful of Kovacs reagent is added to the medium. A red ring indicates production of indole from the breakdown of tryptophan.

corresponding tube no. above (tubes arranged in pairs with Kovacs reagent added to right tube in each pair)	1	2	3
deamination of amino acids (aerobic alkaline rx.)	+	+	+
motility (cloudiness)	–	+	+
decarboxylation of ornithine (anaerobic alkaline rx.)	–	+	+
indole production (red color with Kovacs reagent)	+	–	+
glucose fermentation (acid rx.)	+	+	+
typical examples	Klebsiella oxytoca	Enterobacter aerogenes	Escherichia coli

A special page covering just KIA and MIO is here,
and one for only TSI and LIA is here.

Page last modified on 5/18/10 at 4:00 PM, CDT.
John Lindquist:  new homepage, complete site outline.
Department of Bacteriology, U.W.-Madison

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