Triple Sugar-Iron Agar Test-Dr C R Meera

 

The Triple Sugar-Iron (TSI) agar test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish the Enterobacteriaceae from other gram negative intestinal bacilli.  This differentiation is made on the basis of differences in carbohydrate fermentation patterns and hydrogen sulphide production by the various groups of intestinal organisms. Some bacteria liberate sulfur from sulfur containing aminoacids or other sulfur containing compounds.  The sulfur is used as final hydrogen acceptor leading to the formation of H₂S. 

Aim

To differentiate among members of the Enterobacteriaceae; and also between the Enterobacteriaceae and other groups of intestinal bacilli.

Principle

The TSI agar slants contain lactose (1%), sucrose (1%) and glucose (0.1%) to facilitate observation of carbohydrate utilization patterns. The acid-base indicator phenol red is also incorporated to detect carbohydrate fermentation that is indicated by a change in color of the medium from orange-red to yellow in the presence of acids.  The slant is inoculated by means of a stab and streak procedure.  Following incubation, the fermentative activities of the organisms are determined as follows:

1.      Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in the agar butt): Only glucose fermentation has occurred.  The organisms preferentially degrade glucose first.  Since this substrate is present in minimal concentration, the small amount of acid produced on the slant surface is oxidized rapidly.  The peptones in the medium are also used in the production of alkali.  In the butt the acid reaction is maintained because of reduced oxygen tension and slower growth of the organisms.

2.      Acid slant (yellow) and acid butt (yellow) with or without gas production: Lactose or sucrose fermentation has occurred.  Since these substances are present in higher concentrations, they serve as substrates for continued fermentative activities with maintenance of an acid reaction in both slant and butt.

3.      Alkaline slant (red) and alkaline butt (red) or no change (orange-red) butt: No carbohydrate fermentation has occurred.  Instead, peptones are catabolized under anaerobic or aerobic conditions, resulting in an alkaline pH due to production of ammonia.  If only aerobic degradation of peptones occurs, the alkaline reaction is evidenced only on the slant surface.  If there is aerobic and anaerobic utilization of peptone, the alkaline reaction is present on the slant and the butt.

To obtain accurate results, it is essential to observe the cultures within 18 to 24 hrs following incubation.  This will ensure that the carbohydrate substrates have not been depleted and that degradation of peptones yielding alkaline end products has not taken place. 

The TSI medium also contains sodium thiosulphate, a substrate for hydrogen sulphide (H₂S) production, and ferrous sulphate for detection of this colorless end product.  Following incubation, only cultures of organisms capable of producing H₂S will show an extensive blackening in the butt because of the precipitation of the insoluble ferrous sulphide. 

Requirements

24 hr nutrient broth cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Triple Sugar-Iron Agar slants, Bunsen burner, inoculating needle, glass marking pencil etc.

 Procedure

 1.      Using sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of a stab and streak inoculation. One tube kept as control.

2.      Incubate for 18-24 hrs at 37^oC.

3.    3.  Examine the color of both the butt and slant of all inoculated tubes and determine the type of reaction that has taken place (acid, alkaline or none) and the carbohydrate that has been fermented in each culture.

4.     4.  Examine all cultures for the presence or absence of blackening with in the medium and determine the ability of the organism to produce H₂S.

 Observations    

Bacillus               : Acidic slant, no color change in butt, no gas and no H₂S formation.                            

 Streptococcus     : Acidic slant, acidic butt, gas produced and no H₂S formation.                                     

 Staphylococcus   : Acidic slant, acidic butt, no gas and no H₂S formation.

E.coli                   :  Acidic slant, acidic butt, gas produced and no H₂S formation.                                   

 Pseudomonas     :  Alkaline slant, no color change in butt, no gas and no H₂S formation.

 Result

 Bacillus                :A/ NC                                                                                                                         

 Streptococcus     :   A/A, G                                                                                                                          

 Staphylococcus   :   A/A

 E.coli                    :  A/A, G

 Pseudomonas      :  K/NC

Table 1. Result interpretation of Triple Sugar-Iron Agar test

Results (slant/butt)	Symbol	Interpretation
Red/yellow	K/A	Glucose fermentation only; Peptone catabolized
Yellow/yellow	A/A	Glucose and lactose and/or sucrose fermentation
Red/red	K/K	No fermentation; Peptone catabolized
Red/no color change	K/NC	No fermentation; Peptone used aerobically
Yellow/yellow with bubbles	A/A,G	Glucose and lactose and/or sucrose fermentation; Gas produced
Red/yellow with bubbles	K/A,G	Glucose fermentation only; Gas produced
Red/yellow with bubbles and black precipitate	K/A,G, H2S	Glucose fermentation only; Gas produced; H2S produced
Red/yellow with black precipitate	K/A, H2S	Glucose fermentation only; H2S produced
Yellow/yellow with black precipitate	A/A, H2S	Glucose and lactose and/or sucrose fermentation; H2S produced
No change/no change	NC/NC	No fermentation
A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction

 §  Triple Sugar-Iron Agar slants

Beef extract                                        3.0 g

Yeast extract                                       3.0 g

Peptone                                               15.0 g

Proteose peptone                                 5.0 g

Lactose                                                10.0 g

Saccharose                                          10.0 g

Dextrose                                              1.0 g

Ferrous sulfate                                    0.2 g

Sodium chloride                                  5.0 g

Sodium thiosulphate                           0.3 g

Phenol red                                           0.024 g

Agar                                                    12.0 g

Distilled water                                    1 litre

pH                                                       7.4