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

 

 

Nitrate reduction test- Dr C R Meera

 

Nitrates serve as source of nitrogen for many bacteria.  They can also act as final electron acceptor.  Many organisms can be differentiated and identified by their capacity to reduce nitrates to nitrites.  Most of the enterobacteriaceae reduce nitrates.  This character is useful for the identication of the species in Neisseria, Haemophilus and Branhamella.  Some Pseudomonas and nonfermenters reduce nitrate to nitrite and further down to ammonia or to molecular nitrogen.

The enzyme nitrate reductase possessed by organisms reduces nitrates to nitrites. The reduction of nitrates by some aerobic and facultative anaerobic microorganisms occurs in the absence of molecular oxygen, an anaerobic process.  In these organisms anaerobic respiration is an oxidative process whereby the cell uses inorganic substances such as nitrates (NO₃^-) or sulfates (SO₄ ^2-) to supply oxygen that is subsequently utilized as a final hydrogen acceptor during energy formation.  The biochemical transformation may be visualized as follows:

NO₃^-  +  2H⁺  +  2e^{-        Nitrate Reductase}    NO₂^- + H₂O

Nitrate     Hydrogen    Electrons                                                   Nitrite        Water

Some organisms possess the enzymatic capacity to act further on nitrites to reduce them to ammonia (NH₃⁺) or molecular nitrogen (N₂) and this process is called denitrification.  The reaction may be described as follows:

 NO₂^-    ------->         NH₃⁺

Nitrite                                 Ammonia     

 Or

 2NO₃^-   +  12H⁺  +  10e^{-   -------->} N₂   +  6 H₂O

Nitrate           Hydrogen      Electrons                  Molecular nitrogen and water

 Aim

To determine the ability of some microorganisms to reduce nitrates (NO₃^- ) to nitrites (NO₂^-) or beyond the nitrite stage.

Principle

Nitrate reduction can be determined by cultivating organisms in a nitrate broth medium.  The medium is basically a nutrient broth supplemented with 0.1% potassium nitrate (KNO₃) as the nitrate substrate.  In addition, the medium is made into a semisolid by the addition of 0.1% agar.  Following incubation of the cultures, an organism’s ability to reduce nitrates to nitrites is determined by the addition of two reagents: Solution A, which is sulfanilic acid, followed by Solution B, which is α- naphthylamine.  Nitrites in acid environment immediately produce a cherry red coloration due to the formation of a red diazomium dye, p- sulfo benzene-azo-alphanaphthylamine.

Cultures not producing a color change suggest one of two possibilities: (1) nitrates were not reduced by the organism, or (2) the organism possessed such potent nitrate reductase enzymes that nitrates were rapidly reduced beyond nitrites to ammonia or even molecular nitrogen.  To determine whether or not nitrates were reduced past the nitrite stage, a small amount of zinc powder is added to the basically colorless cultures already containing Solutions A and B.  Zinc reduces nitrates to nitrites.  The development of red color therefore verifies that nitrates were not reduced to nitrites by the organism.  If nitrates were not reduced, a negative nitrate reduction reaction has occurred.  If the addition of zinc does not produce a color change, the nitrates in the medium were reduced beyond nitrites to ammonia or nitrogen gas.  This is a positive reaction.

Requirements

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

Nitrate broth, Solution A (sulfanilic acid), Solution B (α- naphthylamine), zinc powder, Bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1.  Using sterile technique, inoculate each experimental organism into its appropriately labeled tubes by means of loop inoculation. One uninoculated tube kept as control.

2.  Incubate all inoculated tubes at 37^o C for 24-48 hrs.

3. Add 5 drops of Solution A and then 5 drops of solution B to all nitrate broth cultures and observe for the red color development.

4. Add a minute quantity of zinc to the cultures in which no red color developed.  Observe for the color change to red.

 

Observations

Bacillus, Staphylococcus and E.coli produced cherry red coloration immediately after adding Solutions A and B, indicating that they are nitrate positive.  Pseudomonas and Streptococcus produced no cherry red coloration on addition of Solutions A and B.  Addition of zinc powder to these tubes produced a red color in Streptococcus inoculated ones which indicates a negative nitrate reduction result. No colour formation in the tube inoculated with Pseudomonas indicated the conversion of nitrate beyond nitrites to ammonia or nitrogen gas.

Result

Bacillus, Staphylococcus, E.coli and Pseudomonas are nitrate test positive whereas Streptococcus is nitrate negative.

 

§         Nitrate broth

Peptone                                               5.0 g

Beef extract                                        3.0 g

Potassium nitrate                                 5.0 g

Distilled water                                    1 litre

pH                                                       7.2

Citrate utilization test-Dr C R Meera

Aim

To demonstrate the ability of microorganisms to utilize citrate as the sole source of carbon.

Principle

In the absence of fermentable glucose or lactose, some microorganisms are capable of using citrate as a carbon source for their energy.  This ability depends on the presence of a citrate permease that facilitates the transport of citrate in the cell.  Citrate is the first major intermediate in the Kreb’s cycle and is produced by the condensation of active acetyl with oxaloacetic acid.  Citrate is acted on by the enzyme citrase, which produces oxaloacetic acid and acetate.  These products are then enzymatically converted to pyruvic acid and carbon dioxide.  During this reaction the medium becomes alkaline- the carbon dioxide that is generated combines with sodium and water to form sodium carbonate, an alkaline product.  The presence of sodium carbonate changes the Bromothymol blue indicator incorporated into the medium from green to deep Prussian blue.  Bromothymol blue is green when acidic (pH 6.8 and below) and blue when alkaline (pH 7.6 and higher). 

Following incubation, citrate positive cultures are identified by the presence of growth on the surface of the slant, which is accompanied by blue coloration.  Citrate negative cultures will show no growth and the medium will remain green.

Requirements

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

Simmons citrate agar slants, Bunsen burner, inoculating needle, glass marking pencil etc.

 Procedure

1.  Aseptically inoculate each organism into appropriately labeled tubes by means of streak       inoculation.  One uninoculated tube kept as control.

2.  Incubate all inoculated tubes at 37^o C for 24-48 hrs.

 

Observations

In  Streptococcus, Bacillus, Staphylococcus and Pseudomonas inoculated slants, growth was visible on the surface and the medium color changed to blue.  In E.coli inoculated slants there was no growth as well as change in color of the medium.

Result

Among the given cultures, Streptococcus, Bacillus, Staphylococcus and Pseudomonas sp. are Citrate positive whereas and E.coli is Citrate negative.

§        Simmons citrate agar

Ammonium dihydrogen phosphate    1.0 g

Dipotassium phosphate                       1.0 g

Sodium chloride                                  5.0 g

Sodium citrate                                     2.0 g

Magnesium sulphate                           0.2 g

Agar                                                    15.0 g

Bromothymol blue                              0.08 g             

Distilled water                                    1 litre

pH                                                       6.9