Starch Hydrolysis Test

Objective:

-To determine  the ability of certain microorganisms to produce extracellular enzyme  amylase that breaking down starch.

-To identify the starch hydrolyzing bacteria.

Principle:

           

            Starch is a high molecular weight branching polymer. Starch as such cannot be transported to the bacterial cell for energy generation due to its high molecular weight. Therefore starch hydrolyzing exoenzymes are released into the surrounding media which can degrade starch to its glucose monomeric units. These soluble low molecular weight glucose can pass into the cell can be utilized energy generation by microbes.

                                     

Starch agar is nutritive medium with starch. The presence and absence of starch in the medium can be detected by addition of iodine, as iodine reacts with starch to import blue black color.

Materials required:

Bacterial cultures (Bacillus sp., and  E.coli)

Starch agar medium

Procedure :
1.      Inoculate the bacterial cultures on starch agar medium by streaking.
2.      The plates are incubating at 37° C for 24 hrs.
3.      Following incubation, pour the iodine solution on medium and observe the clear zone around the area of growth.

Observation:
- Clear zone around the line of growth of Bacillus sp., 
 In E.coli Plate turns completely blue black color, without zone formation.

Result :
-  Bacillus sp  : Starch hydrolysis positive
- E. coli : Starch hydrolysis negative

Interpretation:

           

            The clear zone of hydrolysis around Bacillus sp., when flooded with iodine solution, indicates that organism produces extra cellular amylase enzyme that hydrolyse starch in the media. E.coli showed negative result  as it did not produce amaylase. Thus could not hydrolyse starch and hence when iodine added it produced blue black color.

  

Starch Hydrolysing organisms:

Bacillus subtilis, Bacillus megatarium

 

Clear zone around the the Bacillus sp.,

(Starch hydrolyzing bacteria)

Below the first plate is positive and the second one is negative

                              

Starch Agar Composition

Ingredients / Liter

                     

Beef extract                             3.0 g   

Starch, soluble                         10. g

Agar                                        12.0 g

pH ( at 25°C) 7.5


Iodine Solution Preparation:

Potassium iodide   2g

Iodine crystals  1g

Distilled water  300ml

Dissolve 2g of potassium iodide in 300 ml of water. Then add 1g of finely ground iodine crystals. Stir at room temperature until completely dissolved.