Table of Contents
- Introduction
- Objectives of Starch Hydrolysis Test
- Principle of Starch Hydrolysis Test
- Requirement for Starch Hydrolysis Test
- Procedure of Starch Hydrolysis Test
- Result and Interpretation of Starch Hydrolysis Test
- Starch Hydrolysis Test Results of Some Common Bacterial Pathogens
- Quality Control
- Precautions
- Applications of Starch Hydrolysis Test
- Limitations of Starch Hydrolysis Test
Introduction
- Starch, a primary food reserve in most plants, is one of the most abundant carbohydrates found in nature.
- It is a polysaccharide composed of two glucose polymers: amylose and amylopectin. Due to its large macromolecular structure, starch cannot be directly utilized by microorganisms in its native form and must be broken down into glucose for metabolism.
- Bacterial extracellular amylase enzymes can hydrolyze starch into maltose and glucose, but not all bacteria can produce these enzymes. The starch hydrolysis test, also known as the amylase test, is a biochemical assay used to determine a bacterium's ability to produce amylase and utilize starch as a carbon source.
- This test is commonly employed to differentiate species within the genera Bacillus, Clostridium, Corynebacterium, Fusobacterium, Streptococcus, Enterococcus, and Pseudomonas.
Objectives of Starch Hydrolysis Test
- To assess the capability of bacteria to produce the amylase enzyme.
- To evaluate the ability of bacteria to hydrolyze starch.
- To differentiate and identify bacteria based on their starch hydrolysis capability.
Principle of Starch Hydrolysis Test
- Bacteria capable of secreting extracellular amylase enzymes have the ability to break down starch into smaller, water-soluble sugar molecules such as glucose, maltose, and dextrose. Conversely, if bacteria do not produce amylase, starch hydrolysis does not occur.
- When amylase-producing bacteria are inoculated, the starch molecules surrounding the colonies undergo hydrolysis, leading to the absence of amylose or starch molecules. Consequently, upon adding iodine solution, no dark blue coloration forms around the colonies.
- The presence of amylase-producing bacteria results in the hydrolysis of starch molecules surrounding the bacterial colony, preventing color development in the surrounding medium upon iodine solution addition.
- Identification of starch hydrolysis is achieved by observing the formation of a clear halo zone around bacterial growth upon adding iodine solution to the inoculated and incubated starch medium.
Requirement for Starch Hydrolysis Test
1. Culture Media
Mueller Hinton Agar (MHA), Starch Agar, and Heart Infusion Agar supplemented with 2% starch are frequently employed for detecting starch hydrolysis.
Composition of Mueller Hinton Agar per 1000 mL
- HM Infusion B (Beef Infusion) weighing 300.00 grams,
- Acicase (Casein Acid Hydrolysate) weighing 17.500 grams,
- Starch weighing 1.5000 grams,
- Agar weighing 17.000 grams,
- With a final pH of 7.3 ± 0.1 at 25°C.
Preparation of MH Agar
- Measure out the appropriate quantity of MHA media powder, which is 38.0 grams per 1000 mL, and dissolve it in the required volume of water in a conical flask or glass bottle. Ensure thorough mixing, either by using a magnetic stirrer or stirring manually, and heat the mixture to boiling to completely dissolve the media powder.
- After dissolution, autoclave the medium at 121°C and 15 lbs. pressure for 15 minutes to sterilize it.
- Once sterilization is complete and the medium has cooled to approximately 40 to 45°C, dispense approximately 20 to 25 mL of the medium into each sterile petri dish with a diameter of 10 cm, and allow it to solidify.
Composition of Starch Agar per 1000 mL
- Meat Extract - 3.00 grams,
- Peptic Digest of Animal Tissue - 5.00 grams,
- Starch - 2.00 grams,
- Agar - 15.0 grams,
- With a final pH of 7.2 ± 0.1 at 25°C.
Preparation of Starch Agar
- Measure out the appropriate quantity of Starch Agar media powder, which is 25.0 grams per 1000 mL, and dissolve it in the required volume of water in a conical flask or glass bottle. Ensure thorough mixing, either by using a magnetic stirrer or stirring manually, and heat the mixture to boiling to completely dissolve the media powder.
- After dissolution, autoclave the medium at 121°C and 15 lbs. pressure for 15 minutes to sterilize it.
- Once sterilization is complete and the medium has cooled to approximately 40 to 45°C, dispense approximately 20 to 25 mL of the medium into each sterile petri dish with a diameter of 10 cm, and allow it to solidify.
2. Reagents
Gram's Iodine Solution is essential for detecting starch hydrolysis.
Preparation of Gram’s Iodine Solution:
- To prepare Lugol’s iodine stock solution, dissolve 25 grams of iodine crystals and 50 grams of potassium iodide in 500 mL of distilled water.
- To make a 5% sodium bicarbonate solution, dissolve 5 grams of sodium bicarbonate (NaHCO3) in 100 mL of distilled water.
- Then, mix 60 mL of Lugol’s iodine solution and 60 mL of the 5% sodium bicarbonate solution with 220 mL of distilled water to produce Gram’s Iodine Solution.
Alternatively:
- Dissolve 1.0 gram of iodine and 2.0 grams of potassium iodide in 300 mL of distilled water.
3. Equipment
- Petri Plate
- Incubator
- Dropper
- Autoclave
- Bunsen burner
- Weighing Machine
- Inoculating loop
4. Test Organism (Sample Bacteria)
5. Control Organisms
- Bacillus cereus ATCC 10876
- Escherichia coli ATCC 25922
- Streptococcus bovis ATCC 33317
- Staphylococcus aureus ATCC 25923
Procedure of Starch Hydrolysis Test
- Using a sterile inoculating loop or cotton swab, collect the sample bacteria from several fresh colonies, typically grown for about 18 to 20 hours.
- Streak the bacteria in the form of short and thick straight lines over the surface of the agar medium. Multiple bacteria can be tested on a single plate by forming multiple lines.
- Incubate the inoculated plates at 35±2°C for a minimum of 48 hours.
- After the incubation period, add a few drops of iodine solution directly over the colonies and observe for the formation of a clear halo around the colonies.
Result and Interpretation of Starch Hydrolysis Test
- A positive result is indicated by the presence of a clear halo around the colonies and the development of a dark blue to purple-blue color in the surrounding medium after the addition of Gram’s Iodine.
- A negative result is indicated by the absence of a clear halo around the colonies and the development of a dark blue to purple-blue color in the surrounding medium after the addition of Gram’s Iodine.
Starch Hydrolysis Test Results of Some Common Bacterial Pathogens
- Starch-hydrolyzing (amylase-producing) bacteria include Streptococcus bovis, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Clostridium perfringens, among others.
- Starch non-hydrolyzing (amylase non-producing) bacteria include Corynebacterium diphtheriae, Clostridium difficile, Clostridium botulinum, bile-esculin positive viridans Streptococci except Streptococcus bovis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas putida, etc.
Quality Control
- Bacillus cereus ATCC 10876 and Streptococcus bovis ATCC 33317 exhibit a positive result, indicated by the formation of a clear halo around their colonies, while the rest of the medium turns dark blue colored upon addition of Gram’s iodine.
- On the other hand, E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 display a negative result, as they do not form a clear halo around their colonies, but the surrounding medium turns dark blue after the addition of Gram’s iodine solution.
Precautions
- It's crucial to add Gram’s iodine only after 48 hours of incubation to avoid false negative results. Testing earlier may lead to inaccuracies.
- When using multiple specimens in a single plate, ensure to streak colonies apart from each other, leaving a gap of about 25 mm or more. This helps prevent cross-contamination and ensures accurate observation of individual colonies.
- Avoid using a medium containing a high amount of glucose, as bacteria may utilize glucose instead of starch, leading to false negative results.
- After adding the iodine solution, it's important to read the result quickly. The blue coloration may fade over time, so prompt observation is necessary to accurately interpret the results.
Applications of Starch Hydrolysis Test
- To distinguish between species of the Bacillus, Clostridium, Corynebacterium, Fusobacterium, Streptococcus, Enterococcus, and Pseudomonas genera.
- To differentiate Streptococcus bovis from other bile-esculin positive viridans Streptococci.
- To distinguish Chryseobacterium indologenes from Elizabethkingia meningoseptica, where the former may test positive initially while the latter may test negative.
Limitations of Starch Hydrolysis Test
- The starch hydrolysis test is not confirmatory on its own and necessitates additional biochemical test results to fully identify the isolated bacteria.
- This test typically requires a longer incubation period, usually at least 48 hours, for accurate results.
- Once Gram’s iodine is added over the medium, the bacteria cannot be utilized for further culture or other tests from the same plate, as the iodine solution may interfere with subsequent tests or cultures.
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