Starch is the reserved meals materials of most vegetation and is among the plentiful carbohydrates in nature. Starch is a polysaccharide made from two glucose polymers –amylose and amylopectin. Being a big macromolecule, starch can’t be utilized in its native state by microorganisms; therefore it have to be damaged down into glucose earlier than metabolism. Bacterial extracellular amylase enzyme can hydrolyze starch into maltose and glucose. Nonetheless, not all micro organism are able to producing amylase enzymes.
The starch hydrolysis check, often known as the amylase check, is a biochemical check that’s used to find out the power of micro organism (microorganisms) to supply amylase and make the most of starch as a carbon supply. It’s generally used to distinguish species of Bacillus, Clostridium, Corynebacterium, Fusobacterium, Streptococcus, Enterococcus, and Pseudomonas genus.
Aims of Starch Hydrolysis Take a look at
- To find out the power of micro organism to supply amylase enzyme
- To find out the power of micro organism to hydrolyze starch
- To distinguish and establish micro organism primarily based on their skill to hydrolyze starch
Precept of Starch Hydrolysis Take a look at
- Micro organism able to producing extracellular amylase enzymes can hydrolyze the starch and produce smaller water-soluble sugar molecules like glucose, maltose, and dextrose. If the micro organism should not capable of secret the amylase enzyme, there received’t be hydrolysis of starch.
- If the inoculated micro organism produce amylase, the starch molecules across the colonies can be hydrolyzed and there received’t be amylose or starch molecules. Therefore, upon the addition of iodine resolution, there received’t be the formation of darkish blue colour across the colonies.
- If the inoculated micro organism produce amylase, starch molecules across the bacterial colony can be hydrolyzed; so there received’t be colour improvement within the medium surrounding the colony upon the addition of iodine resolution.
- Thus, hydrolysis of starch could be recognized by the formation of a transparent zone of halo across the bacterial development after the addition of iodine resolution within the inoculated and incubated starch medium.
Requirement for Starch Hydrolysis Take a look at
1. Tradition Media
Mueller Hinton Agar (MHA), Starch Agar, and Coronary heart Infusion Agar with 2% Starch are generally used for the detection of starch hydrolysis.
Composition of Mueller Hinton Agar per 1000 mL
HM Infusion B type (Beef Infusion) 300.00 grams
Acicase (Casein Acid Hydrolysate) 17.500 grams
Starch 1.5000 grams
Agar 17.000 grams
Closing pH 7.3 0.1 at 250C
Reference: SM173.pdf (himedialabs.com)
Preparation of MH Agar
- Measure the suitable quantity of MHA media powder (38.0 grams per 1000 mL) and dissolve it within the water of the required quantity in a conical flask (or glass bottle).
- Stir properly in a magnetic stirrer or manually and warmth to boil to utterly dissolve the media powder.
- Autoclave the medium at 121°C and 15 lbs. stress for quarter-hour.
- When the medium is cooled to round 40 to 45°C, in a sterile petri dish (of 10 cm diameter) dispense about 20 to 25 mL of the medium and let it 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
Closing pH- 7.2 0.1 at 25°C
Reference: M107S.pdf (himedialabs.com)
Preparation of Starch Agar
- Measure the suitable quantity of Starch Agar media powder (25.0 grams per 1000 mL) and dissolve it within the water of the required quantity in a conical flask (or glass bottle).
- Stir properly in a magnetic stirrer or manually and warmth to boil to utterly dissolve the media powder.
- Autoclave the medium at 121°C and 15 lbs. stress for quarter-hour.
- When the medium is cooled to round 40 to 45°C, in a sterile petri dish (of 10 cm diameter) dispense about 20 to 25 mL of the medium and let it solidify.
2. Reagents
Gram’s Iodine Answer is required for the detection of starch hydrolysis.
Preparation of Gram’s Iodine Answer:
- Dissolve 25 grams of Iodine Crystal and 50 grams of potassium iodide in 500 mL of distilled water to make Lugol’s iodine inventory resolution.
- Dissolve 5 grams of sodium bicarbonate (NaHCO3) in 100 mL of distilled water to make 5% sodium bicarbonate resolution.
- Combine 60 mL of Lugol’s iodine resolution and 60 mL of 5% sodium bicarbonate resolution with 220 mL of distilled water to make Gram’s Iodine Answer.
(Reference: Leber, Amy L., editor in chief. (2016). Scientific microbiology procedures handbook (Fourth version). Washington, DC: ASM Press 1752 N St., N.W., [2016] doi:10.1128/9781555818814.ch3.17.33. Appendix 3.2.1-1; Preparation of Gram Stain Reagents)
Alternatively:
- Dissolve 1.0 grams of iodine and a pair of.0 mL gram of potassium iodide in 300 mL of distilled water.
(Reference: S013.pdf (himedialabs.com))
3. Tools
4. Take a look at Organism (Pattern Micro organism)
5. Management Organisms
Bacillus cereus ATCC – 10876
Escherichia coli ATCC – 25922
Streptococcus bovis ATCC – 33317
Staphylococcus aureus ATCC – 25923
Process of Starch Hydrolysis Take a look at
- Utilizing a sterile inoculating loop (or cotton swab) decide up the pattern micro organism from a number of recent colonies (of about 18 to twenty hours).
- Streak the micro organism within the type of quick and thick straight strains over the floor of the micro organism. (A number of micro organism could be examined in a single plate forming a number of strains.)
- Incubate at 35±2°C for no less than 48 hours.
- Following incubation, add a couple of drops of iodine resolution straight over the colonies and observe for the formation of a transparent halo across the colonies.
End result and Interpretation of Starch Hydrolysis Take a look at
- A optimistic outcome is indicated by the formation of a transparent halo across the colonies and the event of darkish blue to purple-blue colour within the surrounding medium after the addition of Gram’s Iodine.
- A unfavorable outcome is indicated by no clear halo across the colonies and the event of darkish blue to purple-blue colour within the surrounding medium after the addition of Gram’s Iodine.
Starch Hydrolysis Take a look at Outcomes of Some Frequent Bacterial Pathogens
- Starch hydrolyzing (amylase producing) micro organism: S. bovis, Bacillus subtilis, B. cereus, B. megaterium, Clostridium perfringens, and many others.
- Starch non-hydrolyzing (amylase non-producing) micro organism: Corynebacterium diphtheria, Clostridium difficile, C. botulinum, bile-esculin optimistic viridans Streptococci besides S. bovis, E. coli, S. aureus, Pseudomonas aeruginosa, P. putida, and many others.
High quality Management
- Bacillus cereus ATCC – 10876 and Streptococcus bovis ATCC – 33317 provides optimistic outcome i.e. formation of a transparent halo round their colonies is noticed after the addition of the Gram’s iodine whereas the remainder of the medium turns darkish blue coloured.
- E. coli ATCC – 25922 and Staphylococcus aureus ATCC – 25923 provides unfavorable outcome i.e. don’t type a transparent halo round their colonies however the surrounding medium turns darkish blue after the addition of the Gram’s iodine resolution.
Precautions
- Add Gram’s iodine solely after 48 hours of incubation. Testing earlier could give a false unfavorable outcome.
- Whereas utilizing a number of specimens in a single plate, be certain that to streak colonies other than one another making a niche of about 25 mm or extra.
- Don’t use a medium containing a excessive quantity of glucose as a result of micro organism could use glucose as an alternative of starch and provides false unfavorable outcomes.
- Learn the outcome rapidly after the addition of iodine resolution as a result of the blue colour could fade as time passes.
Purposes of Starch Hydrolysis Take a look at
- To distinguish species of Bacillus, Clostridium, Corynebacterium, Fusobacterium, Streptococcus, Enterococcus, and Pseudomonas genus.
- To separate S. bovis from different Bile-esculin optimistic viridans Streptococci.
- To separate Chryseobacetrium indologenes from Elizabethkinga meningoseptica; earlier optimistic whereas later unfavorable.
Limitations of Starch Hydrolysis Take a look at
- It’s not a confirmatory check and therefore requires different biochemical check outcomes to utterly establish remoted micro organism.
- It requires an extended time interval (no less than 48 hours).
- As soon as Gram’s iodine is added over the medium, micro organism can’t be used for additional tradition or different exams from the plate.
References
- Leber, Amy L., editor in chief. (2016). Scientific microbiology procedures handbook (Fourth version) . Washington, DC : ASM Press 1752 N St., N.W., [2016] doi:10.1128/9781555818814.ch3.17.33
- Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth version.) P. 185 – 187. St. Louis, Missouri: Elsevier
- https://microbiologyinfo.com/starch-hydrolysis-test/
- https://www.uwyo.edu/molb2021/additional_info/summ_biochem/starch.html
- https://microbeonline.com/starch-hydrolysis-test/
- https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01percent3A_Labs/1.17percent3A_Starch_Hydrolysis
- https://microbiologynote.com/starch-hydrolysis-test/
- https://www.narajolerajcollege.ac.in/doc/sub_page/20210610_233752.pdf
- https://asm.org/ASM/media/Protocol-Photos/Starch-Agar-Protocol.pdf?ext=.pdf
- https://www.deshbandhucollege.ac.in/pdf/assets/1586748276_BT(H)-VI-IEM-Hydrolysis_of_starch_by_microorganisms.pdf
- https://www.sas.upenn.edu/LabManuals/biol275/Table_of_Contents_files/21-DiagnosticTests.pdf
- S013.pdf (himedialabs.com))
- M107S.pdf (himedialabs.com)
- SM173.pdf (himedialabs.com)
