Practical: Isolation and Purification of Actinomycetes Species for the Production of Antibiotic Metabolites
Aim:
A) To perform crowded plate method for the isolation of antibiotic producing actinomycetes Species.
B) To isolate purify and test the actinomyces Species for the production of antibiotic metabolites.
Theory:
Actinomycetes are ubiquitous prokaryotic Gram-positive bacteria that constitute one of the largest bacterial phyla with Characteristic Filamentous morphology and high G+C DNA. They are classified as bacteria. but are unique enough to be termed as filamentous bacteria. They belong to order of Actinomycetales, whose phylum is Bacterota one of the largest taxonomic units within the bacteria domain. Actinomycete numbers are generally one to two orders of magnitude smaller than total bacterial population. Morphologically, actinomycetes resemble fungi because of their elongated cells that branch into Filamentos or hyphae. These hyphae can be. distinguished from fungal hyphae from on the basis of size with actinomycete hyphae much smaller than Fungal hyphae. The mycellium in some species may break apart to form rod or cocoid-shaped forms. Although orginally recognized as soil microorganism . it is nου being recognized that actinomycetes isolated from water are also important specifically, marine actinomycetes have been shown to posses novel seconddary metabolites that add a new dimension to microbial natural products.
Requirements:
1. Water Sample
2. Nutrent Agar
3. Actinomycetes agar
4. Muller Hinton agar
5. Gentamycin disc (15ug/ml)
6. Pure E.coli Culture
7. Pure Staphyllococcus aureus Culture
Procedure:
- 1 ml of water sample was taken and Senary diluted upto 10-210-2 using distilled water as a dilucent
- 10-2 dilution was further spread on Nutrient agar and allowed to incubate at room temperature for 3 days.
- After an incubation at 3 days the colonies showing visible zone clearance were streaked on Actinomycetes agar.
- The plates were allowed to incubate for 7 days further pure culture of actinomycetes was used for testing secondary antagonistic product formation using actinomycetes broth medium.
- Also the gram Staining and negative staining were done for microscopic characterisation of isolated actinomycetes.
- After an incubation period of to days the actinomycetes culture broth was centrifuged at 8000rpm for 10 minutes.
- The Supernatant was further tested for antibiotics on Muller-Hinton Agar with Standard Gentamycin (Control) and test organisms Staphylococcus aureus and E.Coli
- Next day the result were observed and recorded.
Result:
Using crowded plate method 3 colonies were observed with a clear zone of inhibition. The actinomycetes isolated was observed under microscope as gram positive long filament. with fragmented isolated spores. The filtrate obtained after growing actinomycetes produced an equivalent zone of inhibition when compared to standard Gentamycin antibiotiс
Conclusion:
The isolated actinomycetes is a patent producer of secondary metabolite which inhibited the growth of gram positive Stephalococcus aureus as well as gram negative E. Coli.