Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Influence of Culturing Conditions for Optimum Antimicrobial Metabolite Production by Streptomyces Fulvoviridis

M. M. UDDIN
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh

M. N. ANWAR
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh

M. A. MANCHUR
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh

M. N. MAHMUD
Department of Microbiology, University of Chittagong, Chittagong-4331, Bangladesh

Abstract/ Executive Summary:

Chili field antagonistic actinomycetes Streptomyces fulvoviridis was isolated and tested for optimum antimicrobial metabolite production. Maximum antimicrobial metabolite production was found at temperature 35⁰C and pH 8.0 and on 4th day of incubation in shaking condition. The medium consisting of beef extract - 0.75%, peptone - 0.25%, NaCl - 1.5%, KCl - 1.0%, MgCl₂ - 1.0%, FeSO₄ - 1.0% was screened out as suitable medium for maximum antimicrobial production supplemented individually with four carbon sources of which sucrose was found as the best carbon source. The antimicrobial metabolite was found to be stable at pH and temperature up to 11.0 and 100ºC, respectively. The active agent was best extracted with chloroform. The antimicrobial spectrum of the metabolite was wide and shows activity against - Shigella dysenteriae (AE 14612), Shigella sonnei (CRL, ICDDR, B), Salmonella typhi (AE14296), Vibrio cholerae (AE14748), Pseudomonas aeruginosa (CRL, ICDDR, B), Bacillus cereus (BTCC19), Staphylococcus aureus (ATCC6538), Bacillus subtilis (BTTC17) and Bacillus megaterium (BTTC18).

Keywords: Influence, Antimicrobial metabolite, Streptomyces fulvoviridis

Location: Department of Microbiology, University of Chittagong, Chittagong

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Bio fertilizer

Objectives:

1. The present study was undertaken to find out and optimize the growth of antibiotic producing microbial isolates.

Methodology:

Media used for isolation - The media used for the isolation of antibiotic producing microorganisms were nutrient agar medium, oat meal agar medium and starch casein medium. Plating - One gram of soil sample was mixed with 100 ml of sterile water in a conical flask, shaken well with magnetic stirrer and then allowed to stand for 30 minutes for sedimentation. Necessary dilution (upto 10-5 ) was made with this mother solution. The samples (0.1 ml) were used for inoculating 15 ml molten medium at 45⁰C -50⁰C in petri plates. Then the petri plates were incubated at 37⁰C for about 4 to 5 days. Isolation - Observation was made at 24 hours intervals to detect any colony surrounded by a clear zone of inhibition. Both the organisms (inhibiting and inhibited) involved in antagonistic reaction were isolated aseptically and transferred to slant of the same medium. The inhibiting and the inhibited colonies were marked as X-organisms and Y-organisms, respectively. Primary screening - Three different media namely nutrient agar (NA) medium, starch casein agar (SCA) medium and Czapek Dox Agar (CDA) medium were used for primary screening. In each case, the melted medium was poured into petri plate and allowed to solidify. The isolate was streaked across the surface of the agar medium at the middle position of the plate and incubated at 37⁰C for growth. After growth, the test organisms were streaked perpendicularly. A space of 2-3 mm was kept between two streaks. Then the plates were incubated at 37⁰C for the growth of the test organisms. The plates were then observed for organisms sensitive to the metabolites produced by the isolates. A clear zone of inhibition appeared against those organisms. Antibiotic assay by agar cup plate method - In performing the sensitivity spectrum analyses by agar cup plate method, nutrient agar plates were heavily seeded (2.7×103 cells/ ml) uniformly with the test organisms. Then a hole was made in media by gel cutter in sterile condition. Then one drop of melted agar was poured into hole and allowed to solidify to make a base layer. After that specific amount of culture filtrate (0.1 ml) was poured into the hole. Then plates were kept at low temperature (4ºC) for 2-4 hours to allow maximum diffusion. The plates were then incubated at 37º C for 24 hours at inverted position to allow maximum growth of the organisms. The antibacterial activity of the test agent is determined by measuring the zone of inhibition expressed in milimeter in diameter. The experiment was carried out more than once and mean of reading was recorded. Establishment of suitable culture medium - Four different media (Medium A: Beef extract 3 g, Peptone 5 g, NaCl 5 g, Dist. Water 1000 ml; Medium B :Yeast extract 0.2%, Glucose 1.0%, K₂HPO₄ 0.1%, NaCl 0.5%; Medium C : Beef extract 0.75%, Peptone 0.25%, NaCl 1.5%, KCl 1.0%, MgCl₂ 1.0%, FeSO₄ 1.0%; Medium D: Corn steep liquor 0.2%, Glucose 1.0%, K₂HPO₄ 0.1%, NaCl 0.5%) were prepared for antibiotic production. The pH of media was adjusted at 7.0. 150ml of the medium was taken in each 500ml conical flask. Each type of the medium was prepared in duplicate. Then the sterile media were inoculated with the specific organisms at 10% inoculum potential (15ml) and incubated at 37⁰C. The product was assayed by agar cup method at an interval of three days and continued up to six days. Isolation of antimicrobial agent - The isolation of the antibiotic from the culture broth was achieved by extraction with an organic solvent not completely miscible with water. In this case the solubility of the antibiotic in different organic solvent was first investigated in order to find out a suitable solvent. The solvents used were petroleum ether, nbutanol, chloroform and benzene. For solvent - solvent partitioning of product the fermented product was gently shaken in a separating funnel with almost equal volume of pure petroleum ether, which is immiscible with aqueous alcohol. The mixture was then kept for several minutes for separating of the organic layer from the aqueous phase. The materials of the crude extract were partitioned between the two phases depending on their affinity towards their respective solvents. The organic layer was separated and collected in a conical flask and the process was repeated thrice. After collection of organic phase, the aqueous phase thus obtained was further extracted with other organic solvents like as n-butanol, chloroform and benzene in the same way usually of increasing polarity. Finally all the fractions were collected separately and dried and tested their anti-microbial efficacy. Stability at various temperatures - Twenty five ml of potent culture filtrate was taken in a 50 ml conical flask in duplicates and heated in water bath at different temperature (60⁰C, 70⁰C, 80⁰C, 90⁰C, 100⁰C and autoclave temperature 121⁰C) for half an hour. Then the antibiotic was extracted with the best solvent (chloroform) and assayed by agar cup plate method.

Source of Information: The Chittagong Univ. J. B. Sci.,Vol. 5(1 &2):63-75 , 2010

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

On the basis of their better antimicrobial activity against 10 pathogenic test organisms in three different media, the actinomycetes isolate MU9 was finally selected for detailed study. The sensitivity spectrum analyses test on nutrient agar medium revealed that the antimicrobial agent produced by the isolate MU9 was active strongly against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Shigella sonnei, and weakly active against Shigella dysenteriae, Vibrio cholerae, Pseudomonas aeruginosa, Bacillus megaterium and Salmonella typhi and inactive against Escherichia coli.

Knowledge Management: Journal