Streptomyce Lab Report

Isolation of Streptomyces sp

The soil samples were collected from various agricultural areas which had been serially diluted up to 10-5. The spread plate technique was followed and incubated at 28°C for 7 days. The Streptomyces sp isolates were maintained on starch casein agar slants.

Screening for antimicrobial activity by well diffusion method

The Streptomyces isolates were grown in starch casein broth at 28ºC for 10 days and the activity were observed by well diffusion method. The culture filtrate was loaded into the wells which had been inoculated with test organism, S.aureus, K.pneumonia, P.aeruginosa and E.coli and incubated for overnight and measured the zone of inhibition.

Extraction of the secondary metabolites from culture supernatant

Chitosan was suspended in 1% (v/v) acetic acid to attain a 0.3% (w/v) chitosan solution. Sodium tripolyphosphate (TPP) was dissolved in Millipore water at the concentration of 0.1% (w/v). Chitosan control nanoparticles were obtained by the 1ml of TPP solution was poured drop wise to the 5ml chitosan solution under magnetic stirring at 400rpm for 15 min. The formation of nanoparticles was a result of the interaction between the negative charge of TPP and the positive charge of amino groups of chitosan. The bioactive compound loaded chitosan nanocomposites (BCN) were obtained by the addition of a MIC value of the bioactive compound extract to the chitosan contol nanoparticles before centrifugation and constant stirred for another 45 min. Then the mixture of the solution was treated with sonication for 3min. The nanocomposites were separated by centifugation at 10,000rpm for 40 min. Nanocomposites were frequently rinsed with Millipore water. After centrifugation the chitosan nanocomposites were freeze dried by freeze dry system at -70˚C and used for further analysis (Ali et al., 2010).

Nanoparticles were coated on membrane

The synthesized chitosan loaded nanoparticles (CN) and bioactive compound loaded chitosan nanocomposites (BCN) were coated on 0.4 micron membrane by dipping method. The membrane was placed in synthesized nanoparticles solution and kept in

The diverse mode of vibrations were identified and consigned to conclude the functional groups in the sample.

Morphology study of the nanoparticles

The morphological characteristics (size and shape) of the nanoparticles were examined using a Scanning Electron Microscope (SEM) machine (Phillips C M 12). The sample was placed on a sample holder followed by coating with a conductive metal such as copper, using a sputter coater. The sample is then scanned with a focused fine beam of electrons (Jores et al., 2004).

Membrane filtration technique

The membrane filtration techniques provide a direct count of total coliforms and faecal coliforms in the water sample. This test was carried out in a flow through membrane filtration system on a CN coated membrane, BCN coated membrane and nanoparticles (NCM) non-coated membrane in a sterile condition. . Bacteria are retained on the surface of the membrane which is placed on a suitable selective medium in a sterile container and incubated at an appropriate temperature. If coliforms and/or faecal coliforms are present in the water sample, characteristic colonies form that can be counted directly. The water sample was collected in a sterile container for further analysis. It is clear that the permeability of nanoparticles coated membrane has slightly reduced contrast to non