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(Screening of soil for antibiotics producing organisms), use selective…

- - - - MO that are very less number in the soil sample
        
        Possess specific nutrient requirement
        
        incorporated the required nutrients for MO growth
        
        Adjusting the incubation conditions of required MO
    - - MO capable for producing certain extracellular substance
        
        Eg. amino acids, vitamins
        
        Procedure:
        
        Grow the MO producing desire extracellular substances
        
        Collect the aliquot and spread on the surface of a sterilized agar plate
        
        Spread the suspension of test organism
        
        Incubate
        
        Increase growth of test MO :arrow_right: indicates test MO required the particular extracellular substance
        
        Prepare stock culture of MO which produce extracellular substances
    - - producing acids or amines from natural sources
        
        Incorporating certain pH indicator dyes:
        
        neutral red or bromothymol blue into nutrient agar medium
        
        change in color - the ability of that colony to produce an organic acid or base
        
        calcium carbonate incorporated into agar medium:
        
        production of inorganic acid indicated by the formation of clear zone around the colonies
    - - For detection & isolation of MO capable of utilizing carbon source from volatile substrates
        
        Volatile substrates: hydrocarbons, low molecular weight alcohols, similar carbon sources
        
        Procedures:
        
        Test MO spread on the surface of sterile agar medium containing all nutrients except carbon source
        
        required volatile substrates is applied on the lid of the petri plates
        
        incubated by placing them in an inverted position
        
        enough vapors from volatile substrate spread to the surface of agar within the closed atmosphere to provide the required specific nutrient to the MO:
        
        MO grows and form colonies by absorbing the supplemented nutrient
        
        colonies are isolated, purified and stick cultures are made
    - - Detect MO producing antibiotic
        
        Source: soil
        
        Progressive serial dilutions (10^6 or 10^7)
        
        plated on agar plate (300-400 colonies)
        
        incubation
        
        antibiotic producing activity of a colony, isolated
        
        purified - streaking on a plate containing suitable medium
        
        stock culture
        
        tested for antibiotic spectrum
        
        limitation - unknown effectiveness for desired MO
  - - - used for isolation & detection of antibiotics which diffuse through solid medium
        Example: Streptomyces
        
        It s capable of producing antibiotics during primary screening
        
        Isolated Streptomcyes culture is inoculated into the central area of a sterilized petri plates
        
        Plated are incubated until sufficient microbial growth takes place
        
        Cultures of test organism, are streaked from the edges of plate up-to but not touching the growth of Streptomyces
        
        Further incubated to allow the growth oft he test organisms
        
        Then the distance over which the growth of different test organisms is inhibited by the antibiotic secreted
        
        Streptomyces is measured in millimetres
        
        Inhibition spectrum - the relative inhibition of growth of different test organisms by the antibiotic
        
        MO that grows is inhibited to a considerable distance are considered more sensitive to the antibiotic than those organisms, which can grow close to the antibiotics
        
        Such species of Streptomyces, which have potentiality of inhibition of MO is preserved for further testing
    - - For testing those antibiotics which are poorly soluble in water or do not diffuse through solid medium
        Example: Streptomyces
        
        Streptomyces is grown in a broth
        
        its mycelium is separated by filtration to get culture filtrate
        
        Various dilutions of antibiotic filtrates are prepared and added to molten agar plating medium and allowed to solidify
        
        Later on cultures of various test organisms are streaked on parallel lines on the solidified medium and such plates are incubated
        
        The inhibitory effect of antibiotic agent against the test organism is measured by their degree of growth in different antibiotic dilutions
    - - For further screening to determine the exact amount of antibiotic produced by a MO
        Example: Streptomyces
        
        Erlenmyer conical flask containing highly nutritive medium are inoculated with Streptomyces
        
        Incubated at room temperature
        
        Also aerated by shaking continuously and vigorously during incubation period to allow Streptomyces to produce the antibiotic in an optimum quantity