Screening in Microbiology

SCREENING IN MICROBIOLOGY

It may be defined as use of highly selective procedure which allowed the detection and isolation of only those microorganisms of interest from large population it is of two types

Primary screening

In the primary screening ,we are interested in isolating the microorganism with desirable characteristics but there is no detail analysis ,means the microbes are reacted only on the basis of qualitative analysis.

Primary screening has to be done for 2 purposes

• to discard value less microorganism.

• To select desirable microbes.

Method of screening:

It is mainly done in following ways:

1 Take any natural microbial source, suppose soil is diluted to provide concentration such that aliquots are spread or spray or applied to agar plates. The spread plating technique is mainly used for the soil dilutions in which 0.1 to 0.2 ml portion of soil dilution is applied to agar surface with the help of pipette or spread this dilution with the help of glass rod or spreader. This glass rod or spreader is sterilised by heating or dipping it in alcohol and burning off before spreading.Then this plate is settled on spreader wheel which allow more even spreading of sample.

Examples:

1 Screening of organic acids and amines producers

The microorganism which produces organic acid and amines from belly carbon substrate are detected by incorporation of pH indicating dyes such as neutral red or bromothypol blue into poorly buffered nutrient agar medium. The production of these organic acids is indicated by change in the colour of dye.

Drawbacks

• it does not tell how much organic acid is produced

• it does not tell which type of organic acid is produced

• This method is only applicable to poorly buffered medium because if medium is highly buffered then change in colour is poor.

2 An alternating procedure for detecting organic acid production

It involves the incorporation of calcium carbonate in the medium so that organic acid production is indicated by a clear zone of dissolved calcium carbonate around the colony

Drawbacks:

These procedures are however not a tool for improvement if nitrogen source is nitrogen or ammonium sulfate .The organism in this case may utilise ammonium ions leaving behind the sulfate ions as hydrogen sulfate.A condition indistinguishable from organic acid production occur. thus microbe giving positive reactions require further testing.

Selection for antibiotic production

This screening approach has been employed extensively in search of microbes capable of producing useful antibiotics

Method:

Crowded plate: In this method ,pour the media in petri plates allowed it to solidify and then take the soil solution and make dilute, turn off this stock solution and spread this on agar media to get 20 to 300 colonies . Now antibiotic activity is indicated by zone of inhibited growth of microbes around antibiotic producing colonies

Drawbacks

• We do not know the inhibition spectrum of the isolated microbes.

• The lack of growth may also due to the reason that large collimate of the colony get draw all nutrients ,and other colonies remain off not getting nutrients

• The crowded plate technique does not necessarily producing microbes because the inhibition area along the colony sometimes may be due to change in nutrient or limited supply of nutrients

Improved method

The choice of test organism is the improved method over crowded plate technique . Test organism is an organism used as indicator for the presence of specific antibiotic activity

Procedure

Dilutions of soil are applied to the surface of agar plate so that the isolated colonies will develop roughly 30 to 200 colonies per plate. A suspension of test organism is then sprayed over the surface of agar and the plates are further incubated to allow the growth of test organism . Antibiotic activity is indicated by a zone of inhibited growth of test organism around the antibiotic producing colonies

Examples

Selection of microorganism capable of producing some metabolite , vitamins, amino acids or hormones

We will choose a test organism and we will put the soil suspension on the medium. Then incubate this to get the colony , now we spray the test organism over the colonies if the colonies will produce some vitamins or metabolites then the test organism will grow around the colonies and there will be increased growth around the colonies. And if the colonies producing vitamins and there will be no growth of the bacterial colonies then there will be no growth around the colonies occur.

Drawback

We do not know the amount of vitamin or metabolite produced

To find selection of microorganism capable of utilising a specific carbon or nitrogen source

let us take a source of carbon such as mannitol, then prepared a medium and add this carbon source to the medium. Now spread the diluted cell solution over the medium and incubate this for growth ,only those colonies will grow which can utilise the carbon from mannitol

Secondary screening

It is the detection and isolation of microorganisms which have rare commercial value .Secondary screening may be qualitative or quantitative both

The qualitative secondary screening tells us only whether the microbes producing antibiotics or not

a quantitative secondary screening tell us the yield plus spectrum of antibiotic

Advantages of secondary screening

• Secondary screening yields the information weather the microbe is of true potential value or is of industrial use or not

• It give the information whether the microorganism involved in the screening can be classified at least two families or genera.

• It shows a commercial chemical stability of the product and solubility pictured in various organic solvents

• It show whether the product has a simple complex or even a macromolecular structure

• It revealed whether the product resulting from microbial fermentation occur in culture broth in more than one chemical form or not and whether it is optically active or biologically active material

• It gives us information about whether the microbial fermentation results in some by product or not

• Secondary screening also give information about the microorganism which are chemically alter or even destroyed their own fermentation product.

The primary screening allows only the isolation of Streptomyces which at least on agar media produces substances toxic to growth of sensitive test organism . The next step is secondary screening which is to determine the inhibition spectrum for the antibiotic production by each of Streptomyces isolate i.e the range of types of test organisms sensitive to each antibiotics. The simplest procedure for this alternation is Giant colony inhibition spectrum technique . For this purpose the Streptomyces cultures are inoculated into the central area of petri plate containing nutrient agar medium or any other media or they are streaked in narrow bands across the center of plate . the plates are then incubated until growth and sporulation have occurred. Now the strain of microorganism to be tested for sensitivity of antibiotics test organism are then streaked from edges of petri plate towards center but not touching the Streptomyces growth.

Now again incubate the petri plate for the growth of test organism and observe the inhibition spectrum. The colonies of the test organism which will not grow are sensitive to antibiotic and the microorganism which will grow they are resistant to antibiotics while the test organism which will grow away from the antibiotic products producing microbes they are less sensitive or partially sensitive to anantibiotics . The only those microbes which are sensitive to antibiotics are retained for further testing.

Some antibiotics are poorly soluble in water and do not diffuse through the agar to any extent away from the Streptomyces colonies then an alternate procedure required with more effort is used for this procedure. The streptomyces is first grown in broth culture and then mycelium is separated by filtration. Various dilutions of antibiotic filtrate are then incorporated in melted agar plating media then the various test organisms are streked in parallel lines across the surface of agar plates They are incubated for the growth of test organism and then inhibition spectrum is studied ,the relative activity of antibiotic against various test organisms is then compared with the amount of dilution. The antibiotic culture filtrate is added to plating media then further screening can be carried out using these Streptomyces on agar media