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Saturday, November 20, 2010

primary screening


INTRODUCTION:-
Isolation of Microorganisms - The first step in developing a producer strain is the isolation of concerned microorganisms from their natural habitats. Alternatively, microorganisms can be obtained as pure cultures from organisation, which maintain culture collections, e.g.,
American Type Culture Collection (ATCC). Rockville, Maryland, U.S.A., Commonwealth Mycological Institute (CMI), Kew, Surrey, England, Fermentation Research Institute
(FERM), Tokyo, Japan, U.S.S.R. Research Institute for Antibiotics (RIA), Moscow, U.S.S.R., etc.The microorganisms of industrial importance are, generally, bacteria, actinomycetes, fungi and algae. These organisms occur virtually everywhere, e.g., in air, water, soil, surfaces of plants and animals, and plant and animals tissues. But most common sources of industrial microorganisms are soils, and lake and river mud.
Often the ecological habitat from which a desired microorganism is more likely to be isolated will depend on the characteristics of the product desired from it, and of process development. For example, if the objective is to isolate a source of enzymes, which can withstand high temperatures, the obvious place to look will be hot water springs. A variety of complex isolation procedures have been developed, but no single method can reveal all the microorganisms present in a sample. Many different microorganisms can be isolated by using specialized enrichment techniques, e.g., soil treatment (UV irradiation, air drying or heating at 70120°C, filtration or continuous percolation, washings from root systems, treatment with detergents or alcohols, preinoculation with toxic agents), selective inhibitors (antimetabolites, antibiotics, etc.), nutritional (specific C and N sources), variations in pH, temperature, aeration, etc.The enrichment techniques are designed for selective multiplication of only some of the microorganisms present in a sample. These approaches however take a long time (20-40 days), and require considerable labour and money.
                              The main isolation methods used routinely for isolation from soil samples are: sponging (soil directly), dilution, gradient plate, aerosol dilution, flotation, and differential centrifugation. Often these methods are used in conjunction with an enrichment technique.
Screening of Microorganisms for New Products - The next step after isolation of microorganisms is their screening. A set of highly  selective procedures, which allows the detection and isolation of microorganisms producing the desired metabolite, constitutes primary
screening.
                                                                      
SCREENING
In Microbial Technology Microorganisms holds the key to the success or failure of a fermentation process. It is therefore important to select the most suitable microorganisms to carry out the desired industrial process.
The most important factor for the success of any fermentation industry is of a production strain. It is highly desirable to use a production strain possessing the following four characteristics:
It should be high-yielding strain.
It should have stable biochemical/ genetical characteristics.
It should not produce undesirable substances.
It should be easily cultivated on large-scale.

Def:
Detection and isolation of high-yielding species form the natural sources material, such as soil, containing a heterogeneous microbial population is called Screening
OR
Screening may be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from among a large microbial population.

Thus to be effective, screening must, in one or a few steps allow the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the small percentage of useful microorganisms that are present in the population.
The concept of screening will be illustrated by citing specific examples of screening procedures that are or have been commonly employed in industrial research programs.
During screening programs except crowded plate technique a natural source such as soil is diluted to provide a cell concentration such that aliquots spread, sprayed or applied in some manner to the surface of the agar plates will yield well isolated colonies (30-300).

Primary screening of Organic acid/ amine producer:-
· For primary screening of organic acid or organic amine producers, soil sample is taken as a source of microorganism.
· It is diluted serially to an extent to get well-isolated colonies on the plate when spread or applied in some form.
· After preparation of dilution these dilutions are applied on a media incorporated with a pH indicating dye such as Neutral red (Pink to yellow)or Bromothymol blue (Yellow -blue), into a poorly buffered agar nutrient medium. The production of these compounds is indicated by a change in the color of the indicating dye in the close vicinity of the colony to a color representing an acidic or alkaline reaction.
 Fig.detection of fungi capable of producing organic acids by incorporation of CaCo3 into the agar medium.

· The usefulness of this procedure is increased if media of greater buffer capacity are utilized so that only those microorganisms that produce considerable quantities of the acid or amine can induce changes in the color of the dye.
An alternative procedure for detecting organic acid production involves the incorporation of calcium carbonate (1-2 %) in the medium so that organic acid production is indicated by a cleared zone of dissolved calcium carbonate around the colony. These procedures are not foolproof, however, since inorganic acids or bases also are potential products of microbial growth. For instance, if the nitrogen source of the medium is the nitrogen of ammonium sulfate the organism may utilize the ammonium ion, leaving behind the sulfate ion as sulfuric acid, a condition indistinguishable form organic acid production. Thus cultures yielding positive reactions require further testing to be sure that an organic acid or base actually has been produced.


Primary screening of antibiotic producer (Crowded plate technique):
Fig.cwowded plate screening for antibiotic producing microorganisms .Notice the  inhabition of growth to several of the colonies.

· The crowded plate technique is the simplest screening technique employed in detecting and isolating antibiotic producers.
· It consists of preparing a series of dilution of the source material for the antibiotic producing microorganisms, followed by spreading the dilution on the agar plates.
· The agar plates having 300- 400 or more colonies per plate after incubation for 2-4 days are observed since they are helpful in locating the colonies producing antibiotic activity.
· Colonies showing antibiotic activity is indicated by the presence of a zone of inhibition (arrow in fig) surrounding the colony.
· Such a colony is sub- cultured to a similar medium and purified.
· It is necessary to carry on further testing to confirm the antibiotic activity associated with a microorganism since zone of inhibition surrounding the colony may sometimes be due to other causes. Notable among these are a marked change in the pH value of the medium resulting from the metabolism of the colony, or rapid utilization of critical nutrients in the immediate vicinity of the colony.
· Thus, further testing again is required to prove that the inhibitory activity associated with a microorganism can really be attributed to the presence of an antibiotic.
The crowded plate technique has limited application, since usually we are interested in finding a microorganism producing antibiotic activity against specific microorganism and not against the unknown microorganism that were by chance on the plate in the vicinity of an antibiotic producing organism. Antibiotic screening is improved, therefore by the incorporation into the procedure of a “Test organism” that is an organism used as an indicator for the presence of specific antibiotic activity.
Dilutions of soil or of other microbial sources are applied to the surface of agar plates so that well isolated colonies will develop. The plates are incubated until the colonies are a few millimeters in diameter and so that antibiotic production will have occurred for those organisms having this potential. A suspension of test organism is then sprayed or applied in some manner to the surface of the agar and the plates are further incubated to allow growth of the test organism. Antibiotic activity is indicated by zones of inhibited growth of the organism around antibiotic producing colonies. In addition a rough approximation of the relative amount of antibiotic produced by barious colonies can be gained by measuring in mm the diameters of the zones of inhibited test organism growth. Antibiotic producing colonies again must be isolated and purified before further testing.

Primary screening of growth factor (Amino acid/ Vit) producer (Auxanography):

This technique is largely employed for detecting microorganisms able to produce growth factors (eg. Amino acid and Vitamins) extracellularly. The two major steps are as follows:
Step I
A filter paper strip is kept across the bottom of a petri dish in such a way that the two ends pass over the edge of the dish.
A filter paper disc of petri dish size is placed over paper strip on the bottom of the plate.
The nutrient agar is poured on the paper disc in the dish and allowed to solidify.
Microbial source material such as soil, is subjected to dilution such that aliquots on plating will produce well isolated colonies.
Plating of aliquots of properly diluted soil sample is done.
Step II
A minimal medium lacking the growth factor under consideration is seeded with the test organism.
The seeded medium is poured on the surface of a fresh petri dish and allowed to solidify.
The agar in the first plate as prepared in step- I is carefully and aseptically lifted out with the help of tweezers and a spatula and placed without inverting on the surface of the second plate as prepared in the second step.
The growth factor(s) produced by colonies present on the surface of the first layer of agar can diffuse into the lower layer of agar containing the test organism. The zone of stimulated growth of the test organism around the colonies is an indication that they produce growth factor(s) extracellularly. Productive colonies are sub cultured and are further tested.
OR
A similar screening approach can be used to find microorganisms capable of synthesizing extracellular vitamins, amino acids or other metabolites. However, the medium at makeup must be totally lacking in the metabolite under consideration. Again the microbial source is diluted and plated to provide well-isolated colonies and the test organism is applied to the plates before further incubation. The choice of the particular test organism to be used is critical. It must possess a definite growth requirement for the particular metabolite and for that metabolite only, so that production of this compound will be indicated by zones of growth or at least increased growth of the test organism adjacent to colonies that have produced the metabolite.

Enrichment culture technique:
This technique was designed by a soul microbiologist, Beijerinck, to isolate the desired microorganisms form a heterogeneous microbial population present in soil. Either medium or incubation conditions are adjusted so as to favour the growth of the desired microorganism. On the other hand, unwanted microbes are eliminated or develop poorly since they do not find suitable growth conditions in the newly created environment. Today this technique has become a valuable tool in many screening program for isolating industrially important strains.
 
Refrences:-

Ø  INDUSTRIAL MICROBIOLOGY. By ah Rose. Butterworths,
Ø  Industrial microbiology General account :maintenance of stock cultures, ... Industrial Microbiology - Casida, Wiley Eastern publishers, 1994. ...
Ø  Industrial Microbiology. Wiley Eastern Ltd. New Delhi. Cruckshank R, Dugnid jp .... Industrial Microbiology. Mac Millan Pub. Ltd. Wisconsin. ...

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