Secondary Screening

3.2. Secondary Screening

Primary screening (or preliminary screening) solely enables not only the ‘detection’, but also the ‘isolation’ of such viable microorganisms that essentially possess potentially interesting and com- mercially feasible applications. Nevertheless, this screening is invariably followed by a secondary screen- ing so as to ascertain more useful information about these organisms, besides their actual inherent capabilities.

It is, however, pertinent to state here that the primary screening establishes exclusively the capa- bility of microorganisms that are responsible for producing a compound without giving enough idea either with respect to the yield or production potential for the organisms. On the contrary, the secondary screening categorically enables the further ‘sorting out’ of those specific microorganisms that essen-

tially possess the ‘real inherent value’ for feasible and gainful industrial processes ; and distinctly elimi- nating those devoid of such a potential.

3.2.1. Methodology

The various steps involved are as follows : (1) Secondary screening is usually carried out on agar plates aseptically. (2) It may also be conducted in flasks or small fermentors containing liquid media, or as a

combination of such available procedures. (3) However, one may use ‘liquid culture’ as an alternative to ‘agar plate’ in the ‘secondary

screening’ method. Following are some of the important merits and demerits of these two techniques stated briefly :

S.No. Agar Plate Method Liquid Culture Method

1. It is not so sensitive.

It is very sensitive.

2. More information is obtained. Relatively provides less information.

3. Usually occupies much lesser space in an Occupies relatively larger space in an incu- incubator ; besides, does not require enough bator, and also requires sufficient procedural degree of handling and work up effort.

details.

* An organisms used as an indicator for the presence of specific antibiotic activity.

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4. It essentially provides only a restricted It distinctly provides a much vivid idea with knowledge with respect to the actual

regard to the physical, nutritional and produc- product yield potentials amongst the various tion responses of an organism in comparison isolates obtained.

to the actual fermentation production

parameters.

3.2.2. Salient Features of Secondary Screening

The various vital and important salient features of secondary screening are enumerated below : (1) It may be either qualitative or quantitative in its approach.

(a) Qualitative Approach : Provides valuable information(s) with regard to the wide spectrum or ranges of microorganisms that is significantly sensitive to an altogether

newly discovered antibiotic. (b) Quantitative Approach : Gives authentic information(s) with regard to the specific

yields of ‘antibiotic substance’ that may be expected when the microorganisms is allowed to grow in various media having varying composition.

Special Notes :

(i) There exists no clear cut and necessarily a distinct difference between the qualitative and the quantitative secondary screening.

(ii) In reality, a qualitative screening for the ensuing ‘microbial inhibition spectrm’ of an antibiotic essentially gives an ample idea with respect to the ‘test organisms’ that are found to be sensitive to the antibiotic, whereas it may provide information pertaining to the comparative prevailing sensitivities of these organisms to the respective antibiotic.

(2) It invariably promulgates a wide spectrum of highly valuable and authentic information(s) that are very much needed in order to evaluate the precise and actual (true) potential of a particular microorganisms for industrial application.

Example : It must determine as well as establish the types of microorganisms that are involved, in addition to the fact whether these may be classified at least to various genera or families.

Note : The aforesaid information is of immense value, because it broadly makes possible

a befitting comparison between the newly isolated organisms with those already reported either in the scientific journals or in the patent* literatures showing a adequate logical evidence to produce fermentation products of commercial value and interest.

(3) Classification of Organisms : Interestingly, classification of the organisms due to secondary screening certainly makes a room for the much needed prediction of whether they (organisms) do own any genuine pathogenicity for humans, animals or plants that would necessarily warrant special precautions in the handling of such organsisms. Besides, it gives a probable prediction of the growth characteristic features in the intensive studies of these microorganisms.

(4) Establishing a More Economically Variable Process : The secondary screening must pro- vide adequate information(s) with respect to the fact whether the microorganisms (isolated

* A newly discovered microorganisms invariably helps in obtaining a ‘patent’ as it predominantly adds ‘nov- elty’ or newness to the microbial process.

ANTIBIOTICS

and identified) are virtually giving rise to ‘newer chemical entities’ not reported earlier or, alternatively, for such fermentation procedures which are already reported.

Highlights : A few highlights are as given below : (i) In case, the resulting product happens to be an altogether newly discovered compound

then an attempt must be made to establish its real genuine usage. (ii) In actual practice, the patents are usually granted for exclusively new as well as useful

products*. (5) Real Differences in Product Yield Potentials : The secondary screening must be able to

detect the real differences in product yield potentials amongst the various isolates irrespec- tive of the fact whether the outcome of the ‘ultimate fermentation product’ is or is not a new chemical entity (i.e., compound). Therefore, in order to save on valuable resources, such as : manpower, time, energy and money, it is almost mandatory to allow the organisms to grow upon different media in liquid-culture for various length of time so as to accomplish quantitative assays effectively. Nevertheless, the aforesaid studies may be carried out only after the complete rejection (or elimination) or useless cultures previously ascertained by agar-plate procedures.

(6) Critical Requirements for Specific Microorganisms : The secondary screening should articulately reveal whether the two cardinal phenomena, namely : (a) growth of the organism ; and (b) formation of chemical products, are dependent on pH, aeration or other critical re- quirements associated with specific microorganisms. Importantly, it must also detect ‘gross genetic instability’ present in various microbial cultures.

Therefore, secondary screening essentially expatiates the following relevant facts : • a microorganisms is of practically little importance if it takes care of to either mutate or

change in some manner thereby drastically losing its inheret ability to accumulate high yields of product.

• it must reveal evidently whether some ‘medium constituents’ are either missing or perhaps prove to be too toxic to the growth of the organism or to its in-built capacity to accumulate fermentation products.

• it should exhibit the chemical stability profile of the product ; and consequently, the prod- uct’s solubility profile in different organic solvents.

• it must adequately establish whether the resulting product bears a simple, complex or rather

a macro-molecular structure. • it should demonstrate explicitely whether the ‘ultimate isolated product’ possesses either

typical physical characteristic properties, for instance : UV-absorption, fluorescence ; or typical chemical characteristic properties which may be judiciously exploited to detect the compound via various sophisticated analytical techniques or by the aid of elaborated paper chromatographic studies, and which ultimately be of immense value is predicting and as- signing the most probable chemical structure of the ‘compound’.

• adequate determinations must be made so as to ascertain whether gross human, animal or plant toxicity can be attributed to the fermentation product(s) obtained specifically during

* It is usually ascertained by the help of paper, thin-layer or high performance thin layer chromatogrophic

procedures so as to compare the newly discovered compound with the known reference compounds.

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the secondary screening, in case it is be utilized (e.g., antibiotics) exclusively in the treat- ment of human ailments. Therefore, the compound must be in its purest form so as to obtain both valid and reliable informations. Importantly, for studying the detailed toxicity tests of an impure compound, one should resort to a qualified and educated ‘guess’ with respect to

the various types of contaminating substances which may be intimately associated with the compound, and ultimately provide appropriate experimental parameters for such identified

contaminants in the ensuing toxicity testing.

• it must reveal clearly whether the ‘final product’ obtained from a microbial fermentation occurs in the culture broth in a racemic form i.e., as a mixture of optically active d- and l- forms of which one may turn out to be a biologically active material. Besides, there may also exist two or even more compounds of variant nature that could be obtained from a ‘single fermentation’. Hence, in the ultimate fermentation broth one may come across frequently one to several intermediate chemical entities in the prevailing ‘metabolic pathways’ lead- ing to product formation ; besides absolutely unrelated chemical entities. Importantly, the presence of additional minor as well as major products are of distinct interest, because their subsequent recovery and commercial value as viable by-products may substantially improve upon the economic status of the ‘prime fermentaion’.

• it should adequately ascertain the fact whether the ensuing microorganisms are capable of undergoing changes under the influences of chemical compounds or even cause destruction of their self-generated fermentation products. Thus the microorganisms may, by virtue of the relatively high-level accumulation of product present in the culture broth, generate enough adaptive enzymes that would grossly destroy the potential value and usefulness of the product.

Examples : (a) A microorganisms may be able to produce a ‘racemase enzyme’ which in turn shall alter the L-configuration of an amino acid product to an equimolar mixture of the corresponding D- and L-isomers, with the D-isomers exhibiting almost little biological value. (b) A microorganism may strategically respond to the accumulation of an amino acid by adaptively influencing the production of a ‘decarboxylase enzyme’ which would specifically

remove CO 2 from the resulting molecule, thereby rendering an organic amine at the end. • secondary screening thus may give rise to a broad-spectrum of valuable information, such

as : (i) it helps in deciding precisely which of the various microbial isolates possess probable

useful potentialities as a viable industrial organism. (ii) it immensely helps most articulately in predicting the approaches to be utilized justifi-

ably in persuing further productive, aggressive and meaningful research on the selected microorganism and its corresponding fermentation process.