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SECONDARY SCREENING, ection and isolation of microorganisms, , Primary screening allows the det, industrial applications. This screening is, , that possess potentially in interesting

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60 Industrial Microbiology, , usually followed by a secondary screening to further test the capabilities of, and gain information about these organisms. The primary screening may have, yielded only a few microorganisms, or many microorganisms may have been, obtained. Probably, however, only a very small number of these organisms, will have any real commercial value, because primary screening determines, which microorganisms are able to produce a compound without providing, much idea of the production or yield potential for the organisms. In contrast,, secondary screening allows the further sorting out of those microorganisms, that have real value for industrial processes, and the discarding of those, lacking this potential. Organisms of the latter type must be discarded as soon, as possible from research studies because of the high expense of such studies,, , Secondary screening is conducted on agar plates, in flasks or small, , fermentors containing liquid media, or as a combination of these approaches., The use of agar plates, although not as sensitive as liquid culture, is of, advantage for initial secondary screening, because more information is, obtained with the expenditure of a similar amount of effort. Agar plates in, an incubator take up relatively little space and do not require the amount, of handling and work-up effort associated with liquid cultures. However,, agar plate cultures provide only a limited indication of the actual product, yield potentials among various isolates; to obtain this information, we must, employ liquid culture, because liquid culture provides a much better picture, of the nutritional, physical, and production responses of an organism to actual, fermentation production conditions., , Secondary screening can be qualitative or quantitative in its approach., The qualitative approach, for example, tells us the spectrum or range of, microorganisms which is sensitive to a newly discovered antibiotic. The, quantitative approach tells us the yields of antibiotic which can be expected, when the microorganism is grown in various differing media. However, there, is not necessarily a distinct difference between qualitative and quantitative, secondary screening. Thus, a qualitative screening for the “microbial, inhibition spectrum” of an antibiotic determines which test organisms are, sensitive to the antibiotic, but may, at the same time, yield information on the, relative sensitivities of these organisms to the antibiotic,, , Secondary screening should yield the types of information which, are needed in order to evaluate the true potential of a microorganism for, industrial usage. For example, secondary screening should determine what, types of microorganisms are involved and whether they can be classified at, least to families or genera. This information is of value, because it allows a, comparison of the newly isolated organisms with those already described, in the scientific and patent literatures as being able to produce fermentation

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.., , ening, , 61, , , , cre, $ f commercial interest. Classification of the organisms also allows, of whether they possess any pathogenicity for plants, animals, or, a pre ich would need to be considered in the handling of the organisms. In, mat whl toacertain extent, itallows a prediction of the growth characteristics, ai requirements to be considered in studying these microorganisms., a 8 sification of microorganisms often involves the expenditure of, The a rable time and effort, so that we may wish not to classify them except, coll sad groups until their distinct value as industrial prospects has been, into pert However, the organism should be classified as to species by, seit a patent application is filed. We do not patent a microorganism as, : put the use of a newly described microorganism does help in obtaining, , such, : , tke, apatet because it adds “novelty” or newness to the microbial process (see, f, , Chapter 15)., Secondary screening should determine whether the microorganisms are, , actually producing new chemical compounds not previously described or,, altematively, for fermentation processes that are already known, secondary, screening should determine whether a more economical process is possible., should the product be a newly discovered compound, it must be determined, ifit really has a use. Thus, patents are granted only for useful products. To, determine whether a product actually is a newly discovered, compound, we, can utilize paper, thin-layer, or other chromatographic procedures to compare, the product with known compounds. These chromatographic approaches, are of particular value in comparing new antibiotics with those previously, described. Nevertheless, regardless of whether the fermentation product is or, isnot anew compound, secondary screening should detect real differences in, product yield potentials among the various isolates. The organisms are grown, for various periods of time and on various media in liquid culture so that, quantitative assays may be performed. These studies involve much work and,, ifpossible, théy should not be made until agar-plate procedures have allowed, the discarding of worthless cultures., , Secondary screening should reveal whether there are pH, aeration, or, other critical requirements associated with particular microorganisms, both, forthe growth of the organism and for the formation of chemical products. The, Screening should also detect gross genetic instability in microbial cultures., Thus, a microorganism is of little value if it tends to mutate or change in, i Manner so that it loses its ability to accumulate high yields of product., , ondary screening should show whether certain medium constituents, He missing or, possibly, are toxic to the growth of the organism or to its, ability to accumulate fer é Ct 1a show thing of, the chemi tmulate fermentation products. It shou show some ing 0, , cal stability of the product, and of the product's solubility picture, , products °, diction

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62 Industrial Microbiology, in various organic solvents, It should determine whether the Product has a, simple, complex, or even a macromolecular structure, if this information is, not already available, It should show whether the product possesses physica], properties, such as ultraviolet-light absorption or fluorescence, or chemicaj, properties that can be employed to detect the compound during the use of, paper chromatography or other analytical methods, and which also might be, of value in predicting the structure of the compound., , , , , , , , , , Figure 4.7 Giant colony inhibition spectrum. The growth along the central streak is that of, a nonsporulated streptomycete. The lower group of test organisms (from left to right) and, the upper group (from right to left) are, respectively, Escherichia coli, Staphylococcus aureus,, Pseudomonas aeruginosa, Saccharomyces cerevisiae, and Bacillus cereus. As is apparent, the, antibiotic produced by the streptomycete has demonstrated activity only against the yeast., , During the secondary screening associated with certain kinds of, fermentation products, determinations should be made as to whether gross, animal, plant or human toxicity can be attributed to the fermentation product,, particularly if it is to be utilized (as are antibiotics) in disease treatment.

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|, , screening 63, , obviously, valid information for testing of this type requires that the, compound be ina pure state. However, any indications of toxicity which can, be observed early in the screening program will allow the discarding of poor, cultures, OF possibly the relegation of the compound to use only for topical, treatment. In order to test the toxicity of a compound when it is not in a, ure state, we must make an educated guess as to the types of contaminating, materials that may be associated with the compound, and then provide, able experimental controls for these contaminants in the toxicity testing., Secondary screening should reveal whether a product resulting from a, microbial fermentation occurs in the culture broth in more than one chemical, form, and whether itis an optically or biologically active material. Two or more, different compounds also can be products of a single fermentation, although, one of the compounds usually accumulates in greater quantities. Thus, one to, several intermediate compounds in the metabolic sequence leading to product, formation, as well as completely unrelated compounds, may also accumulate, in the fermentation broth. These additional major or minor products are of, distinct interest, since their recovery and sale as by-products can markedly, improve the economic position of the prime fermentation., , Secondary screening should reveal whether microorganisms are able, to chemically alter or even destroy their own fermentation products. The, microorganisms may, because of a high-level accumulation of product in the, culture broth, produce adaptive enzymes that destroy the usefulness of the, product. Thus, a microorganism might produce a “racemase” enzyme that, will change the L-configuration of an amino acid product to a mixture of, the D- and L-isomers, with the D-isomer being of little biological value. A, microorganism also might respond to the accumulation of an amino acid by, adaptively producing a “decarboxylase” enzyme that removes carbon dioxide, from the molecule, leaving an organic amine., , The preceding discussion emphasizes the fact that secondary screening, can provide a broad range of information which helps in deciding which, of various microbial isolates possess possible usefulness as an industrial, organism. Also it is obvious that secondary screening helps in predicting the, approaches to be utilized in conducting further research on the microorganism, and its fermentation process. There are many possible techniques and, Procedures that can be applied in secondary screening. To illustrate their, application and the sequence of events that may occur in a study of this, type, the search for streptomyces species capable of producing new and, valuable antibiotics will be specifically considered. However, it should be, , Temembered that, while the described methods of screening relate primarily, o the study of antibiotic-producing microorganisms, similar screenmg and, , suit