Screening of Microorganisms: Primary and Secondary Techniques |

Industrial Biotechnology

In this article we will discuss about the primary and secondary screening of microorganisms.
The economics of a fermentation process largely depends upon the type of microorganism
used. If fermentation process is to yield a product at a cheaper price the chosen
microorganism should give the desired product in a predictable and economically adequate
quantity. The microorganism with a desired characters is generally isolated from natural
substrates like soil etc. Such an organism is generally called as a producer strain.

A producer strain should possess the following characters:

1. It should be able to grow on relatively cheaper substrates.

2. It should grow well in an ambient temperature preferably at 30-40°C. This reduces the
cooling costs.
3. It should yield high quantity of the end product.
4. It should possess minimum reaction time with the equipment used in a fermentation
process.
5. It should possess stable biochemical characteristics.
6. It should yield only the desired substance without producing undesirable substances.
7. It should possess optimum growth rate so that it can be easily cultivated on a large scale.

Detection and isolation of a microorganism from a natural environment like soil containing
large number of microbial population is called as screening. It is very time consuming and
expensive process. For example, Eli Lilly & Co. Ltd discovered three species of antibiotic
producing organisms in a span of 10 years and after screening 4,00,000 organisms.
Although there are many screening techniques, all of them are generally grouped into two
broad categories.

They are:

1. Primary screening, and

2. Secondary screening.

1. Primary Screening of Microorganisms:

Primary screening may be defined as detection and isolation of the desired microorganism
based on its qualitative ability to produce the desired product like antibiotic or amino acid or
an enzyme etc. In this process desired microorganism is generally isolated from a natural
environment like soil, which contains several different species. Sometimes the desired
microorganism has to be isolated from a large population of different species of
microorganisms.

The following are some of the important primary screening techniques:

(i) The crowded plate technique

(ii) Indicator dye technique

(iii) Enrichment culture technique

(iv) Auxanographic technique

(v) Technique of supplementing volatile and organic substrates.

(i) The Crowded Plate Technique:

This technique is primarily employed for detecting those microorganisms, which are capable
of producing antibiotics. This technique starts with the selection of a natural substratum like
soil or other source consisting of microorganisms. Progressive serial dilution of the source is
made. Suitable aliquot of the serial dilution is chosen which is able to produce 300 to 400
individual colonies when plated on an agar plate, after incubation. Such a plate is called as
crowded plate.

The antibiotic producing activity of a colony is indicated by no growth of any other bacterial
colony in its vicinity. This region of no growth is indicated by the formation of a clear and
colorless area around the antibiotic producing microorganism’s colony on the agar plate. This
region is called as growth inhibitory zone.

Such a colony is isolated from the plate and purified either by making repeated sub-culturing
or by streaking on a plate containing a suitable medium, before stock culture is made. The
purified culture is then tested for its antibiotic spectrum.

However, the crowded plate technique has limited applications, as it will not give indication
of antibiotic producing organism against a desired organism. Hence, this technique has been
improved later on by employing a test organism to know the specific inhibitory activity of the
antibiotic.

In this modified procedure, suitable serially diluted soil suspension is spread on the sterilized
agar plate to allow the growth of isolated and individual microbial colonies (approximately
30 to 300 per plate) after incubation. Then the plates are flooded with a suspension of test
organism and the plates are incubated further to allow the growth of the test organism. The
formation of inhibitory zone of growth around certain colonies indicates the antibiotic
activity against the test organism.
A rough estimation of the relative amounts of antibiotic produced by a microbial colony can
be estimated by measuring the diameter of the zone of inhibited test organism’s growth.
Antibiotic producing colonies are later on isolated from the plate and are purified before
putting to further testing to confirm the antibiotic activity of a microorganism.

(ii) Indicator Dye Technique:

Microorganisms capable of producing acids or amines from natural sources can be detected
using this method by incorporating certain pH indicator dyes such as neutral red or
bromothymol blue into nutrient agar medium. The change in the color of a particular dye in
the vicinity of a colony will indicate the ability of that colony to produce an organic acid or
base.Production of an organic acid can also be detected by an alternative method. In this
method calcium carbonate is incorporated into the agar medium. The production of organic
acid is indicated by the formation of a clear zone around those colonies which release organic
acid into the medium. The identified colonies are isolated and purified either by repeated sub-
culturing or by streaking methods and a stock culture is made which may be used for further
qualitative or quantitative screening tests.

(iii) Enrichment Culture Technique:

This technique is generally employed to isolate those microorganisms that are very less in
number in a soil sample and possess specific nutrient requirement and are important
industrially. They can be isolated if the nutrients required by them is incorporated into the
medium or by adjusting the incubation conditions.

(iv) Auxanotrophic Technique:

This technique is employed for the detection and isolation of microorganisms capable of
producing certain extracellular substances such as growth stimulating factors like amino
acids, vitamins etc. A test organism with a definite growth requirement for the particular
metabolite is used in this method.For this purpose, spread a suitable aliquot on the surface of
a sterilized agar plate and allow the growth of isolated colonies, after incubation. A
suspension of test organism with growth requirement for the particular metabolite is flooded
on the above plate containing isolated colonies, which are subjected to further incubation.
The production of the particular metabolite required by the test organism is indicated by its
increased growth adjacent to colonies that have produced the required metabolite. Such
colonies are isolated, purified and stock cultures are prepared which are used for further
screening process.

(v) Technique of Supplementing Volatile Organic Substances:

This technique is employed for the detection and isolation of microorganisms capable of
utilizing carbon source from volatile substrates like hydrocarbons, low molecular weight
alcohols and similar carbon sources. Suitable dilution of a microbial source like soil
suspension are spread on to the surface of sterile agar medium containing all the nutrients
except the one mentioned above.The required volatile substrate is applied on to the lid of the
petri plates, which are incubated by placing them in an inverted position. Enough vapors from
the volatile substrate spread to the surface of agar within the closed atmosphere to provide the
required specific nutrient to the microorganism, which grows and form colonies by absorbing
the supplemented nutrient. The colonies are isolated, purified and stock cultures are made
which may be utilized for further screening tests.

2. Secondary Screening of Microorganisms:

Primary screening helps in the detection and isolation of microorganisms from the natural
substrates that can be used for industrial fermentations for the production of compounds of
human utility, but it cannot give the details of production potential or yield of the organism.
Such details can be ascertained by further experimentation.
This is known as secondary screening, which can provide broad range of information
pertaining to the:

i. Ability or potentiality of the organism to produce metabolite that can be used as an

industrial organism.
ii. The quality of the yield product.
iii. The type of fermentation process that is able to perform.
iv. Elimination of the organisms, which are not industrially important.

To evaluate the true potential of the isolated microorganisms both qualitative and quantitative
analysis are generally conducted. The sensitivity of the test organism towards a newly
discovered antibiotic is generally analysed during qualitative analysis, while the quantum
yield of newly discovered antibiotic is estimated by the quantitative analysis.

Evaluation of Potentialities of Microorganisms:

Microorganisms isolated in the primary screening are critically evaluated in the secondary
screening so that industrially important and viable potentialities can be assessed.

They include:

1. To determine the product produced by an organism is a new compound or not.

2. A determination should be made about the yield potentialities of various isolated
microorganisms that are detected in primary screening for that new compound.

3. It should determine about the various requirements of the microorganism such as pH,
aeration, temperature etc.
4. It should detect whether the isolated organism is genetically stable or not.
5. It should reveal whether the isolated organism is able to destroy or alter chemically their
own fermentative product by producing adaptive enzymes if they accumulate in higher
quantities.
6. It should reveal the suitability of the medium or its constituent chemicals for the growth of
a microorganism and its yield potentialities.
7. It should determine the chemical stability of the product.
8. It should reveal the physical properties of the product.
9. It should determine whether the product produced by a microorganism in a fermentative
process is toxic or not.
10. Secondary screening should reveal that whether the product produced in fermentation
process exists in more than one chemical form. If so, the amount of formation of each
chemical formation of these additional products is particularly important since their recovery
and sale as byproducts can greatly improve the economic status of the fermentation industry.
11. The new organism should be identified to the species level. This will help in making a
comparison of growth pattern, yield potentialities and other requirements of test organism
with those already described in the scientific and patent literature, as being able to synthesize
products of commercial value.
12. It should select industrially important microorganisms and discard others, which are not
useful for fermentation industry.
13. It should determine the economic status of a fermentation process undertaken by
employing newly isolated microorganism.

Methods of Secondary Screening:

Secondary screening gives very useful information pertaining to the newly isolated
microorganisms that can be employed in fermentation processes of commercial value. These
screening tests are conducted by using petri dish containing solid media or by using flasks or
small fermenters containing liquid media. Each method has some advantages and
disadvantages. Sometimes both the methods are employed simultaneously.

Liquid media method is more sensitive than agar plate method because it provides more
useful information about the nutritional, physical and production responses of an organism to
actual fermentation production conditions. Erlenmeyer flasks with baffles containing highly
nutritive liquid media are used for this method. Flasks are fully aerated with glass baffles and
continuously shaken on a mechanical shaker in order to have optimum product yield.

There are several techniques and procedures that can be employed for secondary screening.
However, only a specific example of estimation of antibiotic substance produced by species
of Streptomyces, is described in the following paragraph. Similar methods could be used for
the detection and isolation of microorganisms capable of producing other industrial products.

(i) Giant Colony Technique:

This technique is used for isolation and detection of those antibiotics, which diffuse through
solid medium. Species of Streptomyces, is capable of producing antibiotics during primary
screening. The isolated Streptomyces culture is inoculated into the central area of a sterilized
petri plates containing nutrient agar medium and are selected. The plates are incubated until
sufficient microbial growth takes place.

Cultures of test organism, whose antibiotic sensitivity is to be measured are streaked from the
edges of plate’s upto but not touching the growth of Streptomyces and are further incubated
to allow the growth of the test organisms. Then the distance over which the growth of
different test organisms is inhibited by the antibiotic secreted Streptomyces is measured in
millimeters.

The relative inhibition of growth of different test organisms by the antibiotic is called
inhibition spectrum. Those organisms whose growth is inhibited to a considerable distance
are considered more sensitive to the antibiotic than those organisms, which can grow close to
the antibiotic. Such species of Streptomyces, which have potentiality of inhibiting
microorganisms is preserved for further testing.

(ii) Filtration Method:

This method is employed for testing those antibiotics which are poorly soluble in water or do
not diffuse through the solid medium. The Streptomyces is grown in a broth and 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 against the test
organisms is measured by their degree of growth in different antibiotic dilutions.

(iii) Liquid Medium Method:

This method is generally employed for further screening to determine the exact amount of
antibiotic produced by a microorganism like Streptomyces.

Erlenmeyer conical flasks containing highly nutritive medium are inoculated with
Streptomyces and incubated at room temperature. They are also aerated by shaking
continuously and vigorously during incubation period to allow Streptomyces to produce the
antibiotic in an optimum quantity.

Samples of culture fluids are periodically withdrawn aseptically for undertaking the
following routine checks:

1. To check the suitability of different media for maximum antibiotic production.

2. To determine the value of pH at which there will be maximum growth of the
microorganism and antibiotic production.
3. To check for contamination.
4. To determine whether the antibiotic produced is new or not.
5. To check the stability of the antibiotic at various pH levels and temperatures.
6. To determine the solubility of the antibiotic in various organic solvents.
7. To check about the toxicity of the antibiotic against the experimental animals

Genetic Improvement of Strains: Features and Methods

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Read this article to learn about the features and methods of genetic improvement of strains.

In general, the wild strains of microorganisms produce low quantities of commercially

important metabolites, although the yield can be increased by optimizing the fermentation
conditions. The potentiality of the metabolite formation is genetically determined. Therefore,
genetic improvements have to be made and new strains developed for any substantial
increase in product formation in a cost-effective manner.

There are strain development programmes (mutation and recombination) to increase the
product yield by 100 times or even more. The nature of the desired product determines the
success associated with strain improvement. For example, if alterations in one or two genes
(i.e. one or 2 key enzymes) can improve the product yield, it is simpler to achieve the target.

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This type of approach is sometimes possible with primary metabolites. As regards the
secondary metabolites, the product formation and its regulation are quite complex. Hence,
several genetic modifications have to be done to finally produce high-yielding strains.

Features of Genetic Improvement:

Ideally speaking, the improved strains should possess the following characteristics (as many
as possible) to finally result in high product formation:

1. Shorter time of fermentation

2. Capable of metabolizing low-cost substrates

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3. Reduced O2 demand
4. Decreased foam formation

5. Non-production of undesirable compounds

6. Tolerance to high concentrations of carbon or nitrogen sources

7. Resistant to infections of bacteriophages.

It is always preferable to have improved strains of microorganisms which can produce one
metabolite as the main product. In this way, the production can be maximised, and its
recovery becomes simpler. Through genetic manipulations, it has been possible to develop
strains for the production of modified or new metabolites which are of commercial value e.g.
modified or newer antibiotics.

The major limitation of strain improvement is that for most of the industrially important
microorganisms, there is lack of detailed information on the genetics, and molecular biology.
This hinders the new strain development.

Methods of Strain Development:

There are two distinct approaches for improvement of strains-mutation, recombination and
recombinant DNA technology.

1. Mutation:
Any change that occurs in the DNA of a gene is referred to as mutation. Thus, mutations
result in a structural change in the genome. Mutations may be spontaneous (that occur
naturally) or induced by mutagenic agents.

The spontaneous mutations occur at a very low frequency, and usually are not suitable for
industrial purposes. Mutations may be induced by mutagenic agents such as ultraviolet light,
various chemicals (nitrous oxide, nitrosoguanidine, and hydroxylamine). Site-directed
mutagenesis is also important for strain improvement.

Selection of Mutants:

Selection and isolation of the appropriate mutant strains developed is very important for their
industrial use. Two techniques commonly employed for this purpose are briefly described.

Random screening:

The mutated strains are randomly selected and checked for their ability to produce the desired
industrial product. This can be done with model fermentation units. The strains with
maximum yield can be selected. Random screening is costly and tedious procedure. But
many a times, this is the only way to find the right strain of mutants developed.

Selective isolation of mutants:

There are many methods for selective isolation of improved strains:

1. Isolation of antibiotic resistant strains:

The mutated strains are grown on a selective medium containing an antibiotic. The wild
strains are killed while the mutant strains with antibiotic resistance can grow. Such strains
may be useful in industries.

2. Isolation of antimetabolite resistant strains:

Antimetabolites which have structural similarities with metabolites can block the normal
metabolic pathways and kill the cells. The mutant strains resistant to antimetabolites can be
selected for industrial purposes. In the Table 19.5, a selected list antimetabolites used for
screening the metabolites is given.

3. Isolation of auxotrophic mutants:

An auxotrophic mutant is characterized by a defect in one of the biosynthetic pathways. As a

result, it requires a specific compound for its normal growth. For instance, tyr mutants of
Corynebacterium glutamicus require tyrosine for their growth while they can accumulate
phenylalanine. The isolation of such mutants can be done by growing them on a complete
agar medium that can specifically support the biochemically defective mutant.

2. Genetic Recombination:
The strain improvement can be made by combining genetic information from two genotypes,
by a process called genetic recombination. The recombination can be brought out by
transformation, transduction, conjugation and protoplast fusion.

There are many advantages of genetic recombination:

1. By crossing high product yielding mutant strains with wild-type strains, the fermentation
process can be further increased.
2. Different mutant strains with high-yielding properties can be combined by recombination.
3. There is gradual decline in the product yield after each stage of mutation, due to
undesirable mutations. This can be prevented by using recombination.