Screening Techniques

Production Strain
• The most important factor for the success of any fermentation
industry is the production strain. It is highly desirable to use a
production strain possessing the following characteristics:
• High yielding
• Should have stable biochemical characteristics
• Should not produce undesirable side products
• Should be easily cultivable on a large scale
 Screening
• “The use of highly selective procedures to allow the detection &
isolation of only those microorganisms which are of interest from
among a large microbial population”
• Screening techniques allows you to segregate the high yielding species
from natural source material such as soil, water, compost, food or any
other.
• Screening allows the discarding of many valueless microorganisms, at
the same time it allows the easy detection of the useful
microorganisms that are present in the population in very less number
• Two types of screenings are usually carried out
a. Primary Screening and
b. Secondary Screening
 Primary Screening
• Primary screening allows the detection & isolation of microorganisms
that possess potentially interesting industrial application.
• It allows detection and isolation of the desired microorganism based on
its qualitative ability to produce the desired product
• Primary screening separate outs only a few microorganisms having real
commercial value.
• Primary screening determines which microorganisms are able to produce
a compound without providing much idea of the production or yield
potential of the organisms
• Primary Screening is useful technique for detection and isolation of
antibiotics, extracellular enzymes, organic acids, Vitamins, amino acids
etc.
• It is also particularly useful for organisms that are slow growers or utilize
• The following are some of the important primary screening
techniques:
i. Indicator dye technique
ii. The crowded plate technique
iii. Enrichment culture technique
iv. Auxanographic technique
 1. 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.
• pH indicating dyes are chemicals that show colour change in different pH
• For example, Bromothymol Blue (BTB) is:
Yellow – Acidic
Green – Neutral and
Blue – Alkaline pH
• Major types of organic acid produced by microbial activity are citric acid,
succinic acid, lactic acid, itaconic acid, lactobionic acid, gluconic acid,
fumaric acid, propionic acid, and acetic acid
Primary Screening of Organic acid producers
from soil sample
• Collect the soil sample
• Prepare soil Suspension in sterile saline or distilled water
• Prepare nutrient agar(poorly buffered) and add a pH indicating dye(e.g. BTB or
bromocresol green) to it and sterilize it by autoclaving at 15lbs pressure (121
degree C) for 15-20 minutes.
• Pour the medium in sterile petri dishes and allow to solidify
• Spread about 100µL of the soil suspension on the surface of the medium with
the help of a glass spreader
• Incubate at 37 degree C for 24 hrs.
• Observe plates for colonies which have produced acid and therefore changed
the colour of the medium in the vicinity of the colony from green to yellow.
• Subculture the colonies of Nutrient agar slants to make the stock cultures.
• These are then subjected to Secondary screening to ensure these truly have any
potential to be a production strain.
2. Crowded Plate Technique (For
antibiotic producers)
• For screening of antibiotic producing organisms, the simplest
technique is “crowded plate” procedure.
• This technique is used where one is interested only in finding
microorganisms that produces an antibiotic irrespective of its action
against any specific organism.
• Requirements:
Soil sample
Dilution blank
Sterile pipettes
Glass spreader
Sterile Nutrient agar plates
• Procedure
1. Suspend 1.0gram of soil sample in almost about 10 ml of sterile distilled
water.
2. Mix well and allow the soil particle to settle down.
3. Prepare different dilution of the supernatant using dilution blank.
4. Spread 0.1 ml of inoculum on sterile nutrient agar plate with glass
spreader. Incubate the plate at room temperature for 24 to 48 hours.
5. Observe the plate at different intervals. The ability of an organism to
exhibit antibiotic activity is indicated by the presence of zone of inhibition
surrounding the colony. The plates having 300-400 colonies per plate are
considered since they are helpful in locating antibiotic activity.
6. Subculture the colony, purify by streaking and make stock cultures. Then
after its microbial inhibition spectrum is determined.
• Advantages:
• The crowded plate technique is fairly simple -- indeed, the simplest
method to find antibiotic-producing microorganisms in soil samples.
• It's also fairly rapid, taking only a couple of days to produce results.
• Introducing "test organisms" can help to determine whether a specific
kind of microorganism (e.g., a disease-causing germ) is susceptible to
the antibiotic compound. If it does indeed prove useful for this purpose,
the compound can be isolated for further study.
• Drawbacks
• The crowded plate technique only detects microorganisms that produce
compounds to kill bacteria found in their immediate environment.
• The crowded plate procedure also does not necessarily select an
antibiotic producing microorganism, because the inhibition area around
the colony sometimes can be due to other reason like:
(1) Marked change in the pH of the medium resulted due the metabolism
of the colony.
(2) Rapid utilization of critical nutrients in the vicinity of the colony etc.
• These compounds could potentially be toxic to humans, and they may be
lethal only to certain types of bacteria (e.g., soil bacteria), as opposed to
the bacteria that actually cause disease in humans.
Wilkins’s Method

• Another technique mainly used is Wilkins’s method.

• A Wilkin’s medium which contains a pH indicating dyes i.e. Bromo-
thymol Blue which is green colour at neutral pH but yellow at acidic
pH.
• This method differentiates antibiotic producer from the acid producer.
Those colonies that produce antibiotics give zone of inhibition against
sensitive organisms without changing colour surrounding it while in
case of acid producer colony we may find zone of inhibition due to fall
in pH along with yellowish area due high acid production which result
in lower pH , ultimately change the colour of dye from green to
yellow.
• It has some additional advantage that the organism producing
antibiotic against the microorganisms of choice can be secondary
screened
• 1. Spread 0.1 ml of inoculum from each dilution on sterile Wilkins’s agar plate.
• 2. Incubate at 37 degree for 24 hrs.
• 3. Next day observe the plate for growth of different organism.
• 4. Inoculate about 2 ml of test culture into Wilkins’s broth/ sterile melted
Wilkins’s top agar previously cooled at 45 degrees. Mix it well and pour it over
the base Wilkins’s agar spread on previous day.
• 5. Allow it to solidify and incubate at 37 degrees for 24 hrs.
• 6. Next day observe the plates for colonies showing inhibition of growth of
test organism surrounding its vicinity, i.e. antibiotic producing colony.
• 7. Mark the colony write colony characteristics, perform Gram’s staining, and
sub culture it for secondary screening.
 Enrichment Culture Technique
• The microbiologist (and botanist) Martinus Beijerinck is credited with
developing the first enrichment cultures
• Enrichment culture is basically an isolation technique designed to
make conditions of growth very favourable for an organism of interest
while having an unfavourable environment for any competition.
• Enrichment culture techniques are used to increase a small number of
the desired organisms to detectable levels. This allows for the
detection and identification of microorganisms with a variety of
nutritional needs
• This enrichment is generally done by introducing nutrients or
environmental conditions that only allow the growth of an organism
of interest.
• For example, if you wish to isolate a bacterium that is a thermophile
(prefers to grow at a high temperature such as 55 deg C), incubate the
sample at that high temperature.
• Organisms that cannot tolerate that temperature fail to grow, while
thermophiles will grow and increase in number, over time becoming a
large and larger proportion of the total bacterial population in the
sample. This is an example of enrichment by modifying the physical
condition
Image taken from Google
 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.
 AUXANOGRAPHY
Spread a suitable Flood the plate with a
aliquot on the surface suspension of test
of a sterilized nutrient organism (which has
Incubate
agar plate and allow requirement for the
the plates
the growth of isolated particular growth factor
colonies, after which we are looking
incubation. for
Look for colonies
surrounded by
stimulated growth

Isolate , purify and

maintain stock cultures
 Secondary Screening
• 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.
Therefore, Secondary Screening is strictly essential in any systematic screening program
intended to isolate industrially useful organisms.

• Secondary screening allows further sorting out of microorganisms, obtained from primary
screening ,having real value for industrial processes and discarding of those lacking this
potential. This is known as secondary screening, which can provide broad range of
information.
 Secondary Screening
• Secondary screening is performed to/determine/provides information about:

• The ability or potentiality of the organism to produce metabolite that can be used as an
industrial organism.

• Evaluate the true yield potential of the isolated microorganisms both qualitative and
quantitative analysis are generally conducted.

• If the product produced by an organism is a new compound or not.

• Reveal the optimum conditions associated with particular microorganisms, both for the
growth of the organism and for the formation of a product
 .

• The suitability of the medium or its constituent chemicals for the growth of a
microorganism and its yield potentialities.

• Detect gross genetic stability in microbial cultures

• The number of products produced in a single fermentation. Additional minor

products can be of commercial value and sold as by products.

• The chemical stability of the product.

• The Chemical, physical and biological properties of the product.

• The product has a simple, complex, or even a macromolecular structure, if this

information is not already available
• Whether the product occurs in more than one chemical form and
whether it is an optically or biologically active material.

• Whether the microorganisms are able to chemically alter or even destroy

their own fermentation products (by producing adaptive enzymes)

• Secondary screening helps in predicting the approaches to be utilized in

conducting further research on the microorganisms and its fermentation
processes

• Gross animal, plant or human toxicity of the fermentation product,

particularly if it is utilized (as are antibiotics) in disease treatment
Giant Colony Technique to determine
Spectrum of an Antibiotic
E resources and acknowledgments

• [Link]
Theory)_Primary_and_Secondary_Screening_of_Industrially_Important_Microbes.pdf
• [Link]
• [Link]
• [Link]
• [Link]
with_fig2_279635907
• [Link] Giant colony
• [Link] (animation)
• [Link]
• [Link]