Screening of

Microbial
Cultures
 SCREENING

The procedure of isolation, detection , and separation of

microorganisms of our interest from a mixed population by
using highly selective procedures is called SCREENING
 SCREENING – IT REFERS TO THE ISOLATION OF ONLY THOSE
MICROORGANISMS WHICH ARE OF INTEREST FROM AMONG A
LARGE POPULATION OF MICROBES. THEY ARE MAINLY OF TWO
TYPES:
 Primary and Secondary Screening
 Primary Screening : The effective screening must in one or few steps
which 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.
 Secondary Screening : As primary screening allows detection and
isolation of microorganisms that possess potentially interesting
industrial applications. This is usually followed by a secondary
screening to further test the capabilities of and gain information about
these organisms.
 MICROORGANISMS

SOURCE

COMPOST AND
FOOD (MILK, ENVIRONMENT (AIR , OTHER SOURCES
CHEESE etc. ) WATER, SOIL)

SCREENING

PRIMARY SCREENING

SECONDARY SCREEENING
 IMPORTANT THINGS TO BE CONSIDERED WHILE
SCREENING :-

 1.) CHOICE OF SOURCE - Samples from screening is taken

from soil, water, air, milk, compost etc.
 2.) CHOICE OF SUBSTRATE -Nutrients and growth factors
should be supplied for growth of desired microorganism.
 3.) CHOICE OF DETECTION - Proper isolation and
detection of desired microorganisms is important
 TYPES OF SCREENING
SRCEENING

PRIMARY SCREENING SECONDARY SCREENING

ORGANIC ACID ANTIBIOTIC EXTRACELLULAR ENRICHMENT

PRODUCING PRODUCING METABOLITES PRODUCING CULTURE
MICROORGANISMS MICROORGANISMS MICROORGANISMS TECHNIQUE

BY USING DYES BY USING BY BY

CROWDED AUXANOGRAPHY DEFINED
PLATE TECHNIQUE MEDIA
TECHNIQUE
 TYPES OF PRIMARY SCREENING
 Crowded-plate technique – Natural microbial source such as soil is diluted to
provide a cell concentration such that aliquots spread, sprayed or applied in
some manner to the surfaces of agar plates will yield colonies not touching
neighbouring colonies.
 An alternative procedure for detecting organic acid production involves
incorporation of calcium carbonate in medim so tht organic acid production is
indicated by cleared zone of dissolved calcium carbonate around the colony.
 Screening approach has also been employed extensively in search for the
microrganisms capable of producing useful antibiotics. The simplest screening
technique for antibiotic procedures is the “crowded plate” procedure. Colonies
producing antibiotic activity are indicated by an area of agar around the colony
that is free of growth of other colonies. Such a colony is subcultured to a
similar medium and purified by streaking before making stock cultures.
 PRIMARY SCREENING
 It’s a process for detection and isolation of microorganisms of our
interest.
 Determines which microorganisms are able to produce a
compounds.

Does not provide much idea about the production or potential
yield of microorganisms.
 It separate out only a few microorganisms, only few have
commercial value while discards the valueless microorganisms .
 1) PRIMARY SCREENING OF ORGANIC ACID
PRODUCING MICROORGANISMS
The pH indicating dyes may be used for detecting

microorganism that are capable of producing organic acids.

These dyes undergo color changes according to its pH.
Dyes such as Neutral red, Bromothymol blue are added to the

poorly buffered nutrient agar media .

Colonies are subcultured to make stock culture.
Further testing is needed since inorganic acids, bases are also

metabolic products of microbial growth.

 Incorporation of CaCO3 in medium is also used to screen organic acid
producing microbes on basis of formation of clear zone of dissolved
CaCO3 around the colony.
 2) PRIMARY SCREENING OF ANTIBIOTIC PRODUCING
MICROORGANISMS
Crowded plate technique is used for screening of antibiotic producing
microorganisms.
Does not give information about the sensitivity of antibiotics towards
other microorganisms.
Dilutions are made and then pouring and spreading of soil samples that
give 300 to 400 or more colonies per plate.
Colonies showing antibiotic activity are indicated by zone of inhibition
around the colony .
Such colonies are sub cultured and purified by streak before making stock
cultures.
The purified cultures are then tested to find the Microbial Inhibition

Spectrum.
 IMPROVEMENT OF CROWDED TECHNIQUE
 Antibiotic screening is improved, therefore, by incorporation into the procedure of
“test organism”, that is an organism us as an indicator for the presence of specific
antibiotic activity.
 A suspension of test organism is then sprayed or applied in some manner to the
surface of the agar, and then plates are further incubated to allow growth of the test
organism.
 When volatile substrates such as hydrocarbons, low molecular weight alcohols, and
similar carbon sources are being considered. The dilutions from a microbial
sources are applied to plates of agar media containing all nutrients but the specific
substrate and the specific substrate is placed in the lid of petriplate after inversion
of the plate.
 Enough vapours from the volatile substrate rise to the surface of the agar within
this closed atmospheric to provide the specific nutrient for the microorganism.
3) PRIMARY SCREENING EXTRACELLULAR METABOLITE
PRODUCING MICROORGANISM

 Auxanography technique is employed for detecting microorganisms able to produce

growth factors , vitamins , amino acids etc. extracellularly.
 The 2 major steps are:-
• A filter paper strip is put across the bottom of petri dish.
• The nutrient agar is prepared and poured on the paper disc
• and allowed to solidify.
A.) Preparation of
• Soil sample is diluted and proper dilutions are inoculated.
first plate
• A minimal media lacking the growth factors is prepared and seeded with the
test organism.
B.) Preparation of • The seeded medium is poured onto fresh petri plate and the plate is allowed to
set.
second plate
The agar in first plate is then lifted and placed on the second
plate without inverting.
The growth factors produced on agar can diffuse into the lower

layer containing test organism.

The zones of stimulated growth of test organism around
colonies is an indication that organism produce growth factor
extracellularly.
 SECONDARY SCREENING
 Secondary screening allows the further sorting out of those
microrganisms that have real value for industrial processes and
discarding of those lacking this potential.
 Secondary screening is conducted on agar plates, in flasks or
small fermenters containing liquid media, or combination of these
approaches.
 Liquidculture provides a much better picture of the nutritional
physical, and production of responses of an organism to actual
fermentation production conditions.
 SECONDARY SCREENING CAN BE QUALITATIVE
AND QUANTITATIVE IN ITS APPROACH
 Qualitative approach – eg. Spectrum or range of microorganisms which is
sensitive to a newly discovered antibiotic.
 Quantitative Approach – This tells us about the yields of antibiotic which
can be expected when the microorganism is grown in various differing
media.
 Secondary screening should yield types of information which are needed
in order to evaluate the true potential of a microorganism for industrial
usage.
 Secondary screening should determine whether a more
economical process is possible.
 Todetermine whether a product actually is newly discovered sompound,
we can utilize paper, thin layer or other chromatographic procedures to
compare the product with known compounds.
 Secondary screening should detect real differences in product yiels
potentials among various isolates.

 Secondary screening should reveal whether there are pH, aeration, or

other critical requirements associated with particular microorganisms,
both for the growth of the organism and for the formation of chemical
products.
 Screening should show whether certain medium constituents are missing
or possibly are toxic to growth of the organism, or its ability to
accumulate fermentation products.
 Itshould show something of chemical stability of the product and product
solubility.
 It
should determine whether the product has simple, complex, or even a
macromolecular structure.
 Itshould show whether the product possesses physical properties such as
U.V. light absorption or fluorescence, or chemical properties that can be
employed to detect compound.

 Certainkinds 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 in disease
treatment.
 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 it is an optically or
biologically active material.

 Secondary screening should reveal whether microorganisms are

able to chemically alter or even destroy their own fermentation
products. 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- isomer of being little
biological value.
TECHNIQUES USED FOR SECONDARY SCREENING
 Column Chromatography
 Thin Layer Chromatography
 HPLC
 LC-MS
 GC-MS
 UV- VIS Scan
 FT-IR
 NMR
 SEM
 TEM etc.
THANK YOU