The presentation gives details about primary screening of micro-organisms and explains varying techniques for isolation of organisms.

1. SCREENING OF
MICROORGANISMS
Renu NK Jaisinghani
Assistant Professor
Department of Microbiology
Smt.CHM College

2. Screening
It is defined as use of highly selective procedure
for detection and isolation of only those
microorganisms of interest from among large
microbial population and test their real capability
by qualitative and quantitative analysis.

3. Screening
Primary Secondary

4. Primary Screening
It is defined as those elementary techniques
required for isolation and detection of new
microbial species exhibiting desirable or
industrially important properties.

5. Secondary Screening
It is defined as techniques that select only those
organisms amongst hundred of isolates having
real industrial values and test their capabilities by
qualitative and quantitative analysis.

6. General steps involved in primary screening
1. Collection of sample
2. Preparation of sample
3. Carry out 10 fold dilution
4. Isolation on suitable media
5. Using suitable method like streaking, spreading, pour plate or
membrane imprints(water sample)

7. Examples of Primary Screening
Acid/
Amine
producers
Antibiotic producers Isolation of enzyme
producers
Specific C
and N UtilizerDegraders of
synthetic
waste
Isolation of
Vitamin,
aminoacid
producer

8. I Isolation of Acid/ Amine producer
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.
pH Indicator ACIDIC ALKALINE NEUTRAL
NEUTRAL RED PINK YELLOW ORANGE
BROMOTHYMOL
BLUE
YELLOW BLUE GREEN

9. Isolation of Acid/ Amine producer contd….
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.

10. II Isolation of Antibiotic Producer
Crowded plate technique
Principle
Is different organisms present in the sample are made to grow very
close to each other (using LOW DILUTION) so that any antibiotic
producer among them can exert antagonistic effect towards other
Organisms in its close vicinity. Thus antibiotic producer can be easily
detected by visual detection of clearing around the colony

11. Crowded plate technique
•https://youtu.be/Wf4BBYmQD_g

12. Wilkin’s Agar Overlayer Method
Principle
Is to allow different organisms present in sample to grow on a suitable
media as isolated colonies(using HIGH DILUTIONS) and detection of
antibiotic producer among them as colony showing zone of inhibition
of test organisms around it after overlayering colonies with sterile
molten Wilkins’s agar butt seed inoculated with organisms.
Bromothymol blue, a pH indicator in Wilkin’s agar helps in
differentiating zone of inhibition due to antibiotic and or acid/alkali
produced by the isolate.

13. Wilkin’s Agar Overlayer Method
STEP 1
DAY 1
NUTRIENT AGAR
COLONIES

14. Wilkin’s Agar Overlayer Method
• STEP 2
DAY 2
Overlayering with Wilkin’s
agar(10ml) butt seed
inoculated with test
organism

15. Wilkin’s Agar Overlayer Method
Step 3
Day 3
Alkali producer
Antibiotic producer
Acid producer

16. Wilkin’s Agar Overlayer Method
Antibiotic Producer

17. III Isolation of vitamin, aminoacid,
Growth factor producer

18. ENRICHMENT
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.

19. ENRICHMENT TECHNIQUE
Few Examples
• PROTEASE PRODUCER-
MEDIA CONTAINING PROTEIN
• CELLULASE PRODUCER-
MEDIA CONTAINING CELLULOSE
• LYSINE PRODUCER-
MEDIA DEVOID OF LYSINE

20. Auxanography

21. Terminology
• Auxotroph – Requires a particular growth factor for its growth i.e it
cannot synthesize its own growth factor.
It is denoted as name of growth factor and –(minus)
For Example: Lys-, VitB12
-
• Prototroph – produces its own growth factor.
It is denoted as name of growth factor and +(plus)
For Example: Lys+, VitB12
+

22. • Auxanography technique is employed for detecting microorganisms
able to produce growth factors , vitamins , amino acids etc.
extracellularly
A minimal media
lacking the
growth factors is
prepared and
seeded with the
test organism. •
The seeded
medium is
poured onto
fresh petri plate
and the plate is
allowed to se
Preparation of
second plate
• 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. • Soil
sample is diluted
and proper
dilutions are
inoculated.
Preparation of first
plate

23. REPLICA PLATE TECHNIQUE
Principle
Is to transfer large number of colonies from one plate(MASTER PLATE)
to another plate(REPLICA PLATE- usually minimal medium), that will
support growth of any one type of organisms of interest from number
of different types present on master plate, in a single step using sterile
replicator block covered with sterile velveteen cloth whose threads
serve as needles and pick up colonies from one plate to transfer on
another.

24. REPLICA PLATE TECHNIQUE
Source: Researchgate.net

25. REPLICA PLATE TECHNIQUE
https://youtu.be/uYb-_hYSVYw

26. IV ISOLATION OF ENZYME PRODUCERS
• Enzymes have huge demand in varying industry pharmaceuticals,
brewing, textile ,cheese, meat, baking etc.
For isolation of enzyme producer specific substrate is required to be
provided
Eg: PROTEASE- Any media containing protein
AMYLASE- Media containing Starch
LIPASE- Media containing oil or lipids

27. V ISOLATION OF DEGRADERS OF SYNTHETIC
WASTE
Pesticides, detergents and solvents do not get degraded easily.
-STARTER CULTURE
-CONSORTIUM OF ORGANISMS

28. VI ISOLATION OF SPECIFIC ‘C’ AND ‘N’
UTILIZER
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.

29. VI ISOLATION OF SPECIFIC ‘C’ AND ‘N’
UTILIZER
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.

30. VI ISOLATION OF SPECIFIC ‘C’ AND ‘N’
UTILIZER
Source: ResearchGate.net

31. THANK YOU