The document discusses various methods for isolating and preserving microorganisms in pure culture. To isolate microbes, common methods include streak plating, pour plating, and serial dilution. Maintaining pure cultures long-term involves subculturing to fresh media periodically or preservation through lyophilization, low-temperature storage, or overlaying cultures with mineral oil. Lyophilization involves freeze-drying microbes under vacuum to remove water and stop metabolic activity, allowing long-term viability.
Direct methods of measurement of microbial growth includes various methods of enumeration of both viable and non viable cell also includes growth curve. Helpful for UG and PG programs of microbiology
Direct methods of measurement of microbial growth includes various methods of enumeration of both viable and non viable cell also includes growth curve. Helpful for UG and PG programs of microbiology
Preservation of industrially important microorganisms, methods of preservation, periodic transfer, storage in saline suspension, storage in sterile soil, cryopreservation
A pure culture theoretically contains a single bacterial species. There are a number of procedures available for the isolation of pure cultures from mixed populations. A pure culture may be isolated by the use of special media with specific chemical or physical agents that allow the enrichment or selection of one
organism over another.
Microbial interactions are ubiquitous, diverse, critically important in the function of any biological community.
The most common cooperative interactions seen in microbial systems are mutually beneficial. The interactions between the two populations are classified according to whether both populations and one of them benefit from the associations, or one or both populations are negatively affected.
Secondary screening of industrial important microbes DhruviSuvagiya
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.
The term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment. It becomes necessary to maintain the viability and purity of the microorganism by keeping the pure culture free from contamination.
Preservation of industrially important microorganisms, methods of preservation, periodic transfer, storage in saline suspension, storage in sterile soil, cryopreservation
A pure culture theoretically contains a single bacterial species. There are a number of procedures available for the isolation of pure cultures from mixed populations. A pure culture may be isolated by the use of special media with specific chemical or physical agents that allow the enrichment or selection of one
organism over another.
Microbial interactions are ubiquitous, diverse, critically important in the function of any biological community.
The most common cooperative interactions seen in microbial systems are mutually beneficial. The interactions between the two populations are classified according to whether both populations and one of them benefit from the associations, or one or both populations are negatively affected.
Secondary screening of industrial important microbes DhruviSuvagiya
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.
The term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment. It becomes necessary to maintain the viability and purity of the microorganism by keeping the pure culture free from contamination.
Pure Culture Technique
Culture : Act of cultivating microorganisms or the microorganisms that are cultivated.
Mixed culture : more than one microorganism
Pure culture : containing a single species of organism.
Common isolation techniques:
1. Streak plate method
2. Pour plate method
3. Spread plate method
4. Roll tube method
PURE CULTURE TECHNIQUE ISOLATION AND IDENTIFICATION PROCESS .pptxVishekKumar8
Pure culture technique
INTRODUCTION
PURE CULTIURE TECHNIQE
ISOLATION PROCESS
STREAK PLATE METHOD
POUR PLATE METHOD
SPREAD PLATE METHOD
IDENTIFICATION PROCESS
BIOCHEMICAL TEST
MOLECULAR METHOD
SEROGICAL TECHNIQUE
In microbiology, the term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest. Historically, the laboratory techniques of isolation first developed in the field of bacteriology and parasitology (during the 19th century), before those in virology during the 20th century. Methods of microbial isolation have drastically changed over the past 50 years, from a labor perspective with increasing mechanization, and in regard to the technology involved, and hence speed and accuracy.
Much faster rates of growth can be induced in vitro than by traditional means.
Multiplication of plants which are very difficult to propagate by cuttings or other traditional methods.
Production of large numbers of genetically identical clones in a short time
Seeds can be germinated with no risk of damping off/ predation.
Under certain conditions, plant material can be stored in vitro for considerable periods of time with little or no maintenance
Tissue culture techniques are used for virus eradication, genetic manipulation, somatic hybridization and other procedures that benefit propagation, crop improvement, and basic research.
By means of tissue culture it is possible to produce pathogen free plantlets by mass multiplication in a very limited amount of area from a very small sterile part of a mother plant. This method is also used to produce/ multiply plants that are to be transported across national border and so for their faster multiplication.But the establishment of a tissue culturing unit needs huge financial investments, skilled labors/technicians and required areas for its establishment are major constraints. Plant tissues grow and multiply in the labs only when there is an uncompetitive, growing condition with uninterrupted supply of nutrients.
Medium:
It contains all the elements that contribute the required nutrients that aid to the growth of the tissues; it is in liquid state or semi-solid in nature. The tissues are grown on the media. It consists of 95% of water, major and minor nutrients, plant growth hormones, vitamins, sugar rich compounds and chelating agents.
Totipotency:
It is the ability of a tissue or an organ of a plant to produce the whole plant, under the optional laboratory conditions and this is called as Totipotency. This is the baseline over which plant tissue culture relies upon.
Callus Culture:
When the cells divide into an undifferentiated mass it is called as callus. Any part of a plant can be used to produce the calli. It may be a stem, leaf, meristem or any other part. It is used to produce variations among the plantlets.
Suspension culture:
The callus produced from the explants are grown on nutrient solutions (that are semi solid) for a period of time and they are induced to produce plants with new traits.
Embryo Culture:
The method of culturing mature and immature embryos in media is called embryo culture. By this method, it is possible to produce plants from dormant seeds and seeds with metabolites that inhibit germination. This method is very important in crop improvement programs.
Somatic Embryogenesis:
When the plants are grown on nutrient media, calli are formed. When these calli are subjected to growth in cytokinin medium, somatic embryos are formed. They are circular, elongated,
Single cell culture
• As stated earlier, cells derived from a single cell through mitosis constitute a clone and the process of obtaining clones is called cloning (asexual progeny of a single individual make up.
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2. ISOLATION OF MICROBIAL PURE
CULTURE
• Microorganisms are generally found in nature (air, soil and water) as
mixed populations.
• Even the diseased parts of plants and animals contain a great number
of microorganisms, which differ markedly from the microorganisms of
other environments.
• To study the specific role played by a specific microorganism in its
environment, one must isolate the same in pure culture.
The two major steps of obtaining a pure culture are as follows :
• Firstly, the culture has to be diluted until the various individual
microorganisms are separated far apart on agar surface that after
incubation they form visible colonies isolated from the colonies of
other microorganisms.
• Secondly, an isolated colony has to be aseptically picked off the
isolation plate
3.
4. Points to be taken into consideration during the inoculation are :
• The inoculation loop has to be sterilized before every inoculation
of colonies from the agar plate
• Every time the loop is sterilized by heat it must be cooled before
inoculating the next colony
• There are several methods of isolating the pure culture of
bacteria, like, streak plate method, pour plate method, spread
plate method and serial dilution.
Why important ?
Pure cultures are important in microbiology for the following reasons
Once purified, the isolated species can then be cultivated with the
knowledge that only the desired microorganism is being grown.
A pure culture can be correctly identified for accurate studying
and testing and diagnosis in a clinical environment.
Testing/experimenting with a pure culture ensures that the same
results can be achieved regardless of how many time the test is
repeated.
• Pure culture spontaneous mutation rate is low
• Pure culture clone is 99.999% identical
5. COMMON METHODS OF
ISOLATION OF PURE CULTURE
• The process of screening a pure culture by
separating one type of microbes from a mixture is
called Isolation.
Some common isolation methods are;
• I) Streak plate method
• II) Pour plate method-a) Loop dilution technique
b) Serial Dilution technique
• III) Spread plate method
• IV) Micromanipulator method
• V) Roll tube method
6. I. STREAKING OR
STREAK PLATE TECHNIQUE
• In This method the tip of a fine
structure wire loop called
Inoculation needle consist of a
wooden or glass handle with a
Nichrome wire the end of which is
bend to form a loop is used to
transfer microbes from culture.
• The straight wires are similar to wire
loop except they do not have loop.
These are used to transfer culture in
colony formed on solid culture
medium.
• In such cases, the colony from solid
medium is streaked on the surface
of nutrient agar medium in a sterile
petri dish.
7. This technique consist of the following steps-
• A. Hold the broth culture containing tube in left hand and shake it.
• B. Sterilize the wire loop of the inoculation needle on burner flame .
• C. Remove the cotton plug of the broth culture tube by little finger of right hand.
• D. Flame the mouth of the test tube immediately.
• E. Insert the wire loop to form a thin film and replace the cotton plug.
• F. The thin film in the loop is streaked in either a zig-zag manner by removing the
loop backwards and forwards firmly. Care should be taken that loop should not
be firmly pressed against the agar surface.
• G. Incubate the petri dish in incubator at a required temperature.
• H. Growth of the bacteria will be visible (after an overnight incubation)on the
streaked marks.
8. II) Pour plate method
• The bacterial culture and liquid agar medium
are mixed together.
• After mixing the medium, the medium
containing the culture poured into sterilized
petridishes (petriplates), allowed solidifying and
then incubated.
• After incubation colonies appear on the surface.
• Link: https://microbeonline.com/pour-plate-
method-principle-procedure-uses-dis-
advantages/
9.
10. Disadvantages of Pour plate method
1. The microorganisms are trapped beneath the
surface of medium when it solidifies. Hence,
surface as well as subsurface colonies are
developed and it is very difficult to isolate and
count the subsurface colonies.
2. This method is tedious, time consuming and
requires skill.
3. The microorganisms are subjected to hot shock
because liquid medium is maintained at 45°C
temperature.
4. This method is unsuitable for isolation of
psychrophile bacteria.
12. a) SERIAL DILUTION
This method is commonly used to obtain pure cultures of those
microorganisms that have not yet been successfully cultivated on solid media
and grow only in liquid media.
A microorganism that predominates in a mixed culture can be isolated in pure
form by a series of dilutions.
The inoculum is subjected to serial dilution in a sterile liquid medium, and a
large number of tubes of sterile liquid medium are inoculated with aliquots of
each successive dilution.
• Link: https://www.sciencedirect.com/topics/earth-and-planetary-
sciences/dilution
13. • If we take out 1 ml of this medium and mix it with 9 ml of fresh
sterile liquid medium, we would then have 100 microorganisms in 10
ml or 10 microorganisms/ ml.
• If we add 1 ml of this suspension to another 9 ml. of fresh sterile
liquid medium, each ml would now contain a single microorganism.
• If this tube shows any microbial growth, there is a very high
probability that this growth has resulted from the introduction of a
single microorganism in the medium and represents the pure culture
of that microorganism.
14. III) Spread plate method
• Links for further
study:https://vlab.amrita.edu/?sub=3&brch=73&sim=213&cnt
=1
• https://jcm.asm.org/content/jcm/29/7/1462.full.pdf
15. Advantages of spread plate method
1. It is a simple method.
2. In this method only surface colonies are
formed.
3. Micro-organisms are not exposed to
higher temperature.
16. IV-MICROMANIPULATOR METHOD
Micromanipulators
• Micromanipulators have been built, which permit one to pick out
a single cell from a mixed culture. This instrument is used in
conjunction with a microscope to pick a single cell (particularly
bacterial cell) from a hanging drop preparation.
ADVANTAGES OF MICROMANIPULATOR METHOD
• The advantages of this method are that one can be reasonably
sure that the cultures come from a single cell and one can obtain
strains with in the species.
DISADVANTAGES
• Disadvantages are that the equipment is expensive,
• Its manipulation is very tedious, and it requires a skilled operator.
17.
18. Maintenance and Preservation of
Pure Cultures
• Once a microorganism has been isolated and
grown in pure culture, it becomes necessary to
maintain the viability and purity of the
microorganism by keeping the pure cultures free
from contamination.
• Normally in laboratories, the pure cultures are
transferred periodically onto or into a fresh
medium (subculturing) to allow continuous
growth and viability of microorganisms.
• The transfer is always subject to aseptic
conditions to avoid contamination.
19. Objectives of Preservation
To maintain isolated pure cultures for extended
periods (future use) in a viable conditions.
To avoid the contamination
To restrict genetic change(Mutation)
20. Application of Preservation
• Academic Use:
• Research Purpose:
• Fermentation Industry:
• Biotechnological Field:
• Once a microorganism has been isolated and grown in pure culture, it becomes
necessary to maintain the viability and purity of the microorganism by
keeping the pure culture free from contamination.
• Normally in laboratories, the pure cultures are transferred periodically onto or
into a fresh medium (sub-culturing) to allow continuous growth and viability of
microorganisms. The transfer is always subject to aseptic conditions to avoid
contamination.
• Since repeated sub-culturing is time consuming,
• It becomes difficult to maintain a large number of pure cultures successfully
for a long time.
• In addition, there is a risk of genetic changes as well as contamination.
• Therefore, it is now being replaced by some modern methods that do not need
frequent sub-culturing.
• These methods include refrigeration, paraffin method, cryopreservation, and
lyophilization (freeze drying).
21. Methods of Preservation
1. Periodic transfer to fresh media (Sub-
culturing)
2. Storage in sterile soil
3. Storage at low temperature
4. Preservation by overlaying cultures with
mineral oil
5. Freeze dying/Lyophilization
22. 1. Periodic transfer to fresh media
• Strains can be maintained by periodically preparing a fresh culture from the previous
stock culture.
• The culture medium, the storage temperature, and the time interval at which the
transfers are made vary with the species and must be ascertained beforehand.
• The temperature and the type of medium chosen should support a slow rather than a
rapid rate of growth so that the time interval between transfers can be as long as
possible.
• Many of the more common heterotrophs remain viable for several weeks or months on
a medium like Nutrient Agar.
• The transfer method has the disadvantage of failing to prevent changes in the
characteristics of a strain due to the development of variants and mutants.
23. Advantages
1. It is a simple method, any special apparatus
are not required.
2. Easy to recover the culture
Disadvantage
1. Risk of contamination is more
2. It may be possible to change in genetic and
biochemical characteristics
24. 2. Lyophilization (Freeze-Drying)
• Freeze-drying is a process where
water and other solvents are
removed from a frozen product
via sublimation.
• Sublimation occurs when a frozen
liquid goes directly to a gaseous
state without entering a liquid
phase.
• It is recommended using slow
rates of cooling, as this will result
in the formation of vertical ice
crystal structures, thus allowing
for more efficient water
sublimation from the frozen
product.
25.
26. Procedure
• In this process, a dense cell suspension is placed in
small vials and frozen at -60 to -70°C.
• The vial are immediately connected to a high vacuum
line.
• The ice present in the frozen suspension
evaporates(sublime) under the vacuum.
• This results in dehydration of bacterial cell and their
metabolic activities are stopped; as a result, the
microbes go into dormant state and retain viability for
years.
• The vials are then sealed off under a vacuum and
stored in the dark at 4°C in refrigerators.
27. Advantage of Lyophilization
• Only minimal storage space is required; hundreds of
lyophilized cultures can be stored in a small area.
• Remained viable for more than 30 years.
• Frequent sub-culturing is not required
• Maintained without contamination
• Lyophilised strains remains genetically stable
• Small vials can be sent conveniently through the mail
to other microbiology laboratories when packaged in
a special sealed mailing containers.
• This method is employed for the preservaton of sera,
toxin, enzymes and other biologicals.
28. Storage in sterile soil
• It is mainly applied for the preservation
of sporulating microorganisms (a single
spore (endospore) within the cell).
• Fusarium, Penicillium, Alternaria,
Rhizopus, Bacillus, Aspergillus,
Penicillium, etc. proved successful for
store in sterile soil.
• Soil storage involves inoculation of 1ml
of spore suspension into soil
(autoclaved twice) and incubating at
room temperature for 5-10 days.
• The initial growth period allows the
fungus to use the available moisture
and gradually to become dormant.
• The bottles are then stored at
refrigerator.
• Viability of organisms found around 70-
80 years.
29. Storage at low temperature
• Culture medium can be successfully stored in
refrigerators or cold rooms, when the
temperature is maintained at 4˚C.
• Another: liquid nitrogen can provide long term
preservation of culture. In this method, dense
suspension of microbes is prepared in a medium
containing a protective agent(Glycerol or
dimethyl sulfoxide) which prevent cell damage
due to ice crystal formation. Suspension is sealed
into small ampoules or vials and then frozen at -
150°c
• At this temperature range the metabolic
activities of microbes slows down greatly and
only small quantity of nutrients will be utilized.
• This method cannot be used for a very long time
because toxic products get accumulated which
can kill the microbes.
• 10-30 Years without changing the characteristics.
30. Preservation by overlaying culture with
mineral oil or liquid paraffin storage
• In this method sterile liquid paraffin is poured over the
slant culture of microbes and stored upright at room
temperature.
• Where as cultures can also be maintained by covering
agar slants by sterile mineral oil which is stored at
room temperature or preferably at 0-5°C.
• It limit the oxygen access that reduces the
microorganism’s metabolism and growth, as well as to
cell drying during preservation.
• The preservation period for bacteria from the genera
Azotobacter and Mycobacterium is from 7-10 years, for
Bacillus it is 8-12 years.
Adv:
• Simple
• Mainly used anaerobic microorganism
• Cost effective method
• Can preserve 10-15 years