ISOLATION, ENRICHMENT CULTURE AND PRESERVATION

TECHNIQUES

•Micro organisms are present everywhere

•Isolation of microorganisms from particular environment is useful
to study the diversity
•Environments where microbes are predominant- soil, water,
rhizosphere, plants and animals internal and external parts
•Isolation – to separate the microbes of interest from a mixed
population of an environment and habitat
•Methods – Serial dilution and pour plate method, streak plate
and spread plate
 SERIAL DILUTION TECHNIQUES

Serial dilution involves taking a sample and diluting it through a series of standard volumes
of sterile diluent, e.g. distilled water.
 PLATING TECHNIQUES
Pour Plate: a small amount of inoculum from dilution is added by pipette to the
center of a petridish.

• Molten, cooled agar medium in a test tube or bottle, is then poured into the Petri
dish containing the inoculum.

• The dish is gently rotated to ensure that the culture and medium are thoroughly
mixed and the medium covers the plate evenly.

• Pour plates allow micro-organisms to grow both on the surface and within the
medium
POUR PLATING TECHNIQUES
Spread Plate: spreading the culture/sample evenly over the surface of the growth
medium, so that every cell grows into a completely separate colony
• also known as lawn plates,
• should result in a heavy, often confluent growth of culture
 SPREAD PLATE TECHNIQUE

Colony- macroscopically visible growth of individual cell

in the form of cell clusters

Each colony represents a pure culture

STREAK PLATE TECHNIQUE
 ENRICHMENT CULTURE TECHNIQUES

ENRICHMENT- basic principle is selection

• Select for desired organisms through manipulation of medium and
incubation conditions
• To provide growth conditions that are very favourable for the organisms
of interest and as unfavourable for competing organisms
• Usually followed for microbes present in low numbers in an environment
• Martinus Beijerininck developed the enrichment concept
 ENRICHMENT CULTURE TECHNIQUES
• Two types of enrichment

i) Modifying physical conditions - (isolation of thermophiles)

• Incubate the sample at 550C
• Organisms cannot withstand the temperature will die
• Only those able to grow would have a change and build their
numbers
 ENRICHMENT CULTURE TECHNIQUES
ii) Modifying Nutrient conditions of culture medium
Enrichment isolation of Nitrogen fixing bacteria
• Incubating a soil sample in the culture medium with all ingredients necessary for
growth except Nitrogen
• Nitrogen fixing bacteria will have advantage and increase the number

Enrichment isolation of pesticide degrading bacteria

• a man-made, unnatural compound also called as Xenobiotic
• incubate the soil with pesticide as sole source of carbon
• Only organisms capable of degrading pesticide will grow in high number
• After sufficient period of incubation isolate the organisms by streak or pour plate
ENRICHMENT – NIROGEN FIXING BACTERIA (AZOTOBACTER)
 WINOGRADSKY COLUMN

• A simple device for culturing a large diversity

of microorganisms
• Invented in the 1880s by Sergei Winogradsky
• The device is a column of pond mud and
water mixed with a carbon source such as
newspaper (containing cellulose), blackened
marshmallows or egg-shells
(containing calcium carbonate), and
a sulfur source such as gypsum (calcium
sulfate) or egg yolk
• Incubating the column in sunlight for months results in an aerobic/anaerobic gradient
as well as a sulfide gradient.

• Two gradients promote the growth of different microorganisms such as Clostridium,

Desulfovibrio, Chlorobium, Chromatium, Rhodomicrobium, and Beggiatoa, as well as
many other species of bacteria, cyanobacteria, and algae.
 PRESERVATION TECHNIQUES

Preservation-Storing the microorganisms in the state of dormancy (maintaining

the viability for longer period)
Microorganisms are cultured on maintenance media especially
designed to allow low growth rates and extend the culture life

Types of preservation
• Short term storage
• Medium term storage
• Long term storage
 PRESERVATION TECHNIQUES

Short term storage (1-3 months)

• Storing the microorganisms in 1-3 months
• Storing the bacteria in a slant cultures at refrigerator (4-100 C)
is used
• Generally the metabolic activity of the microorganisms are
slowed down
• Growth will be slow nutrients will be utilized and waste
products produced
• Regular subculture is necessary for bacteria 2-3 weeks, fungi
3-4 months
Sub culturing- transferring of microorganisms from one culture
medium to another under aseptic conditions
 MOLD CULTURE ON DISTILLED WATER

• Refrigerated storage cultures while sub culturing leads to sterile mutant

• To prevent that molds are maintained in sterile distilled water
• They remain viable for several years
• Ideally revived every two years
 MEDIUM TERM STORAGE
Overlying the culture in mineral oil (liquid paraffin
method) -1-10 years
- Remain viable for several years at room
temperature
- Sterile liquid paraffin is poured over (1/4 inch)
a slope culture of the microorganisms and
stored up right at room temperature
- Paraffin prevent dehydration (moisture loss)
of the medium by ensuring anaerobic
conditions
- Microorganisms remain dormant state
- Paraffin 1010C for 1h at hot air oven
 LONG TERM STORAGE

Preservation in glycerol at -400C

• 2ml of glycerol is added on the agar slope
• Liquid broth can also be stored 1ml culture +0.5 ml
sterile glycerol
• The ampoule is placed in a mixture of industrial
methylated spirit and CO2 and freezed rapidly to -700C
• The ampoules are removed from the mixture and
stored in -400C
• Glycerol acts a cryo-protectent and slows down the
metabolic activity
 LIQUID NITROGEN METHOD

• FREEZING in liquid nitrogen at temperature

-1960C
• Cell suspension in the presence of cryoprotectant
glycerol and DMSO (di methyl sulfoxide)
• prevents the formation of ice crystals which may
kill the cells
• Most bacteria can remain viable for 10 to 30
years without under going any change in their
characteristics
 FREEZE DRYING

• Rapid dehydration of microorganisms while

they are in a frozen state (lyophilisation)
• Microbes are protected from the damage
caused due water loss by this methods
• Culture is rapidly frozen at -700C and then
dehydrated by vacuum and the tubes
containing freeze dried cultures are sealed
and stored in refrigerators
• Storing methods in national and international
culture collection centres
• They remain viable for more than 30 years
without changing the characteristics
 STORAGE IN STERILE SOIL
 Widely used to preserving spore forming fungi

Sterile soil is used to grow and store the soil microorganisms

Spore suspensions are added to sterile soil (sterilized for 2-3 hrs at intervals
of 1-2 days) and allow to grow for 10 days.

The mixture is dried at room temperature and stored in a refrigeration

Maintained upto 70-80 years

THANK YOU