The document discusses various techniques used to isolate microorganisms from mixed cultures to obtain pure cultures. These include streak plate method, pour plate method, spread plate method, loop dilution technique, serial dilution method, and micromanipulator method. The document also discusses methods for preserving isolated pure cultures long-term, such as storage at low temperatures, storage in sterile soil, preservation using mineral oils, and lyophilization or freeze drying. Lyophilization allows cultures to remain viable for over 30 years without sub-culturing and with minimal storage space requirements.

1. ISOLATION TECHNIOUES

2. • Micro-organism in nature never found in pure population.
• Mixed type of m.o.present in soil, air, food, etc.
• So, to study nature of specific m.o. we need isolate it
from mixture.
• So we use following technique to isolate pure culture
 Streak Plate method
 Pour plate method
– Loop Dilution technique
– Serial dilution method
Spread Plate technique
Micromanipulator method
Roll Tube Method

3. Streak Plate method

4. • Simple method widely used for
isolation of pure cultures.
• Prepared by streaking small amount
of mixed culture over surface of
solid medium with inoculating loop.
• Thin streaking separate m.o.
• We carry out streaking in 4 zones
last streak separate m.o.
• Follow link
• https://www.youtube.com/watch?v
=c6v84FQ36kM
• https://www.youtube.com/watch?v
=_1KP9zOtjXk
• https://www.youtube.com/watch?v
=D9MqxWj6SKI

6. Pour Plate Technique
• In this method mixed culture diluted in tube which
contains nutrient agar
• Medium maintained in liquid state by maintaining
temperature .I
• Innoculated medium transferred to petriplates. Allowed
to solidify and incubated.
• A series of petriplates shows decease no of colonies.
• https://www.youtube.com/watch?v=nViO9Y4Yxfk

9. Spread Plate Method
• In this technique mixed culture not diluted in culture
medium.
• It is diluted in series of tubes containing sterile water.
• Sample removed from each dilution tube (0.1ml)placed
on to surface of agar plate.
• Spread culture by using glass spreader on nutrient agar
plate.
• Incubate plate & observe isolated colonies.
ADVANTAGE
• Simple method.
• Only surface colonies are formed.

11. Micromanipulator method
• It is device which pick up single microbial cell from colony of
mixed culture.
• Single cell sucked into micropipette & transferred to culture
medium.
Roll Tube method
• Used for isolation of strick anaerobes.
• Stoppered anaerobic culture tube is used for isolation which has
been coated with prereduced agar medium with oxygen free
nitrogen.
• When stopper removed tibe kept anaerobic by continuously
flushing oxygen free carbon dioxide from cannula.
• Inoculation done with inoculating loop on agar surface from
bottom to top by motor.
• Then tube is restopered and incubated anaerobically to get
colonies

13. Preservation of bacterial culture
• To maintain isolated pure culture for large period in viable
condition, without any genetic changes refer as
preservation.
• Culture preserve by avoiding contamination.
• Objective of preservation as follows:
Academic use
 Fermentation industry
Biotechnology field
Research Purpose

14. • 1. Periodic transfer on fresh media:
• In lab m.o. preserve on slants. After incubation this slants are
store in refrigerator.
• This culture periodically transfer to fresh medium.
• Time interval at which transfer made varies from species to
species.
• 2. Storage at low temperature:
• Culture stored at low temperature in refrigerators where
temperature maintained 4oC
• But this method used for short preservation sub culturing is
necessary after 4 weeks.
• Another method very low temp , liquid nitrogen provided.
• In this method m.o. suspension made into protective agent e.g.
glycerol, dimethyl sulfoxide it prevents cell damage due to ice
crystal formation.
• By using this method cells viable for 10-30 years.

15. • 3. Storage in sterile Soil:
• This method is used for sporulation m.o.
• Pure culture of m.o. kept in sterile soil & stored for number
of month s under refrigeration.
• 4. Preservation by overlapping culture with
mineral oils:
• Bacterial species preserved by covering growth on agar
slants with sterile mineral oils or liquid paraffin.
• Oil must cover slant completely.
• In this method we can remove some growth by inoculating
loop.
• Simple method used for anaerobic m.o.
• Cost effective
• Preserve cultures for 15-20 years.

16. 5. Lyophilization or Freeze Drying
• It preserve different type of m . o.
• In this method a dense microbial suspension placed into vials &
frozen at -60 to -78oC.
• Vials are immediately connected to high vacuumed line.
• The ice present into suspension get evaporate under vacuum. This
result in dehydration of bacteria. With no damage to bacterial cell
structure.
• The vials are seal & stored into refrigerator.
Advantages:
• Culture preserve by this method viable for more than 30 years.
• Sub culturing not require.
• Strain remain genetically stable.
• Minimum storage space is required.
• Revived by transferring rehydrated culture to suitable growth
medium.
• By using this method we can store vaccines, toxin, sera enzyme etc.

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