Sharing Knowledge is a Great Legacy. MMG

1. Dr. Imran Sajid

2. Experiment: Preservation of industrial
strains (microbial culture) by lyophilization
Materials Required
• 24 hours fresh microbial cultures
• Round Bottom flasks
• Liquid nitrogen or dry ice + acetone
• Glass vials
• Lyophilizer

3. Principle (Theoretical Background)
Preservation of microbial cultures is a basic requirement in
industry. Preservation and storage of industrially important
microbial cultures for long time along with their specific activity
is very important in industries. Industrial strains are stored not
only for few days or months but they need to be stored for many
years to be used in the industries. There are two to theree
objectives of the strains storage.
1. To maintain the isolated pure cultures for a specific period of
time along with the activity or specific industrial function of
the organism
2. To avoid contamination and mixing of cultures
3. To minimize the genetic changes in the cells, as the strains
are used or stored for long time there might happen some
genetic modifications in the strain

4. There are many methods for short term and long term
preservation of microbial strains
1. Storage as agar slants (agar slants are prepared and stored
for few weeks, usually the working culture can be stored like
this)
2. Glycerol stocks (25% glycerol stocks, strains can be kept for
few months to an year or longer based on the storage
temperature, -20 C or -80 C)
3. Preservation by attachment to some solid support like glass
beads etc.
4. Preservation in the vaporous phase of liquid nitrogen (liquid
nitrogen temperature -196 C)
5. Lyophilization

5. Lyophilization (Freeze drying)
It is freeze drying process in which the microbial cultures or any
heat sensitive material to be preserved or concentrated are first
frozen and then are dried under low temperature (-80 C) and high
vacuum pressure. The microbial cultures (broth cultures or cells in
water) are transferred to the round bottom flasks, the flask is
dipped in liquid nitrogen or dry ice/acetone bath and is rotated for
time to freeze the material in the flask. The material inside the
flask freeze and make a shell on the inner walls of the flask (this
provide more surface area to the frozen material). The flasks are
then attached to the lyophilizer which maintains the lyophilization
conditions (ultra low temperature i.e -80 C and high vacuum
pressure). Under these conditions water present in the cells or
solid material sublimes (sublimation process) and is evaporated, so
the cells or solid material is dried. Removal of water from
biological material under these conditions do not damage the
cellular components and the cells can be stored for long time

6. The dried material is shifted to glass vials under aseptic and dry
conditions and vials are closed tightly to avoid moisture
absorbance. The can be stored at room temperature under dry
conditions for many years. For revival the cultures are soacked
in little quantity of water for some time, cells uptake the water
and are transferred to the relevant media for revival and growth.

7. Procedure
1. Take liquid nitrogen in a suitable container use proper PPEs
(Gloves, goggles etc) or alternatively use dry ice/acetone
bath
2. Transfer the 24 h fresh culture in round bottom flask
3. Dip the flask in liquid nitrogen by making an angle of 45 and
rotate the flask un till the material in the flask freezes and
make a shell on the inner walls of the flask
4. Attach the flask with the lyophilizer which is already on and
maintaining the lyophilization conditions (-80 C temperature
and high vacuum pressure).
5. After completion of the process (it may take 10-12 h, the
material in the flask will dry and will appear as like a bird’s
nest) remove the flask and transfer the dried material in to
glass vial, tightly close the vial
Precaution: completely dry the material and avoid moisture
during transferring to glass vials

8. Observations and Results