Preservation of industrial cell culture

PRESERVATION OF
INDUSTRIAL CELL
CULTURE
R.M.Sagalgile.

Preservation of industrially important
organisms
 Process organism as ‘most precious resource’
 Culture needs to be viable and free of
contaminants
 Objectives of Preservation:
i. To maintain culture for long period in pure culture
form (Retains viability)
ii. To restrict the genetic change (mutations)
iii. To avoid the contamination
 ‘Subculturing’ partially fulfill the needs
 Probability of strain degeneration and risk of
contamination
C.K.Thakur A.C.S. College, New Panvel

Preservation of industrially important
organisms
 Definition: To maintain the isolated pure
culture for extended periods in a viable
condition, without any genetic change is
referred as preservation.
 During the preservation:
 Stop to the microbial growth
 At least lower the growth rate
 No toxic chemicals are accumulated
 Promotion of viability of organism
 Reduction in the mutation frequencyC.K.Thakur A.C.S. College, New Panvel

Methods of Preservation
 Factors to be consider for it are listed below :
 Various practical necessities
 Amount of labour involved
 The storage space,
 Period of storage.
 Numbers of methods are used for
preservation:A. Continuous Metabolism :
(Metabolism continues)
i. Periodic Transfer on media
ii. Oil overlay
iii. Storage in sterile soil
iv. Saline suspension
B. Suspended Metabolism :
(Metabolism inactivated/stop)
i. Freezing
ii. Storage in silica gel
iii. Drying in vacuum
iv. Lyophilization

 Other way of classification :
 Storage at reduced temperature
 Storage on agar slopes
 Cryopreservation- storage below -135o C
 Storage in dehydrated form
 Dried cultures
 Lyophilization

 Storage at reduced temperature:
 Storage on agar slopes
 Procedure –
 1. Prepare a slant with suitable medium
 2. Streak a suspension of desired organism on slant.
 3. Incubate the slant at appropriate temperature.
 4. On next day observe the slant for sufficient growth.
 If growth occurs, transfer the slant in refrigerator &
further preserve at 50C or in freezer at -200C.
 Sub culturing interval may be: 6 months, 1 year
(Overlay with St. mineral oil)C.K.Thakur A.C.S. College, New Panvel

 Cryopreservation- storage below -135o C
 Metabolic activities reduced considerably at very
low temperature ( below -135o C, commonly -150 to
-196o C)
 Use of liquid nitrogen or mechanical freezer
 Preferred approach: Storage in the vapor phase of
a liquid nitrogen refrigerator (Smith & Ryan,2012)
 Key issue: Avoidance of intracellular ice formation
 Water becomes amorphous solid, but it’s not a
practical solutionC.K.Thakur A.C.S. College, New Panvel

 Cryopreservation- storage below -135o C
 Role of Cryoprotective agents (CPA):
 Decreases freezing point of water
 Increases its viscosity
 Reduces ice crystal formation
 E.g. 10% glycerol +5-10% Dimethyl sulphoxide (DMSO)
 Revival of cells by rapid thawing in water bath at 37oC
 Advantages:
 Most universally applicable
 Bacteria, fungi, bacteriophages, viruses, algae, yeasts, animal &
plant cells, and animal tissues can be preserved
 Disadvantage:
 ‘General stress response’ when m.o. enters in stationary phase
 Animal cells may undergo Apoptosis.

 Storage in Dehydrated forms:
 Dried Cultures/ Dried Soil Cultures:
 Procedure:
 1. Prepare a spore suspension by using a special medium.
 2. Prepare a mixture of soil (20%), sand (78%) and calcium
carbonate (2%) and distribute it into tubes (a few grams per
tube). and sterilize it for 8 to 15 hours at 130oC.
 3. Add the spore suspension into sterile loam as prepared in step
(2). Allow the growth for about 2 weeks.
 4. Keep the inoculated tubes in a desiccator under vacuum. The
reason behind this is to evaporate the excess water. Then seal
the tubes.
 5. Store the culture tubes in a refrigerator at about 5 to 8 oC
temperature
 1800 Actinomycetes dried on soil about 50% viable after 20
years.
 Silica gel and porcelain beads are also suggested.

 Lyophilization/ Freeze drying:
 It is developed by Raper and Alexander (1945) of national
research laboratory (NRRL), Peoria.
 Procedure:
 1. Grow the bacterial cells on petriplate containing suitable media.
 2. Overlay the growth by 4ml of 10% skimmed milk.
 3. The skimmed milk is a suspending fluid, sterilise it previously by 121oC
for 15min. Instead of this, inositol broth (sterilize for 1210C for 15min) or
inositol serum (sterilize by filtration) may used.
 The role of suspending solution is to prevent over drying and protect
bacteria from mechanical or chemical damage.
 4. Glass ampoules (110mm x 5.5 mm) preciously cotton plugged &
autoclaved are
filled with 0.2 ml of suspension (suspending fluid + bacteria).
 5. Place such a prepared ampoules in rotating mixture of dry ice (solid
CO2) &
o

 Primary drying
Place the ampoules in drying cabinet of freeze dryer. The vacuum pump is
started in order to remove all water from frozen suspension.
 Constriction of ampoules & packing –
Push the cotton plug down to half-length of ampoules with sterile pipette.
Using a narrow flame constrict the ampoules little above to cotton plug to
produce short capillary of 2 mm diameter. Take care to not char the cotton
plug.
 Secondary drying & sealing –
Reduce the moisture content during the secondary drying up to 1%. Take
precaution during handling the ampoules sealing. Seal the ampoules under
vacuum.
 Storage –

 Advantages –
 1. The bacteria can be maintained by lyophilisation up to 20 years.
 2. No sub culturing is required.
 3. The lyophilised strain remains genetically stable.
 4. No risk of contamination.
 5. No need to maintain ultra low temperature.
 6. Ampoules are safe to carry.
 7. Like bacteria, fungi & its spores – proteins, virus and serum are also
preserved for longer time.
 Drawbacks -
 1. Method is costly & needs skilful staff.

Quality Control of Preserved Stock Cultures
 Quality of stocks and Security of storage system
 Smith & Ryan, 2012: Discussed implementation of
Organization for Economic Development and
Cooperation(OEDC)
 Best practice guidelines for validation of preserved
strains.
 Guidelines are intended for national culture collections
as well as collection held by research organizations.

 Key Criteria:
1. Culture should be preserved at least by 2 different methods.
Cryopreserved stocks should be maintained in different
location.
2. The numbers of transfers/generations should be kept to a
minimum, so that reducing the risk of degradation.
3. A bank of identical preserved master cultures must be
established as a definitive source of organism within
organization. (“Crown Jewel” stock by Gershater, 2010)
Working stock culture bank can be souced from master
culture bank (MCB)
4. The inoculum for each fermentation run is provided by an
ampoule from the bank of working stock culture. In some
bank working stock culture is used to generate up to 10 agar
slopes.
5. Documentation of both the cultures should be effective to
track the ancestry of individual fermentation.C.K.Thakur A.C.S. College, New Panvel

 Development of Master Culture Bank:
Development of MCB of
100 Vials.
One of which will be
used to generate
‘Working Stock Culture
Bank’.
Each of which will be
used to start a
fermentation.

 Viability test to ensure reproducibility of the
MCB:
i. Garshater described
this procedure.
ii. Applicable to original
culture, master and
working stocks
iii. Depended on sufficient
replication to enable
the application of
analysis

Reference:
 Principles of Fermentation Technology,
Whitakar & Stanbury, 3rd Edition.
Any?
Thank You

Preservation of industrial cell culture

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