V. Gopal Krishna
Bahagian Mikrobiologi
Institute Sains Biol;ogi, Fak. Sains, U.M.
8 Feb. 2012
Types of preservation techniques
 Vegetative
 Silica gel impregnation with skim milk
suspension
 Glycerol stocks
 Storage under liquid nitrogen
 Lyophilisation – freeze drying
Vegetative stocks
 Very useful for frequent usage
 Easy to prepare
 Fast to transfer
 Easy to store
 However, the duration of survival is rather
short – days to months only, and
 Possibility of mutating (changing form)
Silica gel impregnation with skim
milk suspension
 Technique that involves the storing of cultures in the
form of 10% skim milk suspension absorbed by the
granules of non-dyed silica gel granules
 Granules of silica gel are transferred to small test
tubes, cotton plugged and dry-sterilised (180degrees
Celcius for 2 hours)
 The tubes are immersed in ice cubes in a container to
avoid heat build-up when the suspension is placed
 2 mls of 10% sterilised skim milk (sterilised by wet
sterilization – 121 deg. C for 20 minutes) is transferred
to a slant of actively growing culture and the cells
suspended.
 Eight to ten drops of the suspended cells are dropped,
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one drop at a time (to avoid over-heat buid up), onto
the ice-immersed tubes of silica granules.
The granules are vortexed to break up the granules
from aggulitinating and placed back in the ice for a
short while
The tubes are then stored in a dessicator for a week.
At the end of the week, the granules are again
vortexed, covered with parafilm and stored at 4deg. C
until use.
When required, asceptically one or two of the crystals
are placed onto a growth medium.
Glycerol stock-cultures
 Cultures are ensured to be actively growing
 20% glycerol in 2 ml. Bijou bottles or 1ml. Glycerol in
1.5ml. Microfuge tubes are wet-sterilised (121 deg. C for
20 minutes).
 The sterile glycerol is poured onto a slant of culture
and the cells suspended in the glycerol asceptically.
 The suspended cells are then re-transfered to the Bijou
bottle / Microfuge tube, labeled and stored at 20deg. C
or lower.
 When need to use, the culture is quickly thawed under
running water, a few drops transferred asceptically to
the growth medium, and the balance kept back.
Storage under liquid nitrogen
 The culture to be stored is prepared as for the glycerol
stock, but transferred to a microfuge tube, carefully
labelled and transferred to a vertical microfuge holder.
 The whole holder is immersed into a tank of liquid
nitrogen. Liquid nitrogen is replanished regularly in
the tank
 When need to use, one of the tubes is carefully
removed from the holder, thawed rapidly by placing
under running water and part of the contents placed
on a growth medium.
Lyophilisation – freeze drying
 This is the most difficult and the most
expensive of the storage techniques but is
known to be the most preferred of the
storage techniques in view of the longitivity
of the survival of the micro-organism.
 It is the most expensive technique due to the
requirement of a special equipment called a
freeze-dryer.
 The tubes too are special in nature
Process contd.
 It is nearly always,with regards to long term storage of
cell suspensions, that contain greater than 10 to the
power 8 cells ml–1 (Miyamoto-Shinohara et al., 2000;
Costa et al., 2000).
 The reason for preserving high cell concentrations is
on the premise that the majority of cells die during
long term storage, but a sufficient number survive to
enable the continuation of the strain. Bozoglu et al.
(1987) suggest that survival of 0.1% of the original cell
population is a “sufficient number” of survivors of
freeze drying to allow continuation.
Lyophilisation - contd
 The tube used is as shown aside. A
piece of absorbent filter paper is
placed inside the tube. The neck is
constricted by using flame to avoid
the cotton wool to be place after
the constriction from dropping
into the culture portion – at the
base. The cotton wool prevent cross
contamination from other cultures,
whilst drying as well as when the
culture is reconstituted for growth.
The process continued
 The diagram aside shows the
Constriction
tube after it is wet sterilised.
An aluminium foil is wrapped
over the mouth of the tube to
prevent the cotton from
getting wet.
Cotton plug  A suspension of the culture to
be lyophalised is made, as for
the silica stock.
 About 2 mls. of the
suspension is placed onto the
filter paper asceptically ,
Absorbent filter paper
using a pasteur pippette.
 The tube then is kept at
below -40 deg. C to freeze the
sample
The process continued
 The frozen cultures are then loaded onto a freeze dryer,
which is pre-set at -40deg. C
 The drying process commences, where the liquid is
directly converted into the gaseous state, by-passing the
liquid phase.
 The freeze-dried samples are then paced in an holder of
the lyophilised tube and sealed under vacuum.
 The tubes are then stored at 4deg. C until further use
 It must however be noted that the tube can be used only
once. As such, several tubes of cultures are prepared at a
time.
Summary points
 Among the five methods, the most preferred is the
lyophilised form of storage, where 10%skim milk is the
medium of storage
 The results from bacterial strain recovery efforts
following hurricanes Katrina and Rita are reported.
Over 90% of strains frozen in 10% skim milk were
recovered whereas various recovery rates were
observed for glycerol-stored stocks (56% and 94% of
Escherichia coli, depending upon the laboratory).
References
 Cody L.W, 2008, Skim milk enhances the preservation
of thawed − 80 °C bacterial stocks
 Lech, K., Brent, R.,1993. Growth on Solid Media. In:
Ausubel, F.M., Brent, R., Kingston, R.E.,Moore, D.,
Seidman, J.G., Smith, J.A., Struhl, K. (Eds.), Current
Protocols in Molecular Biology. John Wiley and Sons,
New York, NY. 1.3.5.