This document discusses several methods for preserving microbial cultures over long periods of time, including their key features. It describes agar stabs/slants which are inoculated and stored at 5-20°C or with mineral oil, being sub-cultured every 6 months to 1 year. Salt concentrations of 1% are used to preserve bacterial cultures at room temperature. Lyophilization involves drying cultures under vacuum and storing at 4°C for up to 10 years. Cryopreservation uses cryo-protectants like glycerol and dimethyl sulfoxide with storage in liquid nitrogen at -176 to -196°C for 30+ years. Quality control testing is needed to ensure the viability of preserved cultures.

1. COURSE: B.SC. (MICRO)
SUBJECT: ELEMENTARY MICROBIOLOGY
UNIT 4.3
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2.  When you have developed good strains of microorganisms, it
must be preserved properly, so that it could be used in future
also.
 If the cultures have not properly been preserved, their
characteristic features may decline after certain period.
 Therefore, several methods have been developed for
maintaining the organisms in viable conditions over a long period
of time. These methods vary according to strains.
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3.  Agar stabs or slants -prepared after
autoclaving the medium.
 inoculated carefully with individual culture
and incubated overnight.
 stored at 5 to -20°C.
 sub-cultured at an interval of six months.
 Then the cultures are covered with mineral oil
to a depth of 1 cm above the slant. They are
sub-cultured after the storage of about 1
year.
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4.  Bacteria -salts for various metabolic
activities.
 But at high concentration, salts act as
bacteriostatic (temporary growth inhibitor).
 Hence, bacterial culture is preserved in 1%
salt concentration in screw-capped tubes to
prevent evaporation.
 The tubes are stored at room temperature.
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5.  The cultures - dried over CaCl2 in a vacuum,
then stored in a refrigerator.
 At such conditions, the organisms survive for
longer period than the air dried cultures.
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6.  microbial suspension is put in small ampoules and frozen
through drying under vacuum.
 Vacuum drying results in sublimation of cell water.
 Then the ampoules are sealed off in vacuum.
 The lyophilised cultures are stored at 4°C.
 The cultures remain viable for about 10 years.
 By this method a large number of cultures are maintained
without variation in their characteristics.
 Easy to transport
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7.  The cultures are grown in culture tubes.
 10-30% of a cryo-protective agent (e.g. Glycerol or dimethyl
sulfoxide) is added into it so that it could protect the culture from
cold sock.
 The culture is dispensed into ampoules.
 The ampoules are properly sealed and kept in liquid nitrogen at -176
to -196°C.
 The liquid nitrogen method has been successful with many species
that cannot be preserved by Lyophilization and most species can
remain viable under these conditions for about 30 years or more
without undergoing change in their characteristics.
 Expensive
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8.  Whichever technique is used for the preservation, maintenance
of industrially important organisms it is essential to check the
quality of the preserved organisms stocks.
 Each batch of newly preserved cultures should be routinely
checked to ensure their quality.
 A single colony is transferred into a shake-flask to ensure
growth of particular kind of microorganism; further shake-flask
subculture is used for the preparation of huge quantity of vials.
 For the assessment of purity, viability and productivity of
cultures few vials are tested. If samples fail any one of these
tests the entire batch should be destroyed. Thus, by the use of
such a quality-control system stock cultures many be retained,
and used, with confidence.
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