This document discusses various methods for preserving microorganisms. Moist storage involves serial subculture in nutrient-poor media like agar every 1-6 months depending on the organism. Cold storage maintains organisms at refrigeration or freezing temperatures from 4-80°C. Dry storage methods include impregnating organisms in filter paper or gelatin discs then drying and sealing them to preserve organisms for long periods without mutation or contamination risks of moist storage. Freeze drying in vials with cryoprotectants at -70°C or in liquid nitrogen at -196°C also enables long-term preservation of viable microbial cultures.

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 Microorganisms are kept for:
 Control the culture media (testing
media).
 Control the biochemical &
sensitivity testing.
 For teaching purpose.
 For further identification.
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 The bacteria are vary in their ability
to remain a life after they complete
their growth. There are different
methods for preservation of
microorganisms:
 Moist storage.
 Cold storage.
 Dry storage.
 Freeze drying storage (The best one).
 In this method keep the organisms
by serial subculture (interval time)
to keep it is life.
 Use different types of media
according to the type of
microorganisms
 Choice media with low nutritional
substances.
 Semi-solid media (Nutrient agar).
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 Cooked meat media (CMM).
 Blood broth.
 Dorset egg.
 Egg saline.
 Skimmed milk.
 Semi-solid media:
 Staphylococci: Subculture every 1-2 month.
 Enterobacterieace: Subculture every 3
month.
 Blood broth:
 Streptococci: all of them except
pneumococci every one month.
 Dorset egg, Egg saline & skimmed
milk:
 Used for many organisms at least every 6
month subculture.
 CMM:
 Clostriia: subculture every 6-12month.
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 Method:
 Use screw capped container.
 After inoculation (stabbing), incubate
over night.
 Sealing by paraffin wax – fill the
container.
 Make at least duplicate every organism.
 Advantage:
 Easy.
 Practical.
 Disadvantage:
 Cannot prevent mutation.
 There is chance for drying &
contamination.
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 Use low temperature for
preservation the microorganisms.
 Use different degree of temperature:
 4 - 8 C° domestic refrigerator.
 -20 C° - -40 C° deep freezer.
 -70 C° - -80 C° ultra deep freezer.
 -70 C° solid CO2 (Micro-bank methods).
 -196 C° liquid nitrogen methods.
 Some organisms like Niesseria and H.
influenzae cannot preserve by this method.
 4 - 8 C° or -20 C° - -40 C° keep the organisms
for short time.
 -70 C° culture the organism in tube and then
inserted in container CO2.
 Adv. : keep organism for long time – no change
and no need for subculture.
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 Disadv. : expensive – regular filling with
CO2.
 -80 C° ultra deep freezer:
When used it need certain precautions to
avoid ice crystals and concentration of
salt (destroy the organisms) by using:
 Glycerol – Sugar – Dimethyl sulphoxide
(DMSO).
 -196 C° (liquid nitrogen):
 Rapid freezing and rapid rewarming will
avoid formation of ice crystal, and viability
does not affected.
 Disadv: have some disadvantage of
CO2.
 Micro-bank system:
 Commercial supplied cryovials which
are screw capped 2ml container.
 Method: Bacterial suspension add to
the vial which contain
cryopreservative. The organism
absorbed by the glass beads in the vial,
the excess fluid is absorbed and
discarde (storge at -70 C°).
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 Done by several method:
 Filter paper disc methods: Thick
suspension of organism is prepared,
and impregnate the disc of filter paper
with it. Drying take place in a desiccant
under vacuum, over reducing
substance such as phosphorus peroxide
(P2O5), and lastly impregnated with
paraffin oil.
 Slamp method: Similar to previous
method
with some modification in the
reducing substance which is
incorporated in the medium (10%
gelatin with ascorbic acid – paper is
impregnated with the paraffin oil
before the culture – dry as describe
above).
L. method (La page method):
Drying in bottle or prefer tubes or
capillary tube seal it and kept for long
time.
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