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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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