PRESERVATION METHODS

SHORT-TERM PRESERVATION METHODS

OF YEASTS, FUNGI AND BACTERIA

MANAJEMEN KONSERVASI MIKROORGANISME 2020

OUTLINE

➢ Introduction.
➢ Short-term preservation.
➢ Subculturing: Subculturing in broth, Subculturing on
agar slants.
➢ Storage under liquid paraffin.
➢ Storage in glycerol in deep freezer.
➢ Storage in distilled water.
 INTRODUCTION

➢ Preservation of microbial culture is to maintain pure

culture for extended period in a viable condition
without any genetic change.

➢ During preservation, most important factor is to stop

microbial growth or at least lower growth rate.

➢ Due to this, toxic biochemicals (metabolic wastes) are

not accumulated and hence viability of
microorganisms is not affected.
 OBJECTIVES OF PRESERVATION

➢ To maintain pure cultures for extended periods in

a viable condition without any genetic change.

➢ To avoid contamination.

➢ To restrict genetic change (mutation).

 SHORT-TERM PRESERVATION
➢ Short-term preservation involves maintenance of
cultures for up to 1 year.
➢ Cell division and metabolism is not completely
arrested.
➢ The method is simple, inexpensive and widely used.

➢ Most fungal cultures can be maintained by serial transfer.

➢ Periodic transfer is a good option for small collections with
cultures in constant use for short periods (less than 1 year).

➢ The method has several disadvantages; time consuming, labor

intensive, cultures must be checked frequently for contamination
by other microorganisms and for drying, the morphology and
physiology of a cultured fungus may change over time.
 SUBCULTURING
It is applicable to a wide range of microorganisms.

This method consists of:

✓ inoculation of a suitable medium contained in a tube or bottle.
✓ incubation at an appropriate temperature to obtain growth.
✓ storage under suitable conditions.

➢ The process is repeated at intervals that ensure the preparation

of a fresh culture before the old one dies → periodic transfer to
fresh medium.

➢ Subculturing method is inexpensive in terms of equipment but

may be labor-intensive if organisms that require frequent
subculture are maintained.
Storage period can be extended by reducing the metabolic
rate of the microorganisms:

➢ Lowering the storage of temperature, i.e. storage at 4°C is

widely used for a variety of microorganisms.
➢ Restricting the availability of air to the culture, i.e. the entire
agar surface and fungal culture should be submerged
completely in the oil, or water.

Fresh cultures in UICC Fresh cultures in

BIOTEC, Thailand
 RISK OF PERIODIC SUBCULTURING

➢ Contamination is a major problem and this risk occurs with

each subculture.
➢ Contaminants may outgrow and kill the original culture.
➢ Mislabeling.
➢ Loss of viability.
➢ Risk of change of characters → Loss of stability of characters.
 SUBCULTURING IN BROTH
Inoculation, incubation, storage and viability check

➢ A loopful from the old stock culture is transferred aseptically to

each of the broth medium in test tubes.
➢ The cultures are incubated, without shaking, at 25-28⁰C for 48
hr (for bacteria and yeasts; until late log phase), 4-5 days for
filamentous fungi.
➢ Each culture is examined visually for growth.
➢ The duplicate cultures (stock cultures) are stored at 4⁰C. Stock
culture should only be opened once.
 SUBCULTURING ON AGAR SLANTS
Inoculation, incubation, storage and viability check

➢ A loopful from the old stock culture is transferred aseptically to

each of the agar slants in test tubes.
➢ The cultures are incubated, without shaking, at 25-28⁰C for 48
hrs (for bacteria and yeasts; until late log phase), 4-5 days for
filamentous fungi.
➢ Each culture is examined visually for growth.
➢ The duplicate cultures (stock cultures) are stored at 4⁰C. Stock
culture should only be opened once.
 STORAGE UNDER LIQUID PARAFFIN

➢ Living cultures are maintained in a reduced metabolic state by

overlaying the cultures with sterile medicinal-grade liquid paraffin.

➢ The average storage life for fungi is about 5-6 years and for
bacteria about 2-4 years, depending on the strains.

➢ This method has been successfully used for fungi, yeasts and some
bacteria.

Smith, 2004, CABI. UICC

 PREPARATION

➢ Preparation of cultures: Cultures are grown on any suitable media.

➢ Sporulating fungi are incubated until sporulation is achieved.

Yeasts and bacteria are grown until late log phase of growth.

➢ Preparation of liquid paraffin: 25 ml amounts of liquid

paraffin/paraffin oil is poured into 100 ml-erlenmeyer flasks and
autoclaved twice at 121⁰C for 15 min. Sterilize filter paper for
draining off excess paraffin oil.
Each culture slant needs about 7-11 ml.

➢ Liquid paraffin/paraffin oil is poured into culture slants showing

good growth. The paraffin oil should extend at least 1 cm above
the upper end of the agar to prevent it drying out.

➢ The culture is stored at 4⁰C.

 Revival of Cultures

➢ Place one sterile filter paper onto a new sterile empty plate.
➢ One loopful of cultures is taken out with an inoculation needle
or loop and is touch aseptically onto sterile filter paper to drain
off paraffin oil.
➢ The oil-free inoculum on the loop is used to inoculate fresh
agar medium (slant or plate).
➢ The slant, or plate, is incubated at 25-28⁰C for several days until
good growth is obtained.
 STORAGE IN DISTILLED WATER

➢ Living cultures are maintained in a reduced metabolic state by

overlaying the cultures with sterile distilled water.
➢ The average storage life is about 5 years.
➢ This method is very simple and inexpensive and is recommended
for fungi, yeasts, plant pathogenic bacteria and actinomycetes.

➢ Preparation of cultures: Cultures are grown on any suitable media.

➢ Sporulating fungi are incubated until sporulation is achieved.
➢ Yeasts and bacteria are grown until late log phase of growth.

Smith, 2004, CABI.

 PREPARATION

➢ Preparation of sterile water: 5 ml amounts of distilled water is

poured into test tube and autoclaved at 121⁰C for 15 min.

➢ For use in the preservation of sporulating fungi, 0.1% Tween 80

is added to the distilled water before autoclaving.

➢ Sterile distilled water (4-5 ml) is added to slants showing good

growth and a thick cell suspension is prepared.

➢ The suspension is transferred to the sterile Eppendorf tubes

and stored at 4⁰C.
 STORAGE IN GLYCEROL IN DEEP FREEZER
Preparation of (4-10%, v/v) glycerol
➢ 87% glycerol is added with distilled water until final
concentration is reached (4-10%, depending on use).

➢ Amount of 25 ml of (4-10%) glycerol is poured into 100-ml

Erlenmeyer flask and autoclaved at 121⁰C for 15 min.

➢ Sterile glycerol (1 ml) is dispensed into each sterile Eppendorf

tube (for storage at 4°C), or cryotube (for storage at -20 °C or
80°C).

➢ A loopful from fresh culture is transferred to each sterile tube.

The tube is sealed with parafilm (for Eppendorf tube). The
tubes are stored at 4⁰C, or -20⁰C or -80⁰C (deep-freezing
method).
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 Short-Term Preservation Versus Long-Term Preservation

Advantages of Short-term Preservation

➢ The method is simple, inexpensive and widely used.
➢ It is applicable to a wide range of microorganisms.

Advantages of Long-term Preservation

➢ Very long periods of storage and allow low maintenance.
➢ The method is reliable and useful: less risk of contamination
and desiccation.
➢ Viability of cultures is maintained.
➢ No change of morphology, physiology /biochemistry, and
genetic of a culture→ stability of characters.
➢ In the long run, the methods is inexpensive and provide
permanent preservation.
(Nakasone et al. 2004. Preservation and distribution of fungal cultures. Elsevier CRC Press. p. 37-47).
 Disadvantages of Long-term Preservation

➢ Need specialized equipment.

➢ High-cost equipment.
➢ The methods is not appropriate for a wide range of
microorganisms.
(Nakasone et al. 2004. Preservation and distribution of fungal cultures. Elsevier CRC Press. p. 37-47).