Maintenance of pure cultures
Contents
• Methods for maintenance of laboratory cultures
– Periodic transfer in fresh media
– Overlaying the culture with sterile mineral oil
– Soil stock cultures
– Lyophillization
– Storage at low temperatures
Intended
learning objectives
At the end of this lecture the student will be able to:
• Outline the need for maintenance of laboratory cultures
• Explain the methods for maintenance of lab cultures
• List the common fungal media
Introduction
Once a microorganism has been isolated and grown in pure
culture, it becomes necessary to maintain the viability and purity of the
microorganism by keeping the pure cultures free from contamination.
Maintenance
of pure cultures -methods
1. Periodic Transfer to Fresh Media
2. Refrigeration
3. Paraffin Method/ preservation by overlaying cultures with
mineral oil
4. Cryopreservation
5. Lyophilization (Freeze-Drying)
6. Preservation in sterile soil
Periodic
Transfer to Fresh Media
• Periodically preparing a fresh culture from the previous
stock culture
• The culture medium, the storage temperature, and the time
interval at which the transfers are made vary with the species
• The temperature and the type of medium chosen should
support a slow rather than a rapid rate of growth so that the time interval
between transfers can be as long as possible
• Disadvantage – failing to prevent changes in the
characteristics of a strain due to the development of variants and mutants.
Refrigeration
• Pure cultures can be successfully stored at 0-4°C either
in refrigerators or in cold-rooms
• Method is applied for short duration (2-3 weeks for
bacteria and 3-4 months for fungi)
• Metabolic activities of the microorganisms are greatly
slowed down but not stopped
• Growth continues slowly, nutrients are utilized and waste
products released in medium
Paraffin
Method/ preservation by overlaying cultures with mineral oil
• This is a simple and most economical method
• Sterile liquid paraffin in poured over the slant (slope)
of culture and stored upright at room temperature.
• The layer of paraffin ensures anaerobic conditions and prevents
dehydration of the medium.
• Microorganisms remain in a dormant state
• The culture can be preserved form months to years
• Advantage – we
can remove some of the growth under the oil with a transfer needle, inoculate a
fresh medium, and still preserve the original culture.
• Disadvantage –
changes in the characteristics of a strain can still occur.
Cryopreservation
• Cryopreservation (i.e., freezing in liquid nitrogen at
-196°C or in the gas phase above the liquid nitrogen at -150°C) helps survival
of pure cultures for long storage times.
• Microorganisms of culture are rapidly frozen in liquid nitrogen
at -196°C
• Stabilizing agents such as glycerol or Dimethyl Sulfoxide
(DMSO) are added that prevent the cell damage due to formation of ice crystals
and promote cell survival.
• This 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 10 to 30 years without undergoing change in
their characteristics, however this method is expensive
Lyophilization
(Freeze-Drying)
• Culture is rapidly frozen at a very low temperature
(-70°C) and then dehydrated by vacuum
• Under these conditions, the microbial cells are dehydrated
and their metabolic activities are stopped
• The microbes go into dormant state and retain viability
for years.
• Lyophilized or freeze-dried pure cultures are then sealed
and stored in the dark at 4°C in refrigerators.
• Freeze-drying method is the most frequently used technique
by culture collection centers.
• Many species of bacteria preserved by this method have remained
viable and unchanged in their characteristics for more than 30 years.
Advantage
of Lyophilization
• Only minimal storage space is required; hundreds of
lyophilized cultures can be stored in a small area
• Small vials can be sent conveniently through the mail to
other microbiology laboratories when packaged in a special sealed mailing
containers
• Lyophilized cultures can be revived by opening the vials,
adding liquid medium, and transferring the rehydrated culture to a suitable
growth medium.
Preservation
in sterile soil
• Spores of bacteria can be preserved in sterile soil
• Spore suspension is added to sterile soil
• Moisture is quickly removed and the cap is replaced
Fungal
media
Two general types of culture media are essential to ensure
the primary recovery of all clinically significant fungi from clinical
specimens.
1. One medium should be nonselective (such as Brain Heart
Infusion Agar; that is, one that will permit the growth of virtually all
clinically relevant fungi
2. Other medium should be selective, specially tailored to
isolate specific pathogenic fungi.
• Brain-heart
infusion (BHI) agar: It is a nonselective fungal culture medium that
permits the growth of virtually all clinically relevant fungi. It is used for
the primary recovery of saprophytic and dimorphic fungi.
• Inhibitory mold
agar (IMA): Primary recovery of dimorphic pathogenic fungi. Saprophytic
fungi and dermatophytes will not be recovered.
• Mycosel/Mycobiotic
agar:
– It is generally Sabouraud’s dextrose agar with
cycloheximide and chloramphenicol added.
– It is used for the primary recovery of dermatophytes.
• Niger Seed Agar:
It is used for the identification of Cryptococcus neoformans.
• Potato Dextrose
Agar (PDA): It is a relatively rich medium for growing a wide range of
fungi.
• Sabouraud’s Heart Infusion (SABHI) agar: Primary recovery
of saprophytic and dimorphic fungi, particularly fastidious strains.
• Potato flake agar:
Primary recovery of saprophytic and dimorphic fungi, particularly fastidious
and slow growing strains
Sabouraud’s dextrose
agar (SDA):
• Sabouraud’s agar is sufficient for the recovery of
dermatophytes from cutaneous samples and yeasts from vaginal cultures.
• Not recommended as a primary isolation medium because it
is insufficiently rich to recover certain fastidious pathogenic species,
particularly most of the dimorphic fungi.
• Sabouraud’s dextrose agar (2%) is most useful as a medium for
the subculture of fungi recovered on enriched medium to enhance typical sporulation
and provide the more characteristic colony morphology.
Summary
• Cultures should be maintained in their original state to
study their characteristics
• Methods for maintaining cultures are – periodic transfer,
overlaying with mineral oil, lyophilisation, low temperature storage
• Depending on the requirement each of the above method is
chosen
• Some common fungal media include Sabouraud’s dextrose agar
(SDA), Potato Dextrose Agar (PDA), Brain-heart infusion (BHI) agar, Inhibitory
mold agar (IMA)
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