Maintenance of pure cultures

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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