Bacterial and Fungal Preservation Methods

  • Biopreservation is the process of preserving the integrity and functionality of cells.
  • Most bacteriological laboratories maintain stock cultures of microorganisms for educational, research, bioassay, industrial, or other purposes.
  • A wide variety of techniques are available for the preservation of bacteria and it may be difficult to choose a method for a particular strain, which not only assures survival, but which also makes certain that the genotype and hence the unique characteristics do not change. 
  • The primary aim of culture preservation is to maintain the organism alive, uncontaminated, and without variation or mutation, that is, to preserve the culture in a condition that is as close as possible to the original isolate.

Bacterial and Fungal Preservation Methods

Agar Slant Cultures

  • All microbiology laboratories preserve micro-organisms on agar slant.
  • The agar slants are inoculated and incubated until good growth appears.
  • They are then covered with sterile mineral oil to a depth of 1 cm above the tip of slant surface.
  • The slants are incubated for 24hr or more and are then stored in a refrigerator.
  • These cultures are periodically transferred to fresh media.
  • Transfers are made by removing a loop full of the growth, touching the loop to the glass surface to drain off excess oil, inoculating a fresh medium and then preserving the initial stock culture.
  • Time intervals at which the transfers are made which varies with the origin and condition of growth.
  • This is a simple and most economical method of preserving bacteria and fungi where they remain viable for several years at room temperature.


  • Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms.
  • This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped.
  • Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime.

Paraffin Method

  • This is a simple and most economical method of maintaining pure cultures of bacteria and fungi.
  • In this 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.
  • This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.

Saline Suspension

  • Sodium chloride in high concentration is frequently an inhibitor of bacterial growth.
  • Bacteria are suspended in 1% salt solution (sublethal concentration in screw cap tubes to prevent evaporation).
  • The tubes are stored at room temperature. Whenever needed the transfer is made on agar slant.


  • Cryopreservation (i.e., freezing in liquid nitrogen at-196°C) helps survival of pure cultures for long storage times.
  • In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol, that prevent the formation of ice crystals and promote cell survival.

Lyophilisation (Freeze-Drying)

  • In this method, the culture is rapidly frozen at a very low temperature (around -70°C) and then dehydrated by vacuum.
  • Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years.
  • Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators.
  • Freeze- drying method is the most frequently used technique by culture collection centres.

The Lyophilisation Process

  • In this process the microbial suspension is placed in small vials.
  • A thin film is frozen over the inside surface of the vial by rotating it in mixture of dry ice (solid carbon dioxide) and alcohol, or acetone at a temperature of −78oC .
  • The vials are immediately connected to a high vacuum line. This dries the organism while still frozen.
  • Finally, the ampules are sealed off in a vacuum with small flame.
  • These cultures can be stored for several years at 40°C.
  • This method is also employed for preservation of toxins, sera, enzymes and other biological material.
  • To revive microbial cultures it is merely necessary to break open the vial aseptically, add a suitable stale medium, and after incubation make further transfers.
  • The process permits the maintenance of longer number of culture without variation in characteristics of the culture and greatly reduces the danger of contamination.

Preservation at Very Low Temperature

  • The organisms are suspended in nutrient broth containing 15% glycerol.
  • The suspension is frozen and stored at -15°C to -30°C.

Preservation by Drying in Vacuum

  • The organisms are dried over calcium chloride in the vacuum and are stored in the refrigerator.



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