|Preservation of microbes|
Microbes are required for the production of fermentation products. They are very valuable for specific product. One product produced efficiently by specific microbe will not be given by all the microbes.
The isolation of a desired organism for a fermentation process may be a time consuming and very expensive procedure and it is therefore essential that it retains the desirable characteristics that led to its selection. Also, the culture used for the fermentation process should remain viable and free from contamination. Thus, industrial cultures must be preserved and maintained in such way as to eliminate genetic change, protect against contamination and retain viability.
Different techniques are used for maintenance and preservation of different organisms based on their properties. Selected method should also conserve the properties of the organisms.
Techniques for the Preservation of microbes are broadly divided into two :
Methods where organisms are in continuous metabolic active state
Methods where organisms are in suspended metabolic state
Continuous metabolic active state preservation technique :
In this technique organisms are preserved on nutrient medium by repeated sub-culturing. In this technique any organisms are stored by using general nutrient medium. Here repeated sub-culturing is required due to depletion or drying of nutrient medium. This technique includes preservation by following methods :
Periodic transfer to fresh media :
Organisms are grown in general media on slant, incubated for particular period of time at particular temperature depending on the characteristics of the selected organisms, then it is stored in refrigerator. These cultures can be stored for certain interval of time depending on the organism and its growth conditions. After that time interval again these organisms are transferred to new fresh medium and stored in refrigerator
Overlaying culture with mineral oil :
Organisms are grown on agar slant then they are covered with sterile mineral oil to a depth of 1 cm. above the tip of the surface. This method is simple; one can remove some organisms in aseptic condition with the help of sterile wire loop and still preserving the initial culture. Some species have been preserved satisfactorily for 15 – 20 years by this method.
Storage in sterile soil :
This method is widely used for preserving spore forming bacteria and fungi. In this method organisms will remain in dormant stage in sterile soil. Soil is sterilized then spore suspension is added to it aseptically, this mixture is dried at room temperature and stored in refrigerator. Viability of organisms has been found around 70 – 80 years
Saline suspension :
Normal Saline is used to provide proper osmotic pressure to organism’s otherwise high salt concentration is inhibitory for organisms. Organisms are kept in screw cap bottles in normal saline and stored at room temperature, wherever required transfer is made on agar slats and incubated
Suspended metabolic state preservation technique :
Organisms are preserved in suspended metabolic state either by drying or storing at low temperature. Microbes when dried or kept at low temperature care should be taken so that their revival is possible.
Drying in vacuum :
In this technique organisms are dried over chemical instead of air dry. Cells are passed over CaCl2 in a vacuum and then stored in refrigerator. Organisms survive for longer period of time.
Lyophilization is vacuum sublimation technique. Cells are grown in nutritive media and then placed in small vial, which are then immersed in a mixture of dry ice and alcohol at -78oC. These vials are immediately connected to a high-vacuum line, and when they are completely dried each vial is sealed under vacuum. This is most effective and widely used technique due to long time survival less opportunity for changes in characteristics of organisms and small storage area. Organisms can survive for period of 20 years or more.
Use of liquid nitrogen :
Culture of Microorganisms are grown in nutritive media and then frozen with Cryoprotective agents like Glycerol and Dimethyl Sulfoxide. Frozen culture is kept in liquid Nitrogen refrigerator. Organisms can remain alive for longer period of time.
Storage in silica gel :
Both bacteria and yeast can be stored by this method. By this technique organisms can survive for 1 – 2 years. Finely Powdered Heat sterilized Silica powder is mixed with thick suspension of cell at low temperature.
- Cells should be harvested when actively growing (mid logarithmic phase)
- One method may be used for few organisms or specific organism; all the organism cannot be preserved by anyone technique mentioned above.
Quality control of the preserved stock culture:
Whichever technique is used for the preservation and maintenance of industrially important organisms it is essential to check the quality of the preserved organisms stocks. Each batch of newly preserved cultures should be routinely checked to ensure their quality. A single colony is transferred into a shake-flask to ensure growth of particular kind of microorganism; further shake-flask subculture is used for the preparation of huge quantity of vials. For the assessment of purity, viability and productivity of cultures few vials are tested. If samples fail any one of these tests the entire batch should be destroyed. Thus, by the use of such a quality-control system stock cultures many be retained, and used, with confidence.