Preservation of Microbial cultures

 PRESERVATION OF MICROBIAL CULTURES

·       Different techniques are used for maintenance and preservation of different organisms based on their properties.  

·       Techniques for the Preservation of microbes broadly divided into two categories.

I. Continuous metabolic active state preservation technique.

II. Suspended metabolic rate preservation technique.

I) CONTINUOUS METABOLIC ACTIVE STATE PRESERVATION TECHNIQUE

In this technique, organisms 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.

1) Periodic Transfer

·       Organisms grown in general media on slant, incubated for particular period 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 transferred to new fresh medium and stored in refrigerator.

·       Variables of periodic transfer to new media include transfer frequency, medium used, and holding temperature; this can lead to increased mutation rates and production of variants.

2) Mineral Oil Slant

·       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 and stored at refrigerator temperature.

·       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 preserved satisfactorily for 15 - 20 years by this method.

3) Storage in Sterile soil

·       Storage in Sterile soil is widely used for preserving Spore forming Bacteria and Fungi.

·       In this method, organisms will remain in Dormant stage (a period in an organism's life cycle when growth and development are temporarily stopped) in sterile soil.

·       Soil sterilized then spore suspension added to it aseptically, this mixture dried at room temperature and stored in a Desiccator at refrigeration temperature, or frozen to improve viability.

·       Viability of organisms found around 70 – 80 years.

4) Storage in Saline suspension

·       Normal Saline (0.9 % NaCl) used to provide proper osmotic pressure to organism’s otherwise high salt concentration is inhibitory for organisms.

·       Organisms kept in screw cap bottles in normal saline, stored at room temperature, wherever required transfer made on agar slats, and incubated.

II) SUSPENDED METABOLIC RATE PRESERVATION TECHNIQUE

Organisms preserved in suspended metabolic state by either drying or storing at low temperature. Microbes are dried or kept at low temperature carefully so that their revival is possible.

1) Drying in Vacuum

·       In this technique, organisms dried over chemical instead of air dry.

·       Cells passed over CaCl₂ in a vacuum and then stored in refrigerator.

·       Organisms survive for longer period.

2) Lyophilization (Freeze drying)

·       Lyophilization is Vacuum sublimation Freeze drying technique.

·       Water is removed by sublimation (converting solid into gas), in the presence of a Cryoprotective agent (substance used to protect biological tissue from freezing damage, Example - Glycerol and Dimethyl Sulfoxide); sealing in an Ampule (a small sealed glass capsule containing a liquid) can lead to long-term viability, with 30 years having been reported.

·       Cells grown in nutritive media and then this culture distributed in small vials. These vials culture then immersed in a mixture of Dry ice and Alcohol at -78 °C. These vials immediately connected to a high-vacuum line, and when they are completely dried, each vial sealed under vacuum.

·       This is most effective and widely

3) Refrigeration

·       Washed cultures are stored under refrigeration; these cultures can be viable for 3 to 5 months or longer.

4) Freezing in Growth media

·       Not reliable; can result in damage to microbial structures; with some microorganisms, however, this can be a useful means of culture maintenance.

 

5) Ultrafreezing

·       Liquid nitrogen at -196 °C is used, and cultures of fastidious microorganisms have been preserved for more than 15 years.

·       Microorganisms grown in Nutritive media and then this culture frozen with Cryoprotective agents like Glycerol and Dimethyl Sulfoxide. Frozen culture kept in liquid Nitrogen refrigerator.

6) Storage in Silica gel

·       Both bacteria and yeast stored by this method.

·       By this technique, organisms can survive for 1 – 2 years. Finely Powdered Heat sterilized Silica powder mixed with thick suspension of cell at low temperature.

QUALITY CONTROL OF PRSESERVED CULTURES

Whichever technique 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 routinely checked to ensure their quality. A single colony transferred into a shake-flask to ensure growth of particular kind of microorganism; further shake-flask subculture 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 destroyed. Thus, by the use of such a quality-control system stock cultures retain and used with confidence.