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Maintenance and Storage of DH5alpha E.coli cells
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Objective:


To maintain and store the E.coli DH5 alpha cells for further studies.

 

Theory:

 


Effective storage means that the organism is being maintained in a viable state without contamination and changes in genotypic or phenotypic characteristics. The organism must be easily restored to the same condition it was in prior to preservation. Several methods are available for the preservations of the microorganisms but there are two criteria for selecting a  preservation method for a given culture. They are: i) period of preservation desired and ii) nature of culture to be preserved. For different physiological groups of bacteria, specific maintenance requirements exist. Preservation techniques are broadly classified into two types: short term preservation methods and long term preservation methods.

 

1.    Short Term Preservation Methods

 

a)    Direct Transfer to Subculture

 

The simplest method for maintaining short term viability of organisms, most often used for bacteria, is periodic subculture to fresh medium. The interval between transfers varies among organisms. Issues that must be addressed with direct transfer include the medium to be used, the storage conditions and the frequency of transfer. The medium used should support survival of the organism but minimize metabolic process and slow the rate of growth. A medium with too high a nutrient content will induce rapid replication that requires more frequent transfers.   In this method, transfer organisms into screw –top test tubes and to store them in an organized location away from light and significant temperature changes. Storage at low  temperatures slows metabolic processes and maintains viability for longer periods. Usually 5 to 10 representative colonies were used when performing transferring process.



b)    Freezing at -20 ºC


Refrigeration or freezing in ordinary freezers at -20 ºC is sometimes used to preserve organisms for longer than can be accomplished by repeated transfers. The   media used for storage appear important, since preservation times vary from a few months.

 c)    Drying

 

Most of the organisms do not survive drying, some; especially bacteria that form spores and many molds can be dried and stored for prolonged periods. Soil can be stored as a storage medium if it is autoclaved and air dried. Soil should be autoclaved for several hours on two successive days. It is then transformed into sterile glass tubes. Commercial silica gel can also be used in small cotton plugged tubes after heating in an oven to 175 ºC for 1.5 to 2 h. Alternatively, a suspension of 108 organisms can be inoculated onto sterile filter paper strips or disks. The paper is dried in air or under vacuum and is placed in sterile vials. These vials can be stored in the refrigerator for up to 4years, and then single strips or disks can be removed as needed. This method is commonly used for quality control organisms.
 

 

2.    Long Term Preservation Methods



Ultra low temperature freezing and freeze drying (lyophilization) are the methods used for long term storage. There are several steps are involved in this process.

 

a)    Ultra Low Temperature Freezing


Microorganisms can be maintained at temperature of -70°C or lower for prolonged periods. Systems for achieving these temperatures include ultra low temperature electric freezers and liquid nitrogen store units. Storage vials must be able to withstand very low temperature and maintain a seal for their concerns. Polypropylene or glass tubes may be used. Plastic vials with screw tops and silicone washers are much easier to use than glass vials that must be sealed with a flame and then scored and broken to open. To protect microorganisms from damage during the freezing process, during storage, and during thawing, cryoprotective agents are often added to the culture suspension. There are two types of cryoprotective agents: one that enter the cell and protect the intracellular environment and others that protect the external milieu of the organism. Glycerol and dimethyl sulfoxide (DMSO) are most often used for the former; sucrose, lactose, glucose, mannitol, sorbitol, dextran, polyvinylpyrrolidone and polyglycol are used for the latter. Combinations of agents are sometimes used. Cryoprotectants that enter the cell usually provide better protection for bacteria. Of the internally incorporated agents, glycerol usually provides better organism survival than DMSO. Glycerol is added at a concentration of 10% (vol/vol). Prior to use, glycerol is sterilized by autoclaving. Once prepared, it can be stocked at room temperature for months. Organisms are inoculated in a medium that adequately supports maximal growth. Cultures are allowed to mature to late growth or stationary phase before being harvested. Once at this point, broth specimens are centrifuged to create a pellet of organisms.

 

b)    Freeze –Drying (Lyophilization)

 

 Freeze –drying is considered the most effective way to achieve long term storage of most bacteria. The term “lyophilization,” which means “to make solvent loving,” Freeze drying is defined as a controllable method of dehydrating labile products by vacuum desiccation. Better preservation occurs because intracellular ice crystallization contributes greatly to organism loss in the frozen state. Removal of water from the specimen effectively prevents this damage. Among bacteria, the relative viability with lyophilization decreases from spore formers to gram positive bacteria to gram negative bacteria. In addition, dried organisms take up little space, large numbers of vials of organisms can be stored, and organisms preserved in this way can be easily transported long distances at room temperature. Glass vials are used for all freeze dried specimens. Skim milk and sucrose are the two most commonly used cryoprotective agents for lyophilization process.


Cryopreservation is a storage method  to preserve structurally intact living cells and tissues at very low temperature. Cryopreservation of microorganisms in liquid nitrogen at -196 ºC or -150°C is a very reliable method and is generally considered superior to other preservation methods. These methods preserved viability and productive capacity of the microorganisms
 



 

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