Preservation Of Bacteria By Lyophilization
Microorganisms are prone to pollution, mutation, and even death in the process of use and passage, which often cause the decline of strains and may cause the loss of fine strains.
Lyophilization of bacteria is the most effective way for preserving bacteria, and the significance of preservation of bacteria by lyophilization is to maintain the stability of their original traits and vitality as much as possible, to ensure that the bacteria do not die, mutate, or be contaminated, so as to meet the needs of research, exchange, and use.
The procedure of preservation of bacteria by lyophilization
1)Prepare the ampoule tube. The ampoule tube used for the preservation of freeze-dried strains should be made of neutral glass. The tube can have a long-necked spherical bottom, which is also known as a teardrop ampoule tube. Request sizes: outer diameter: 6-7.5mm; length: 105mm; diameter of the spherical bottom: 9-11mm, and wall thickness: 0.6-1.2mm. A tubular ampoule tube without such a bottom can also be used.
(2)Prepare strains. Lyophilization of bacteria can keep the bacteria for several years to ten years. In order to make no mistakes during the preservation of bacteria by lyophilization, the strains used must be paid special attention to. First, the strains should have high purity, and then the strains need to be cultivated in the most suitable medium under the most suitable temperature to make a good culture. Lyophilization of bacteria also requires the age of bacteria — which should exceed the logarithmic growth period. If the bacteria within the period are used for preservation, the survival rate will be reduced. Generally, bacteria require 24-48 hours of culture.
(3)Prepare bacterial suspension and sub-packaging for the lyophilization of bacteria. Take the bacterial slope as an example, add about 2ml of defatted milk to the inclined test tube to make a concentrated bacterial solution, and 0.2ml of the solution is dispatched to each ampoule tube.
(4)Freezing: The preservation of bacteria by lyophilization needs equipment. There are complete sets of equipment sold for the lyophilization of bacteria, which are expensive. The methods and devices described here are simple and much cheaper, and they also can achieve the same purpose.
Place the packed ampules in a low-temperature refrigerator for freezing. Dry ice (solid CO2) alcohol or dry ice acetone, of which the temperature can reach -70 ℃, can replace the refrigerator. Insert the ampoule tube into the refrigerant and freeze it for only 4 to 5 minutes.
(5) Vacuum drying: Vacuum drying is an important part of the preservation of bacteria by lyophilization. In order to keep the sample frozen during vacuum drying, a freezing tank needs to be prepared. Place the crushed ice cubes and table salt in the tank, mix them evenly, and cool to -15 ℃. Install the instrument and put the ampoule tube into the dry bottle in the freezing tank. Generally, if the vacuum degree can reach 93.3Pa (0.7mmHg) within 30 minutes, the dry matter will not melt. Continue pumping for a few hours, and you can observe with the naked eye that the dried object has become dry.
(6) Sealing: Sealing is the last step for the preservation of bacteria by lyophilization. After drying, take out the ampoule tube and connect it to the glass tube for sealing. You can use the L-shaped five-way tube to continue pumping, which can reach 26.7Pa (0.2mmHg) in about 10 minutes. Under the vacuum state, a fine flame of a gas blowtorch is used to seal the center of the ampoule neck. After sealing, store the tube in the refrigerator or in a dark place at room temperature.
Lyophilization of bacteria is one of the most effective methods for the preservation of bacteria. It is applicable to microorganisms with strong viability, even for some pathogenic bacteria that are difficult to preserve, such as meningococcus and gonorrhea. It is suitable for long-term preservation of strains, which can be stored for several years to more than ten years, but the equipment and operation are relatively complicated. Hawach’ s vacuum freeze drying machine can be used for the preservation of bacteria by lyophilization, contact us for more information about our products and services.