Vaccine strains, mostly refer to the finished vaccine strains produced by China’s specific strain preservation centers, stored in ampoules under liquid nitrogen. According to Chinese regulations, it is necessary to purchase the strain from the specific strain preservation center of China, and it is not allowed to use the vaccine strain from elsewhere.

Common strain preservation methods

1. Basic principles of strain preservation

According to the physiological and biochemical characteristics of microbial strain vaccines, under the standard conditions created by human efforts, try to reduce the metabolic intensity of microbial strain cells, so that the cells are basically in a dormant mode, the growth and breeding are inhibited but will not die, to reduce the mutation rate of the vaccine strain. Ultra-low temperature, drying, hypoxia, and vitamin A deficiency are all inhibitory to the metabolic activity of microbial strains.

Ultra-low temperature, drying, and vacuum pump are the key methods for culture medium preservation. When selecting a storage method, the first consideration should be whether the method can maintain the original characteristics of the vaccine strain for a long time, and at the same time, it should also take into account the economy and simplicity of the method. In specific work, usually use a variety of standards at the same time to improve the storage effect.

2. Culture medium preservation process

Select the characteristic pure bacterial bodies;

Identify the suitable bacterial shape for storage;

Supplement the selection of the most suitable storage method;

Regularly inspect the stored vaccine strains to check for changes, if any changes, the storage method must be changed, and the transplant should be carried out immediately upon expiration of the storage period;

3. Agar slope ultra-low temperature storage method

(1) Method:

Vaccinate the typical bacterial bodies of often-used vaccine strains on an inclined slope (some unique vaccine strains can use liquid culture medium), shape according to the specified temperature and time, wait for sufficient growth and development, and then pack the freshly shaped vaccine strain well with packaging paper. To alleviate the evaporation of the culture medium, increase the storage time, you can replace the cotton plug of the culture medium storage tube with a rubber plug. Store in a refrigerator at about 4℃. Transplant once every 2 to 3 months and then start storage again. If semi-solid high-rise culture medium piercing technique is used for shaping, it can generally be stored for half a year to one year. Some even longer.

(2) Application fields: storage of pathogens, yeasts, and molds.

(3) Characteristics:

The advantages are simple, easy for marketing and promotion; generally does not require another selection of storage culture medium; applicable to most microbial strains;

The disadvantage is that the storage period is too short; the frequency of passages is high, which is prone to genetic variation and environmental pollution.

(4) Common problems:

The stored vaccine should be a fresh culture with sufficient vitality, at least the third-generation culture.

During storage, Clostridium tetani can be used with meat broth culture medium; Clostridium sporogenes can be used with fluid thioglycolate culture medium. It is necessary to regularly maintain the temperature, humidity, and cotton plug of the vaccine storage refrigerator, and if any abnormalities occur, they should be dealt with properly.

After each tube transplant, it should be checked one by one with the original vaccine number and name, and confirmed that the characteristics and purity of the culture medium are accurate before resuming storage.

The culture medium for storing vaccines should be low in sugar, and the sugar content of other edible fungi should be less than 2% to prevent excessive acid production, which may affect the survival of the vaccine.

Pseudomonas aeruginosa is prone to autolysis and death in the refrigerator, and is not suitable for this storage method.

4. Liquid paraffin storage method

This method uses paraffin to isolate the culture from the gas, to reduce the physiological and biochemical level of the vaccine, and can prevent water evaporation, thereby extending the storage period of the vaccine:

(1) Method

Vaccinate the vaccine on the slope or puncture it into 0.3~0.5% agar semi-solid high-rise culture medium, prepare the reserve. Take pure liquid paraffin and put it in a tube, 10~15mL per tube, add cotton plug, wrap the bottle cap with paper, sterilize at 121℃ for 30Min, remove it and place it in a 37℃ incubator or 110~170℃ oven for 1~2h or in an air dryer to remove the moisture in the liquid paraffin.

Add the liquid paraffin to the culture tube of the prepared vaccine, the liquid level of the liquid paraffin should be 1cm higher than the top of the culture medium, stand the tube, and store it in a 4℃ refrigerator.

(2) Application field: Suitable for storing some molds, yeasts, and actinomycetes, but has a poor effect on bacterial storage.

(3) Characteristics: This method is simple and easy to use, and is a common storage method in the laboratory. This method mainly isolates the vaccine from the gas.

(4) Common problems:

When storing blood, the vaccine should be applied with fresh culture, and after checking the purity and characteristics, it can be stored;

During the whole process of blood storage, it is necessary to frequently check whether the slope is dry, if it is dry, it needs to be transplanted again;

When supplementing the vaccine, first tilt the vaccine tube so that the liquid paraffin flows to one side, then use the inoculation needle to pick up the culture to vaccinate it on a fresh slope, and after the culture grows a new culture, transplant it once to the new slope before use;

Soak the inoculation needle with a small amount of liquid paraffin in 95% ethanol for a while, then burn it to sterilize to prevent liquid paraffin from splashing during burning under the alcohol flame lamp, which may cause environmental pollution;

The height-to-width ratio of liquid paraffin in the vaccine tube should be strictly controlled, as too much can affect the gas exchange of the vaccine, resulting in poor storage effect; too little will make the slope very easy to dry out, reducing the storage period. Generally, it is better to be 1cm higher than the slope;

When obtaining sterile detection liquid paraffin with soft tubes, the amount of liquid in each tube should not be too much, otherwise it is easy to contaminate the culture medium of the vaccine during unpacking.