Do you want the bacteria to be stable? Is glycerol cryopreservation reliable?

The long-term stable storage of bacterial strains is the basis for microbial research and production applications. If improper storage leads to bacterial inactivation or genetic variation, the results of previous experiments may be wasted. The glycerol cryopreservation method has become one of the commonly used microbial preservation methods in laboratories due to its simple operation, low cost, and long storage period.

As a professional service provider of laboratory consumables, Aijin Biotech has always been committed to providing scientific researchers with high-quality, stable and reliable consumable support, covering many aspects such as strain preservation, liquid operation, cell culture, etc., to help the efficient conduct of scientific research experiments.

PART 01

Advantages of glycerin cryopreservation method

glycerol cryopreservation

01 Maintain bacterial viability for a long time

In an environment of -80°C or lower, adding glycerin can prolong the survival time of microorganisms (such as bacteria, yeast, etc.) and can usually be stored for several years or even ten years, providing a stable source of bacteria for scientific research and industry.

02 Reduce ice crystal damage to bacteria

During the freezing process, ice crystals formed by water can damage cell membranes and organelles, causing bacterial death. As a cryoprotectant, glycerin can play a role in the following three aspects: first, it lowers the freezing point of the solution so that the solution freezes at a lower temperature; second, it reduces the formation of ice crystals and reduces the growth rate and size of ice crystals; third, it combines with water molecules to reduce the free water content that can be used to form large ice crystals, while stabilizing cell membranes and protein structures, thereby effectively avoiding cell damage due to the formation of ice crystals.

03 Maintain genetic stability of strains

Ultra-low temperature almost blocks microbial metabolism, which can effectively reduce the risk of genetic mutation or drift, effectively maintain the original genetic characteristics of bacterial strains, and is an important guarantee for ensuring experimental repeatability.

04 Easy operation and wide applicability

The materials required for the glycerin cryopreservation method are common, and the operating steps have been standardized, so novice experimenters can quickly master it. This method is suitable for most bacteria (including aerobic and anaerobic bacteria), yeast, some filamentous fungi and some phages. However, for sensitive bacterial species, the glycerol concentration can be adjusted or used with other protective agents (such as DMSO).

05 Easy to use and cost effective

Bacteria are usually packaged and frozen in cryovials in small portions of 1-2 mL. When used, only one tube needs to be taken out for recovery without repeated passaging or streaking operations, which not only reduces the risk of contamination but also reduces the possibility of bacterial strain variation. Compared with liquid nitrogen preservation or commercial strain preservation services, the glycerin cryopreservation method is lower in cost and has a higher cost performance.

PART 02

Operation procedure of bacterial glycerin cryopreservation

Operating procedure

All operations must be performed under sterile conditions, and it is recommended to perform them in a biological safety cabinet or clean workbench.

01 Preparation

Preparing a 50% (v/v) sterile glycerol solution is the first step. Thoroughly mix 50 mL of analytically pure glycerin (high pressure sterilization or 0.22 μm membrane filtration sterilization is recommended) and 50 mL of sterile distilled water (normal saline or culture medium can also be used). Although glycerin itself has certain bacteriostatic properties, sterilization or filtration is necessary to ensure the sterility of the solution.

02 Culture preparation

plate method

Use a sterile cotton swab or inoculating loop to pick an appropriate amount of bacterial lawn (about the size of a pea) from a fresh, healthy plate that is in the late logarithmic growth phase, and suspend it in 0.5 - 1mL sterile saline or corresponding liquid culture medium to make a concentrated bacterial suspension.

liquid culture

Inoculate the bacterial strain into a suitable liquid culture medium (such as LB broth), and culture it with shaking under suitable conditions. When the OD600 of the bacterial liquid reaches about 0.6 - 1.0 (there are slight differences among different bacterial strains), that is, when the bacterial strain is in the late logarithmic growth phase, it can be used for preservation. The bacterial cells at this stage are in good growth condition and have a higher success rate of preservation.

03 Bacterial liquid mixed with glycerin

Mix the prepared concentrated bacterial suspension or log-phase bacterial solution with a sterile 50% glycerol solution to maintain the final glycerol concentration at 15% - 20% (v/v). For example, if you mix 0.6 mL of 50% glycerol with 1.4 mL of bacterial liquid, you can get 2 mL of bacterial suspension containing 15% glycerol; if you mix 0.8 mL of 50% glycerol with 1.2 mL of bacterial liquid, you can get 2 mL of bacterial suspension containing 20% ​​glycerol. When mixing, gently turn up and down or vortex to ensure that the bacterial solution and glycerin are fully mixed.

04 Packaging and labeling

Quickly dispense the evenly mixed bacterial solution-glycerin mixture into sterile cryopreservation tubes, with 0.5 - 1.0 mL in each tube. At the same time, the strain name/number, storage date, glycerol concentration, operator name and other information should be clearly marked on the tube wall and tube cap to facilitate subsequent accurate access and management.

Aijin Biotech provides cryopreservation tubes of various specifications, all made of imported medical-grade polypropylene (PP) material, which is extremely transparent, non-toxic and odorless, and has excellent sealing and chemical resistance. Not only can it withstand high temperature and autoclave sterilization at 121°C/15psi, but it can also remain stable within the temperature range of -196°C to 121°C, ensuring safe use in experiments.

05 Cryopreservation

The cryovials can be pre-cooled in a -20°C refrigerator for 1-2 hours, and then transferred to a -80°C ultra-low temperature refrigerator for quick freezing; you can also use a special programmed cooling box (such as Mr. Frosty). Slow freezing (such as overnight freezing at -20°C) needs to be avoided because slow freezing will form larger ice crystals and cause damage to cells. If long-term storage is required, the cryovials can be placed in a liquid nitrogen gas phase environment, but repeated freezing and thawing should be avoided.

PART 03

Bacterial recovery

Strain resuscitation

01 Fast defrost

After taking out the target cryovial from the -80°C refrigerator, immediately place it in a 37°C water bath (or a water bath suitable for the growth temperature of the bacteria) and shake gently to allow the ice crystals to completely melt within 30 seconds to 1 minute. Avoid slow melting at room temperature to prevent loss of activity.

02 Disinfection and vaccination

Wipe the outer wall of the cryopreservation tube with 70% alcohol for disinfection, open the tube cover in a sterile environment, use a sterile tip or inoculation loop to take a small amount of the thawed bacterial solution (usually 1-2 loops or 10-50 μL), streak it onto a suitable solid plate culture medium, and then culture it under appropriate conditions. It should be noted that the thawed bacterial solution should not be frozen again. A new cryogenic tube must be taken for each use to ensure the quality of the bacteria.

PART 04

Things to note

Points for Attention

01 Strict aseptic operation

Aseptic operation is the key to successful preservation, and contamination at any stage may lead to preservation failure.

02 Control glycerol concentration

15%-20% is a commonly used glycerol concentration range, but different strains may require optimization. If the concentration is too low, the protective effect will be insufficient; if the concentration is too high (more than 25%), it may cause cells to become dehydrated due to osmotic pressure imbalance.

03 Choose the appropriate bacterial age

Be sure to use healthy bacteria in the late logarithmic growth phase. Cells in the senescent phase have a lower survival rate and will affect the preservation effect.

04 Control freezing and thawing speed

Rapid freezing (direct freezing at -80°C or programmed cooling) and rapid thawing can significantly improve the survival rate of bacteria. Slow operation will cause irreversible damage to cells.

05 Stable storage environment

-80℃ is the standard storage temperature. The number of times the refrigerator is opened and closed should be minimized to avoid temperature fluctuations. If conditions permit, liquid nitrogen storage has better effects, but the cost is relatively high.

06 Consider strain characteristics

Not all microorganisms are suitable for glycerol preservation. Some strains that are sensitive to low temperature or osmotic pressure (such as certain archaea, obligate anaerobic bacteria, and some pathogenic bacteria) need to use special methods or protective agents, and strictly abide by biosafety regulations and operate in biosafety laboratories of the corresponding level.

PART 05

High-quality preservation starts with the selection of consumables

Consumables Selection

Strain preservation not only tests operational standards, but also relies on high-quality experimental consumables. The performance of consumables such as cryovials, pipette tips, and reagent bottles will directly affect the activity of bacterial strains and the repeatability of subsequent experiments.

Since its establishment in 2009, Aijin Biotech has focused on the research, development and production of laboratory consumables! The current product system covers more than 900 categories, fully covering scientific research scenarios such as strain preservation, molecular experiments, cell culture, etc., and continues to provide stable and reliable overall solutions for experimental consumables for global scientific research institutions.