Decision No. 54/2000/QD-BNNPTNT/KHCN Issuing the industry standard on: Long-term preservation method for agricultural microorganism genetic resources using liquid nitrogen (416-2000)

 

Pursuant to …;

Issuing industry standards on: Long-term preservation method

for agricultural microorganism gene sources using liquid nitrogen (416-2000)

THE MINISTER OF AGRICULTURE AND RURAL DEVELOPMENT

Pursuant to Decree No. 73/CP dated November 1, 1995 of the Government stipulating the functions, tasks, powers, and organizational structure of the Ministry of Agriculture and Rural Development;

Pursuant to Decree No. 86/CP dated December 8, 1995 of the Government stipulating the division of responsibilities for state management over product quality.

Considering the proposal of the Director of the Department of Science, Technology, and Product Quality

Pursuant to …;

Article 1: Now promulgates the following industry standard:

10TCN: 416-2000 Long-term preservation method for agricultural microorganism gene sources using liquid nitrogen consists of three sections with thirteen points.

Article 2: This Decision shall take effect fifteen days from the date of signature.

Article 3: The Directors of the Office, Heads of the Department of Science and Technology and Product Quality, and related organizations and individuals are responsible for implementing this decision.

LONG-TERM PRESERVATION METHOD FOR AGRICULTURAL MICROORGANISM GENE SOURCES USING LIQUID NITROGEN 10TCN 416-2000

TECHNICAL REQUIREMENTS

Hanoi - 2000

Drafting agency: Microbiology Department

Agency proposing issuance: Institute of Science and Technology

Vietnam Agriculture

Reviewing agency: Department of Science and Technology & CLSP

Issuing agency: Ministry of Agriculture and Rural Development

Issued together with Decision No.: /2000-QĐ/BNN-KHCN

date month year 2000

 

STANDARDS

LONG-TERM PRESERVATION METHOD FOR AGRICULTURAL MICROORGANISM GENE SOURCES USING LIQUID NITROGEN 10TCN 416-2000
(Issued together with Decision No.: 2000/QĐ-BNN-KHCN date month year 2000)

Article 1. Scope of Application:

This standard applies to the long-term preservation of aerobic and facultative anaerobic agricultural microorganisms (excluding those in the veterinary field) in their vegetative cell and spore forms under sterile conditions using the liquid nitrogen method.

2. Terms and Definitions:

2.1. Agricultural microorganisms: Various types of microorganisms belonging to bacteria, actinomycetes, cyanobacteria, yeasts, and molds that are subjects of study and use in agriculture, approved by the Ministry of Agriculture and Rural Development.

2.2. Preservation of microorganisms: The storage of pure culture samples after selection under suitable conditions to ensure high survival rates and maintain initial characteristics.

2.3. Long-term preservation: Storage of culture samples for more than two years.

2.4. Biological characteristics: The ability of microorganisms through their living activities to affect plant growth and development, animal husbandry, biological control, and ecological environment.

2.5. Selective culture medium: A medium suitable only for the growth and development of a particular species or group of microorganisms.

2.6. Diagnostic medium (indicator medium): A medium allowing rapid differentiation between one type of microorganism and others.

2.7. Culture sample: A strain collected, selected, bred, or taken from a stable genetic gene pool by the author.

2.8. Transfer interval: The time period between two transfers to maintain the characteristics of microorganisms.

2.9. Survival rate of culture samples: The percentage ratio of surviving cells in preserved culture samples compared to the original culture sample when first cultured and developed stably.

2.10. Strain recovery: The process of transferring microorganisms from preserved samples into appropriate nutrient media to restore initial characteristics.

3. Content:

3.1. Equipment:

- Drying oven (dry sterilization equipment) and autoclave (wet sterilization equipment)

- Incubator

- Sterile culture hood

- Deep freezer

- Sample container for microorganisms containing liquid nitrogen

- Liquid nitrogen storage tank

- Polyolefin gloves

- Polypropylene test tubes with screw caps, volume 2ml, low temperature resistant, sterilizable or neutral glass ampules

- Analytical balance accurate to 0.01g

- Inoculation loop

- Glass spreader

- Glass test tube

- Measuring cylinder

- Triangular flask

- Petri dish (shaker box)

- Graduated pipette, pipetman

- Alcohol lamp or gas lamp

3.2. Procedure:

3.2.1. Preparation of tools:

- Tools used for identifying microorganisms must be sterilized by one of the following methods:

+ In a drying oven at 160 - 175°C for not less than 2 hours.⁰+ In an autoclave at 1atmotphe (121°C) for not less than 20 minutes.

- Preparation of polypropylene test tubes (or ampules): Soak in 2% HCl solution for 12 hours. Rinse thoroughly with tap water 3-4 times and rinse again with distilled water, then sterilize in an autoclave at 1atmotphe (121°C) for 15 minutes.⁰3.2.2. Preparation of media:

- The culture medium for biomass cultivation and checking the survival rate of culture samples is a selective medium (section 2.5).⁰3.2.2. Preparation of media:

Weigh and dissolve the components of the medium in distilled water according to the given order. Distribute the medium into triangular flasks. Cover with cotton plugs and sterilize under appropriate conditions for each type of medium.

- Protective medium: Contains 10% (w/v) skim milk or 10% (v/v) glycerol or 5% (v/v) Dimethyl sulfoxide (DMSO). The protective medium needs to be sterilized by pasteurization, membrane filtration, or at 121°C for 15 minutes.

3.2.3. Preparation of culture samples:

- Each material sample to be retained should have at least three repeated samples.⁰- Do not select individual colonies during transfer cultures as strains (to avoid growth mutations).

- Sampling should be conducted such that the test sample is pure. The sampler must be trained and experienced in sampling.

- During sampling, transportation, and handling of samples, contamination from outside must be avoided and the initial integrity must be maintained.

3.2.4. Preparation of suspension:

3.2.4.1. Preparation of suspension from culture samples grown on slant agar:

Culture samples are grown on appropriate slant agar medium. After cells develop well and stabilize (growth phase), collect the microbial biomass for preservation.

Use a sterile pipette to draw 5ml of nutrient broth (NB) containing 10% (v/v) glycerol or 5% (v/v) DMSO or 10% (w/v) skim milk into each slant agar tube.

Use sterile tools to mix the entire biomass on the surface of the slant agar into the medium, then draw the entire cell suspension, ensuring a cell density of at least 10

cells/ml.

Use a sterile pipette to divide 1ml of the cell suspension into polypropylene test tubes, tighten the cap. Ampules can also be used, sealed with a blowtorch. Do not pour the microbial suspension onto the edge or wall of the polypropylene test tube or ampule.

Use a sterile pipette to divide 1ml of the cell suspension into polypropylene test tubes, tighten the cap. Ampules can also be used, sealed with a blowtorch. Do not pour the microbial suspension onto the edge or wall of the polypropylene test tube or ampule.

Place the ampules in a refrigerator at 5°C for 30 minutes to achieve stability between cells and the medium.⁸ 3.2.4.2. Preparation of suspension from culture samples grown in liquid medium:

Culture samples are grown in appropriate liquid medium. After cells develop well and stabilize, proceed with liquid nitrogen preservation.

Place the ampul in the refrigerator at -5°C for 30 minutes to achieve stability between the cells and the environment.⁰C in 30 minutes to achieve stability between the cells and the environment.

3.2.4.2. Preparation of the suspension solution from the culture sample on liquid medium:

The culture samples are grown on suitable liquid medium. After the cells have developed well and stabilized, they are preserved using liquid nitrogen.

Using a sterile pipette, supplement a volume of 20% (v/v) glycerol or 10% (v/v) DMSO or 20% (w/v) sterilized skim milk to the volume of microbial culture to achieve a final concentration of 10% (v/v) glycerol or 5% (v/v) DMSO or 10% (w/v) skim milk.

Gently shake to obtain a homogeneous cell suspension, ensuring a cell density of at least 10 cells/ml.⁸ If the microbial strain grows in small colonies or large clusters, a small tissue grinder or a sterile mortar can be used to break them down.

Culture samples are grown in appropriate liquid medium. After cells develop well and stabilize, proceed with liquid nitrogen preservation.

Place the ampul in the refrigerator at -5°C for 30 minutes to achieve stability between the cells and the environment.⁰C in 30 minutes to achieve stability between the cells and the environment.

3.2.5. Cryopreservation process with liquid nitrogen: carried out as follows:

Place polypropylene test tubes (ampules) in aluminum trays. Then place them in the cooling chamber of the freezing machine.

Adjust the cooling rate to 1-2 C/min until the temperature reaches -30 C.⁰Then continue adjusting the cooling rate to 1 C/min until the temperature does not exceed -50 C.⁰Quickly transfer the polypropylene test tubes (ampules) to their final storage position in the liquid nitrogen container (temperature from -156 C to -196 C).⁰Gloves made of polyolefin must be worn during handling to avoid burns.⁰

3.3. Strain testing:⁰Annually check the survival rate and biological characteristics of the strain. Only strains that maintain stable survival rates and biological activity similar to the original strain are allowed to be retained.⁰3.3.1. Survival rate testing:

Remarks: 3.3.1.1. Testing environment:

Use selective culture medium for testing. The medium is prepared according to the given chemical components in sequence. Afterward, it is distributed into pre-prepared glassware and sterilized under appropriate conditions. Cool the medium to 45-50 C and then distribute it into sterile Petri dishes. This operation is performed under sterile conditions.

3.3.1.2. Dilution solution: physiological saline (0.85% NaCl) without nitrogen compounds, with a pH of 7.0 after sterilization.

Distribute the dilution solution into suitable test tubes containing 9 ml each, after sterilization. Cap with cotton plugs and sterilize at 1 atmosphere (121 C) for 30 minutes.

If not used immediately, the dilution solution should be stored in a refrigerator at 4-10 C, with a storage period not exceeding one month from the preparation date.

Note: To avoid affecting microorganisms due to sudden temperature changes, adjust the temperature of the dilution solution to room temperature before use.⁰3.3.1.3. Preparation of cell suspension:

Remove the preserved strain tube from the liquid nitrogen container, allow the ice to melt naturally (in laboratory conditions).

Wipe the outer surface of the polypropylene test tube (ampule) with sterile gauze soaked in 70% (v/v) ethanol, paying special attention to the area between the cap and the body of the tube.⁰Open the cap of the polypropylene test tube (ampule) under sterile conditions. Use a sterile syringe to draw 1 ml of suspension from the tube and add it to 9 ml of dilution solution, avoiding contact between the syringe and the dilution solution. Mix thoroughly by using another sterile pipette to draw up and down 10 times or by mechanical mixing for 5-10 seconds. Repeat these steps to obtain a 10-fold dilution solution.

Sample inoculation: use a sterile pipette to take 0.05 ml (one drop) of diluted sample and inoculate it onto a Petri dish containing the prepared medium (see 3.3.1). Each diluted sample is inoculated onto three Petri dishes (each dilution uses a separate sterile pipette).⁰Use a sterile spreader to evenly spread the sample on the agar surface, wait for the surface to dry, then invert the Petri dish and incubate at the appropriate temperature and time.

Count the number of characteristic colony-forming units (CFUs) of the microbial strain on each Petri dish.

The microbial density per unit tested (ml) is calculated using the following formula:

a x 20

A =

A: number of CFUs/ml of suspension^-3, 10^-4, 10^-5, 10^-6, 10^-7.

a: average number of CFUs in Petri dishes

d: dilution factor

20: number of drops in 1 ml of suspension

Note: The average number of CFUs is calculated as the arithmetic mean of the CFUs in Petri dishes inoculated from the same dilution, where only Petri dishes containing between 30-300 CFUs are counted. Alternatively, the average number of CFUs can be calculated as the arithmetic mean of the CFUs in Petri dishes inoculated from two consecutive dilutions, by calculating the average of the CFUs at each dilution, where the CFUs at the higher dilution are multiplied by 10, then taking the arithmetic mean of the two values if the ratio between the larger and smaller value does not exceed 2. If the ratio exceeds 2, the smaller value is taken as the result. The microbial density per unit tested is expressed as a number between 1.00 and 9.99 multiplied by 10^n, where n is the appropriate exponent.

Where:

3.3.2. Determination of biological characteristics:

Conducted according to specific methods depending on the biological characteristics of each microbial strain.

d: dilution concentration

20: number of drops in 1 ml of solution

Note: The average colony count is calculated as the arithmetic mean of the colony counts from Petri dishes taken from the same dilution, where only Petri dishes containing between 30-300 colonies are counted. The average colony count can also be calculated as the arithmetic mean of the colony counts from Petri dishes taken from two consecutive dilutions by calculating the average colony count at each dilution, with the colony count at the higher dilution multiplied by 10, then taking the arithmetic mean of the two values if the ratio between the larger value and the smaller value does not exceed 2. If this ratio is greater than 2, the smaller value is taken as the result. The microbial density per unit tested is represented by a number between 1.00 and 9.99 multiplied by 10ⁿ, n being the appropriate exponent.

3.3.2. Determination of biological characteristics:

Conduct according to specific methods depending on the biological characteristics of each microorganism strain.