How are microbial strains preserved? Long term
Microbial knowledge and strain preservation methods
1. Preparation, sterilization and disinfection of commonly used media
Nutrition: A physiological process that extracts energy and substances necessary for its life activities from the external environment to meet the needs of growth and reproduction. It is the material basis of life activities and the starting point of life activities.
Nutrients: substances with nutritional functions. Also includes light energy. Provide biological structural substances, energy, metabolic regulators and a good physiological environment.
Nutrients: carbon, nitrogen, energy, growth factors, inorganic salts, and water
Medium: mixed nutrient that is artificially prepared and suitable for the growth and reproduction of microorganisms or the production of metabolites. Requirements: There should be 6 major nutrients; the proportion of substances is appropriate; it must be sterilized.
2. Principles, methods and preparation process for selecting and designing culture media
Principles: clear purpose, coordinated nutrition, and economic savings
The physical and chemical conditions are suitable
Methods: Ecological simulation and literature review
Careful design, experimental comparison
Steps: weighing, melting, adjusting PH, filtering, sub-packaging,
stopper, bandage, sterilization, cooling, sterility check
Type of medium:
First, according to the different components
Natural medium, synthetic medium, semi-synthetic medium
According to the physical state of the culture medium
Solid medium, liquid medium, semi-solid medium
III. By medium use
Basic medium, selectionmedium Enrichment medium identification medium
Disinfection and sterilization:
Disinfection: a nutrient that destroys pathogens and harmful microorganisms
Sterilization: the killing of all microbial vegetative bodies, spores and spores.
Method:
First, physical methods: heating, filtration, radiation
Chemical methods: use chemical reagents to inhibit or kill microorganisms. Mainly destroy the metabolic function of bacteria. Such as heavy metal ions, sulfonamides and antibiotics.
Heating method:
(1) Dry heat method: flame burning and hot air sterilization.
1. Flame burning is suitable for inoculation rings, inoculation needles and metal utensils such as forceps, test tube mouth and bottle mouth, and glass rods for coating.
2. Hot air sterilization uses high temperature dry air (160-170C) to heat sterilize for 1-2h, suitable for glassware and petri dishes, etc. Principle heating denaturates proteins, which is related to the content of water. When the environment and cell water content are greater, the solidification is faster.
3. Precautions:
1) Media, rubber products, and plastic products cannot be used in this manner.
2) The temperature is controlled at < 180 ° C.
3) Items should not be too crowded.
4) Open the box door only when the temperature drops to 70 ° C.
(2) Damp heat method: Principle: Because the bacteria absorb water in the humid heat, the protein is easy to solidify. Due to the increase in the water content of the protein, the required solidification temperature is reduced; the humid heat vapor has latent heat. So the effect is better than dry heat at the same temperature.
Autoclave Sterilization:
1. Equipment: autoclave
2. The length of time varies depending on the type and quantity of sterilized items.
3. It is suitable for the sterilization of culture media, work clothes, rubber items, etc., and can also be used for the sterilization of glassware.
4. Precautions:
1) Complete removal of cold air; 2) Add water; 3) Open when the pressure drops to "0".
Atmospheric pressure steam sterilization: This method can be used for media that are not suitable for autoclaving, such as gelatin medium, milk medium, sugar-containing medium, etc. Complete sterilization is batch sterilization.
Boiling sterilization method: suitable for syringes and dissecting utensils, time 10-15min,
Ultra-high temperature sterilization: 135-150 ° C and 2-8s for milk and other liquid foods. Advantages: Maintains food value and nutritional content.
Filter and sterilize:
Principle: The medium blocks microorganisms under pressure.
Scope of application: Many materials such as serum, antibiotics, and sugar solutions use this method.
Equipment: Chua filter, membrane filter.
Advantages and disadvantages: does not damage the composition of the medium, only a small amount of filtrate is suitable. A lot of sterile air and a purification table are required.
Notes: 1. Appropriate pressure;
2. Carry out under sterile conditions;
3. Prevent infiltration.
Radiation sterilization:
Principle: The UV wavelength is 200-300nm, which has a bactericidal effect and is strong at 265-266. This wavelength is easily absorbed by nucleic acids in cells; ozone and hydrogen peroxide can be produced. The bactericidal efficacy is proportional to the product of intensity and time.
Disadvantages: The penetration is not large. It is advisable to be less than 1.2m away from the irradiated object.
Applications: sterile rooms, hospitals.
Attention: It is irritating to the eyes and skin.
Radiation sterilization: R-ray, with strong penetration, suitable for sterilization of stacked items.
Chemical sterilization:
Principle: Use chemical agents to destroy bacterial metabolism.
Fungicides such as heavy metal ions;
Bacteriostatic agents such as sulfonamides and most antibiotics.
Attention: It is related to the concentration of the drug, the duration of time, the type of microorganisms, and the environment in which the microorganisms are located.
Commonly used chemical fungicides are: ethanol, acetic acid, carbolic acid
Formalin, mercury,potassium permanganateNew Jierme, etc
3. Isolation, purification and culture technology of microorganisms
Isolation and purification: The process of obtaining microorganisms containing only one or more strains from a mixed population of microorganisms.
Why a pure culture is necessary: A single colony on a plate does not necessarily guarantee a single bacterium.
Commonly used separation and purification methods:
Single cell extraction, dilution coating plate method
Dilution mixed plate method, plate scribing method
Dilution coating plate method:
Steps:
1. Inverted plate: beef paste peptone medium, Gao's No. 1 medium, when the Cha's medium is cooled to 55-60 ° C, mix well and pour the plate, beef paste peptone medium pour 4 dishes, Gao's No. 1 medium and Cha's medium each pour 3 dishes.
2. Prepare wastewater dilution: 10g soil sample, add 90ml of sterile water, and shake in a triangular bottle for 20min. Take 0.5ml and add it to a test tube containing 4.5ml of sterile water, and so on to make different dilutions.
3. Coating: Mark the dilution level on the bottom surface of each of the above medium plates, and then use a sterile straw to extract 0.1 ml of the check mark from the last three dilutions, namely 10-4, 10-5, and 10-6 tubes. Place it on the plate and coat it with a glass rod.
4. Culture: Gao's I and Cha's inverted culture were cultured in an incubator at 28 ° C for 3-5days, and beef paste peptone agar medium was cultured at 37 ° C for 1-2days.
5. Pick colonies: turn to the inclined plane, check for consistency, and store.
Flat scribing method:
1. Invert the plate and label the medium name, number, and date of experiment.
2. Scribing method
Dilution mixed plate method:
1. Add bacteria first
2. Pay attention to the medium temperature when inverting the plate
3. Mix well
4. Microbial culture technology
1. Bevel inoculation
2. Inoculation in liquid medium
3. Puncture vaccination
4. Place the inoculated bevel, semi-solid and liquid media in an incubator for cultivation
5. Wrap the growing strains in kraft paper and store them in the refrigerator at 4 ° C.
Several commonly used methods of preservation:
1. Subculture method
PreservedstrainAfter culturing through bevel, puncture or blister medium (for anaerobic bacteria), store it in a 4C? refrigerator, and then store it regularly.
You can use rubber stoppers instead of tampons or cover the inclined surface with a layer of sterile liquid paraffin to further extend the shelf life.
2. Vector method
The suitable microorganisms are adsorbed on a certain carrier for drying.
Commonly used carriers include soil, sand, silica gel, gelatin, bran, magnetic beads and filter paper.
3. Suspension method
The bacteria and yeast cells are suspended in a certain solution and preserved.
The solution includes distilled water, sugar solution, phosphoric acid buffer, salt water, etc.
4. Freezing method
A method of preserving bacteria in a low temperature environment. Including low temperature method and liquid nitrogen method.
The key to this method is to overcome the cold damage of cells. Pay attention to control the cooling rate and the use of protective agents.
5. Vacuum drying method
Such methods include freeze-vacuum drying and L-drying.
The freeze-vacuum drying method is that the microbial samples to be preserved are first pre-frozen at low temperature, and then dried under reduced pressure at low temperature.
The L-drying method does not require low-temperature pre-freezing of the sample, but only maintains the sample in the range of 10-20 ° C for vacuum drying.
Operation method
1. Bevel preservation method
Transfer the strain to a suitable solid inclined medium, and when it is fully grown, wrap the tampon part with kraft paper (it is better to replace the tampon with a rubber plug), and store it in a 4C? refrigerator.
The storage time depends on the type of microorganisms. Mold, actinomycetes and spores are stored for 2-4 months and transplanted once, yeast is separated by two months, ordinary bacteria are separated by one month, and Pseudomonas are passed on every two weeks.
The advantages of this method are simple operation and convenient use, but the disadvantages are short storage time and easy to be contaminated.
2. Liquid paraffin preservation method
Add sterile paraffin wax to the inclined surface where the bacteria have grown, and the dosage is 1CM higher than the top of the inclined surface to isolate the bacteria from the air.
Store the test tubes upright at low or room temperature.
This method is practical and effective. Mold, actinomycetes and spores can be stored for more than two years, yeast can be stored for 1-2 years, and ordinary bacteria can also be stored for about one year.
3. Puncture preservation method
Cultivate the strains according to the puncture inoculation method. After the strains grow, seal them with rubber plugs and store them in a refrigerator at 4 ° C.
4. Sand pipe preservation method
(1) River sand treatment
Take some river sand and add 10% hydrochloric acid, heat and boil for 30min to remove organic matter. Pour off the hydrochloric acid solution, rinse with tap water until neutral, and rinse with distilled water for the last time. After drying, sieve with a 40-mesh sieve, discard the coarse particles, and set aside.
(2) Soil treatment
Take the lean loess or laterite without humus in the non-tilled layer, soak and wash it several times with tap water until neutral. After drying, crush it, sift it with a 100-mesh sieve, and partially discard the coarse particles.
(3) Sand mixing
After the properly handled river sand is mixed with the soil in a ratio of 3:1 (or other proportions are used as needed, or even all can be made into sand or soil), it is evenly packed into 10%.X100mm small test tube or ampoule tube, each tube is divided into about 1g, plugged with tampons, sterilized (usually using intermittent sterilization 2-3 times), and finally dried.
(4) Sterility test
Every 10 sand tubes are randomly drawn, the sand is poured into the broth medium, and incubated at 30 ° C for 40 hours. If microbial growth is found, all sand tubes need to be re-sterilized and then tested for sterility until proven sterile.
(5) Preparation of bacterial suspension
Take the robustly growing fresh bevel strains, add 2-3ml sterile water (every 18x180mm test tube bevel strains), gently wash the moss with an inoculation ring, and make a bacterial suspension.
(6) Packed samples
Add 0.5 ml of bacteria suspension (just wet the sand) to each sand tube (after marking), mix well with inoculation needle.
(7) Dry
Place a sand tube containing the suspension in a desiccant dryer with desiccant at the bottom of the desiccant. Flame seal after pumping the water with a vacuum pump (or plug the tube mouth with a rubber or tampon).
(8) Preservation
Put it in a refrigerator at 4 ° C or in a dry place at room temperature and test it at regular intervals. This method is mostly used for spore-producing bacteria, spore-producing molds and actinomycetes. It is widely used in the industrial production of antibiotics and has good effect. It can be stored for several years, but it has poor effect on vegetative cells.
5. Freeze vacuum drying
(1) Preparation of lyophilized tubes: neutral hard glass, drying, sterilization.
(2) Strain culture: bacterial spores shed; release, fungus spores plump.
(3) Preparation of protective agents: preparation, sterilization, and inspection.
(4) Preparation of bacterial suspension: 2-3 ml of protective agent.
(5) Separate sample: 0.2 ml of bacterial suspension per tube.
(6) Pre-freezing: programmed temperature controller to cool down.
(7) Freeze vacuum drying: vacuum at -50 ° C.
(8) Take out the sample: tap loose to reach the standard.
(9) The second drying.
(10) Melting and sealing.
(11) Survival testing: Extract a tube for survival testing.
(12) Storage: 4 ° C storage.
