How culture media, culture utensils, and plant material are typically sterilized

Culture medium. Once the preparation is complete, it must be sterilized immediately to prevent bacterial contamination of the medium. Once the bacteria contaminate the medium, because the medium is rich in nutrients, the bacteria will be produced in large quantities for a period of time, and will compete with the culture for nutrients, on the other hand, the bacteria will produce toxic substances during the growth process, resulting in the death of the culture, if it cannot be sterilized immediately, it must be re-sterilized aseptically and non-toxic.

Generally, in the case of relatively mature conditions, the pH value of the medium will be increased by a certain amount before autoclaving, so as to adapt to the problem of the pH value dropping after sterilization.

Sterilization method of culture medium.

1. Autoclave sterilization

Autoclave sterilization is suitable for the sterilization of high-temperature media, inoculation equipment, and distilled water. The medium is mixed with various miscellaneous bacteria in the preparation process, and it should be sterilized immediately after packaging, and the sterilization should be completed within at least 24 hours. Sterilization is generally under pressure, when the temperature is 121, sterilization can be done for 15 30min.

The disinfection time should not be too long, nor should it exceed the specified pressure range, otherwise organic substances, especially vitamins, will decompose at high temperatures, lose their nutritional effects, and will also make the medium deteriorate, discolored, and even difficult to coagulate. Pack distilled water or tap water in a triangular bottle, the amount of water generally does not exceed 2 3 of the bottle, wrap and seal it with kraft paper or sulfuric acid paper, and then put it in a pressure cooker to sterilize it is sterile water. The sterilized medium can be pre-incubated in the culture room for 3 days, and if there is no contamination, it proves that the sterilization is thorough and can be used.

It is best to store the medium that is not used for the time being at 10. Medium containing IAA or GA3 should be used up within 1 week, and no more than 1 month for other media.

2. Filtration and sterilization

Some plant growth regulators and organic matter, such as IAA, GA3, ZT, CM, etc., are easy to decompose when exposed to heat, and cannot be sterilized with the medium at high temperature, but should be filtered out by bacterial filters. Bacterial filters and membranes (pore size microns) are autoclaved before use. The filtered solution should be added to the medium immediately, if it is a liquid medium, it can be added when the medium has cooled to 30; In the case of solid media, it must be added before the medium solidifies (50-60) and shakes to mix the solution with other components.

Choose a sterilization method based on the composition of your medium.

1.If all the components in your medium can withstand high temperatures, autoclave them with autoclaving: 121 or 115 for 30 minutes.

2.If some of the components in your medium are not resistant to high temperatures, you can sterilize them by sterilizing the medium by filtering it through a sterilized filter.

3.How not to be demanding, some test medium can be sterilized by boiling.

I don't know what kind of medium you are.

The sterilization of ordinary medium should be done by batch sterilization.

Fractional sterilization uses repeated circulating steam heating to kill all microorganisms, including spores. The method is the same as the flow steam sterilization method, but it should be repeated more than 3 times, and each interval is to put the object to be sterilized into the potato paimin 37 incubator overnight, so that the spores can develop into propagules. If the sterilized material is not resistant to 100 °C, the temperature can be reduced to 75 80 °C, the heating can be extended to 30 60 minutes, and the number of times can be increased.

It is suitable for medium with sugary or milk that is not tolerant to high heat.

Culture medium refers to the nutrient matrix prepared by the combination of different nutrient and lead substances for the growth and reproduction of microorganisms, plants or animals (or tissues). Generally, it contains carbohydrates, nitrogenous substances, inorganic salts (including trace elements), vitamins and water. The medium is not only the basic material that provides cell nutrition and promotes cell proliferation, but also the living environment for cell growth and reproduction.

There are many types of culture media, which can be divided into natural medium, synthetic medium and semi-synthetic medium according to the preparation of raw materials; According to the physical state, it can be divided into solid medium, liquid medium and semi-solid medium; According to the culture function, it can be divided into basic medium, selection medium, enrichment medium, identification medium, etc. According to the scope of use, it can be divided into bacterial culture medium, actinomycete medium, yeast culture medium, fungal culture medium, etc.

After the medium is prepared, the pH is usually tested and adjusted, and sterilization, usually autoclaved and filter-sterilized. Culture media are susceptible to contamination or deterioration due to their nutrient content. It should not be left for a long time after matching, and it is best to use it now.

Petri dish sterilization methods include dry heat sterilization and moist heat sterilization, dry heat sterilization method: wrapped in an iron container, baked at high temperature. Wrap it in kraft paper and autoclave it in a pressure cooker over moist heat.

The medium sterilization methods include moist heat sterilization, boiling sterilization and filter sterilization, boiling sterilization can be boiled in an induction cooker or microwave oven, and filtration sterilization is suitable for cultures that cannot be treated at high temperatures.

Petri dishes are generally sterilized by dry heat, and are generally sterilized by dry hot air: wrap the petri dish in kraft paper and put it in a drying oven for 170 hours for 2 hours.

The medium is generally sterilized with moist heat, and the autoclave is generally used: put the medium in an Erlenmeyer flask, wrap it in a cotton stopper and kraft paper, put it in an autoclave, and set 121 for 20 minutes.

1.Damp heat sterilization.

By increasing the steam temperature by pressurization, sterilization with autoclave, strong penetration, high temperature, the best sterilization effect.

Notes:1Completely remove the cold air inside the autoclave.

In the presence of cold air, the temperature value reached at the same gauge pressure is lower, and the less cold air is discharged, the lower the temperature is. In autoclaving, cold air must be completely removed in order to ensure that the specified temperature is reached. Otherwise, although the pressure is reached, but the temperature does not meet the specified requirements, the sterilization will not be complete.

2.Ultraviolet light has a wide bactericidal spectrum, but the penetration is weak, there are many influencing factors, and the sterilization efficiency is limited to a certain extent.

The effect of ultraviolet disinfection is related to factors such as ultraviolet intensity, irradiation time, temperature and humidity.

China stipulates that the irradiation intensity of ultraviolet lamps should not be less than 70 W cm2 at a distance of 1mGenerally, the ultraviolet lamp should be replaced if it is used for more than 100h. There is dust on the surface, which reduces the sterilization effect.

The temperature of ultraviolet lamp sterilization should be 20 40, and the relative humidity is 40% 60%.

3. Dry heat sterilization. Flame sterilization is usually used in aseptic operation, the test tube mouth, glass bottle mouth, silicone fluorine plastic stopper, etc. are repeatedly passed through the flame several times, and the nozzle is sterilized by using the flame to prevent the nozzle from being polluted, as an auxiliary sterilization means in the process of aseptic operation.

4. Dry baking and sterilization: using thermal radiation and dry hot air for sterilization. Generally, the items to be sterilized such as metal, glass, and ceramic products can be dried and sterilized in the oven after packaging.

Usually heated to 160 °C, keep warm for 2h can be completely sterilized. But it should not exceed 170, and the glass measuring tool is easy to deform. If the cooling is too fast, the quenching can easily cause the glassware to burst.

The items in the dry oven during dry heat sterilization should not be tight, there should be a gap, which is conducive to the flow of hot air, and it is too dense, resulting in uneven temperature and incomplete sterilization of some items.

A CO2 incubator is not required for plant tissue culture.