A New Sterilization Method for Edible Mushroom Cultivation
Release date:
2021-09-28
As everyone knows, in edible mushroom cultivation, microorganisms and bacteria are present everywhere—in the substrates, air, water, and equipment. When cultivating a particular fungus, the culture medium, containers, and equipment must be sterilized before use; otherwise, the culture could become contaminated by other fungi or microorganisms, thereby affecting the actual cultivation process. There are many methods for disinfection and sterilization, and the following are some of the commonly used ones.
I. Dry heat sterilization.
Since general glassware—such as petri dishes and pipettes—contains sterilized materials, wrap them carefully in newspaper and place them in an oven. Gradually raise the temperature to 150–160℃ and maintain this temperature for 2 hours, then turn off the power and allow the oven to cool down slowly. (If the temperature drops too quickly, the glass is likely to break.) Sterilization is now complete.
II. Moist-heat sterilization.
Moist-heat sterilization uses steam to kill microorganisms. It does not require the high temperatures needed for dry-heat sterilization, because heating sterilizes by causing microbial proteins to coagulate. The degree of protein coagulation is influenced by factors such as water content and temperature: when the water content is high, lower temperatures are sufficient to induce coagulation; conversely, when the water content is low, higher temperatures are required for coagulation. Moist-heat sterilization can be further divided into two types: atmospheric pressure sterilization and high-pressure sterilization.
1. Atmospheric Pressure Method
Autoclaving at atmospheric pressure is a method that involves steaming in a steam cooker (or a regular pot). When the temperature reaches 100°C, it typically takes about 4 to 6 hours; if the temperature does not exceed 100°C, the duration generally ranges from 8 to 24 hours, depending on the volume of material being sterilized, to achieve a sterile outcome. This method is often employed when high-pressure sterilization equipment is unavailable or when the culture medium is sensitive to high temperatures and might be damaged by such conditions. Another method used for atmospheric-pressure sterilization is intermittent sterilization. This method is relatively more complicated and usually requires three separate cycles, each lasting one hour. After the first steam cycle, vegetative cells are killed, but their spores remain viable. Therefore, after the first sterilization, the culture medium is placed in an incubator for 24 hours to allow the spores to germinate. Then, the medium is subjected to a second steam cycle for another 24 hours, followed by a third cycle. Only through this three-step process can thorough sterilization be achieved.
2. High-pressure method
This method involves sterilization using a high-pressure steam sterilizer. When sterilizing with steam, if the pressure inside the sterilizer is increased, the temperature will also rise accordingly. For example, at a pressure of 0.56 kg/cm² (0.55 bar), the temperature reaches 112.6°C; at a pressure of 1 kg/cm² (0.98 bar), the temperature can reach 120–121°C. Typically, ideal sterilization conditions involve maintaining a temperature of 120°C for 20 minutes to achieve the desired results. Alternatively, a temperature of 115°C for 30 minutes can also suffice. When using a high-pressure sterilizer, two points should be carefully noted: First, before increasing the pressure, all cold air must be completely expelled from the sterilizer; otherwise, although the pressure may rise, the temperature will fail to reach the required level. Second, after sterilization is complete, the pressure inside the sterilizer should be allowed to decrease gradually; otherwise, liquids in the containers could spurt out. Cotton plugs might also come loose easily, rendering all previous efforts futile. In the absence of an oven, other glassware can also be sterilized by warm-water methods—just be sure to wrap them in paper before sterilization.
3. Flame sterilization.
For sterilizing inoculation needles or other metal instruments, they can be directly heated to red-hot over the flame of an alcohol lamp. Additionally, during the inoculation process, the openings of test tubes or Erlenmeyer flasks are also sterilized by passing them through the flame.
4. Drug sterilization.
There are many types of medications used; here are just three examples.
1. 70% alcohol, used for cooling the inoculation needle after it has been heated red-hot, disinfecting the surfaces of hands or instruments before operation, and soaking slides and coverslips, among other uses.
2. New Jel-M, commonly available on the market as a 5% solution, should be diluted to a concentration of one in ten thousand to one in a thousand when used for sterilizing work environments and surfaces of instruments.
3. 0.1% Mercuric Chloride Solution: Used for disinfecting material surfaces and for the disposal of utensils or discarded cultures after experiments.
4. Formalin (40% formaldehyde solution): Used for fumigation and sterilization of enclosed spaces. Heat the solution or add potassium permanganate to completely release formaldehyde gas. Be sure to seal the space tightly and maintain this condition for 24 hours.
V. Gas Sterilization Without Medication
High-concentration ozone sterilization is used for air sterilization in inoculation boxes, inoculation rooms, and similar environments. In particular, the environmentally friendly ozone sterilization method is currently the most scientifically sound and widely adopted sterilization technique. Its principle involves using a novel PEM membrane to electrolyze deionized water at low-voltage DC through solid-state membrane electrodes connected to both positive and negative terminals. At the special anode solution interface, water is separated into hydrogen and oxygen molecules via proton exchange. Hydrogen is directly released from the cathode solution interface, while oxygen molecules, upon being excited by electrons generated under high-density current at the anode interface, gain energy and combine to form ozone molecules. High-concentration ozone is then utilized to sterilize and disinfect inoculation spaces and inoculation utensils. The sterilization efficiency reaches as high as 99.99%.
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