Sterilization is required in mushroom cultivation in order to kill any potentially harmful or disease-causing microorganisms. It is an important practice as it prevents contamination during the growth cycle.
As the temperature is a key factor in sterilization, there are three main ways to achieve sterilization: autoclave, pressure cooker, and microwave.
Sterilize Mushroom Substrate step by step Guide
Preparation of the substrate mixture (composition)
For wood waste (sawdust, chips, bark) sterilization is the most suitable treatment method. Mostly sawdust of hardwoods and some conifers (pine, cedar) is used in a mixture with nutritional (bran, grain, legume seed flour, etc.) and mineral (chalk, gypsum, lime) additives.
Humidification of the substrate
After thorough mixing of the components, the substrate is moistened with water, but in such a way that there is no excess of free water. When squeezed in the hand, water should not flow out of the substrate. Since the incubation of the substrate takes place in a closed container and the evaporation of water is minimal, the optimal level of substrate moisture is 60-65%. Excess water very often leads to the development of a bacterial infection.
See How to pasteurize bulk substrate complete guide.
The moistened substrate is immediately packaged in heat-resistant containers: jars or polypropylene bags. If the moistened mixture is left for a long time (12-24 hours), then the development of competitive microflora will begin in the substrate, which can greatly complicate the sterilization process later. Glass jars (2-Zl) are filled with the substrate completely up to the neck. Then, with a peg, a channel with a diameter of 25-30 mm is made in the substrate to the bottom of the jar. The density of the substrate is obtained in the range of 0.4-0.6. Banks are closed with heat-resistant lids (polypropylene, rubber) with an air filter (holes for air exchange). There are two types of polypropylene bags: 1) with a glued microporous filter and 2) without a filter. The bags are filled with substrate to 2/3 of the volume. The substrate is not compacted, but only the desired shape is given to the block. Self-compacting of the substrate occurs during incubation. However, if narrow cylindrical bags are used, it is possible to compact the substrate by hand or by a mechanical press. The top of the bag is passed through a ring made of heat-resistant material (diameter 30-50 mm) and closed with a cotton plug. For bags with a filter, the top of the bag is wrapped several times and secured with a clothespin for reliability.
Placement of containers
Containers with the substrate are placed in the sterilizer not close, but at a small distance from each other in tiers on the grids so that air can circulate between them. This arrangement will ensure uniform distribution of steam and heating of the substrate and will significantly reduce the sterilization time. If the containers are placed close to each other, then not a separate container weighing 1-3 kg will be sterilized, but the entire batch of substrate weighing 50-100 kg or more. It takes a lot more time to warm up such a mass. Containers located at the edges will sterilize normally, and those in the center will not be sterilized. It will be necessary to significantly increase the sterilization time, and this may adversely affect the quality of the substrate.
After loading the autoclave, the nature of the redistribution of the cold air of the chamber when steam is launched into it becomes most important. If the chamber is not well purged, the remaining cold air can give the false impression of excess steam pressure. Therefore, it is very good to have a temperature sensor in the autoclave chamber. At a pressure of 1.5 atmospheres, the temperature in the chamber should reach 121°C.
The relationship between temperature and pressure obeys Boyle’s law, but only if the air in the chamber is heated evenly. Sensors allow you to control the temperature both in the chamber and in the substrate. In fact, the countdown of the start of sterilization should begin when the difference in air and substrate temperatures does not exceed 10°C. The substrate temperature reaches the chamber air temperature only after 1-2 hours! With an increase in the mass of the block, the temperature equalization slows down even more.
Thus, a good purge before sterilization is an important and necessary technique. Carrying out sterilization in small volumes in a pressure cooker, you can count the time from the moment of boiling. And after that, you can close the lid and start counting the time.
For complete sterilization of solid media, it is sufficient to process them at a temperature of 120 – 125 ° C and a pressure of 1.5-2.0 atmospheres for 1-3 hours, depending on the volume of the sterilizer and the mass of the substrate container. The larger the sterilizer and the mass of the substrate block, the longer the treatment.
The processing time also has to be increased with an increase in the nutritional value of the substrate or increased infection of the raw material. The minimum processing time is 1 hour (for small blocks), the maximum is 3-4 hours. Longer processing leads to oversterilization. In this case, the sawdust becomes dark brown, their smell changes.
The substrate becomes toxic to the mycelium. Prolonged sterilization causes complex chemical reactions to convert plant terpenes into volatile oils and toxic products such as furfural. For small quantities placed in a pressure cooker, this time can be up to 45 minutes from the start of boiling. Want to know Chemical Sterilization of Mushroom.
After turning off the autoclave the pressure and temperature will slowly come down. Ideally, this should create a vacuum in the chamber. If the autoclave does not hold a vacuum, then when it cools down, it sucks in cold, non-sterile outside air .to be filtered. Autoclave with a pressure swing of +2 atm. up to -2 atm. (vacuum) provides a good quality of sterilization, since with such a pressure drop (4 atm.)
Biological structures are more actively destroyed. Before opening the autoclave, equalize the pressure by letting outside air into the chamber through a cotton filter. The autoclave room must be clean to prevent contamination of sterile containers (when there is no walk-through autoclave). In small volumes, you can leave the substrate to cool in a pressure cooker.
Open the hatch of the sterilizer. The substrate in the containers is still hot. If the autoclave is a walk-through, you can wait until the substrate has cooled down and unloaded it into a clean area. If the autoclave is not continuous and it is unloaded in a dirty area, then it is better to unload the substrate hot and place it to cool in a sterile box under UV lamps.
The autoclave room should be as clean as possible. The substrate is sometimes left to cool in the autoclave (overnight). In this case, air must enter the autoclave through a cotton filter.
Substrate inoculation is carried out in a sterile box in compliance with the rules of sterile work. Persons working in the sterile area should not be used in dirty work before that. When working in the box, they wear a sterile white coat, cap, gauze mask.
Daily disinfection of the desktop in the box and tools. The box room is disinfected with hydrogen peroxide (3-6% solution) or sodium hypochlorite (2-5% solution), or irradiation with UV lamps. The control of air purity in the sterile zone is carried out once a week, in the non-sterile zone – once a month (sedimentation of colony-forming particles on a nutrient agar medium in Petri dishes).
Good sterile conditions are created in the working area of laminar flow cabinets due to fine air purification through HEPA filters or Petryanov cloth.
On a sterile substrate, in the absence of competitors, the development of mycelium proceeds rapidly, and this makes it possible to reduce the rate of sowing mycelium from 5% to 1-2% of the wet weight of the substrate.
Mycelium must be in sterile packaging, not overcooked. Mycelium is either scattered over the surface of the substrate (surface inoculation), poured into the channel (deep inoculation), or mixed evenly with the substrate, or in layers. After inoculation, the containers are transferred to a clean thermostatically controlled incubation room.
With sterile technology, good results are obtained on woody substrates enriched with nitrogen nutritional supplements. In this case, the yield of oyster mushrooms reaches 100% of the dry weight of the substrate.