(Ran Yanzhu)
The basic equipment and operation techniques for cultivation of medicinal fungi are the same as those for general microbial culture.
I. General equipment
(I) aseptic room (inoculation room)
This is the place for aseptic operation, the aseptic room is required to be tightly sealed, generally 4-5m2 size is appropriate, the four walls are best painted with white paint, the room should be neat, clean, do not have convective air, indoor installation of ultraviolet lamps for sterilizing and lighting of fluorescent lamps, and have an operating table and stools, the room should be equipped with a sterilizing lamp and lighting.
(B) aseptic box
Aseptic box, also known as inoculation box (cabinet), is a wooden frame glass cabinet, is instead of the sterile room. Inside the box on both sides of the sliding door can be opened and closed, in the box on the bottom of the front side of the upper part of the two round hand holes, so that the inoculation of the hand into the operation. Inside the box on the upper wall is best to press a ultraviolet light and fluorescent tubes. The corners of the sterile box should be tight, there should be no gaps, so as to avoid the invasion of stray bacteria.
Aseptic room and sterile box must be sterilized before use.
(C) ultra-clean bench
This is also aseptic operation of the device, there are various models and specifications, some suitable for single-person operation, some suitable for two-person operation. It can be set up in aseptic rooms or general room, but the requirements of the environment in which the air flow is small, dry, dust sparse, it is best not to have people walking back and forth. This device is commonly used in current microbiology and tissue culture research work.
(D) sterilization equipment
There must be high-pressure steam sterilizer, the conditions are not enough, can also be replaced with a steamer. Mainly used for culture medium and glassware sterilization.
(E) temperature box and culture room
For heat preservation culture. According to different purposes to choose different equipment. General culture of the mother species of medicinal fungi is carried out in the warm box, while the cultivation of the substrate needs to utilize the culture room. Growing child stage, the air humidity should be high, so the best culture room is a concrete pool, water and sewerage and ventilation conditions. In addition, there are bottle racks, lighting, and temperature and humidity meters.
(F) drying equipment
Oven, baking room.
(VII) commonly used glassware
1. test tubes
For the culture of slant strains. Commonly used for 180 × 20mm and 150 × 15mm two kinds.
2. Triangle bottle
100ml, 500ml, 1000ml and 5000ml and other specifications. Used to contain sterile water and culture medium.
3. Petri dishes
For plane culture. Commonly used for the diameter of 9cm Petri dish.
4. Pipette
For dilution of isolated strains. There are 1ml and 10ml.
5. Glass funnel
For filtration and dispensing medium.
6. Alcohol lamp
For flame sterilization of inoculation tools.
7. Other supplies
Still need gauze, cotton, tweezers, scissors, string, test tube racks, balances, slides, coverslips, microscopes and so on.
If the deep fermentation is carried out, in addition to the above equipment, there are still need to have a shaker, seed tanks, fermentation tanks, filtration of plate and frame or centrifuge and other related equipment.
Two, the basic operating techniques
(a) the preparation and cleaning of glassware
Glassware clean or not directly affects the results of the experiment, in the use of all need to be washed and brushed clean or then sterilized, dried or dried ready for use. Newly purchased glassware have free alkaline substances, the best way to wash is to use 1% HC1 solution treatment once, and then wash with water.
1. Potassium dichromate washing solution preparation
Concentrated preparation:
Potassium dichromate (industrial) 40g
Concentrated sulfuric acid (industrial) 800ml
Tap water 160ml
Dilute preparation:
Potassium dichromate (industrial) 50g
Sulfuric acid (industrial) 100ml
Tap water 250ml
Dissolve potassium dichromate in water, slowly add concentrated sulfuric acid, stirring while adding (note that the potassium dichromate solution should not be poured into the concentrated sulfuric acid to avoid burns). Store in a wide-mouth bottle with a tight-fitting stopper. When applying this solution, the vessel must be dry, and care should be taken not to bring in a large number of reducing substances. The prepared solution is red, thick and has small red crystals.
2. Washing of various glassware
Bacteria-contaminated or contain medicinal fungi of the vessel, the first into the autoclave sterilization 20-30 minutes, while hot, pour out the culture and then use hot soapy water to wash and rinse clean. The wall of the bottle does not hang beads of water shall prevail.
Vessels containing petroleum jelly and wax need to be pre-degreased and, if necessary, boiled in 5% soda solution and then washed with hot soapy water. If for precision tests or when containing chemicals, they are first soaked in a washing-up liquid, rinsed in tap water, distilled water and then dried and set aside.
Bacterial pipette, should be immediately put into 5% carbolic acid solution sterilization for one day, and then rinse with tap water.
New slides and coverslips should be immersed in soapy water (or hydrochloric acid alcohol) for 1 hour, rinsed with water and dried with a soft cloth and stored in 95% alcohol.
(B) the preparation of culture medium
Culture medium is based on the growth and reproduction of medicinal fungi need a variety of nutrients with artificial preparation of organic matter. In addition to containing water, carbohydrates, nitrogenous compounds and a small number of inorganic salts, sometimes also need a variety of essential vitamins.
Carbon is an important source of nutrients for medicinal fungi, it is not only the synthesis of carbohydrates and amino acids, but also a source of energy for life activities.
Nitrogen is an important raw material for the synthesis of proteins and nucleic acids, and the different stages of growth and development of medicinal fungi require different carbon and nitrogen ratios. In the nutrient growth stage, the requirements of carbon and nitrogen ratio of 20:1 or so; in the reproductive growth stage, the requirements of carbon and nitrogen ratio of 30:1-40:1. different medicinal fungi, the requirements of carbon and nitrogen ratio are different. There are two factors that determine the carbon and nitrogen ratio: one is the carbon and nitrogen ratio of the intracellular components, and the other is determined by the assimilation of carbon and nitrogen elements in different species of fungi.
Certain inorganic salts such as potassium, sodium, calcium, magnesium and other phosphates and sulfates, although required in small amounts, also contribute to the growth and development of medicinal fungi. Thiamine (vitamin B1) is an important component of the enzyme transcarboxylase in carbon metabolism, so the nutritional requirements of medicinal fungi must be taken into account when preparing the culture medium.
There are many types of culture media, where the use of natural organic substances containing rich nutrition made of culture media is called natural media, such as potatoes, bran, soybean cake flour, etc.. The use of known ingredients and the number of chemicals formulated for the synthetic medium, such as Tsa's medium. However, generally a part of natural substances are used as a source of carbon, nitrogen and growth factors, and then add some inorganic salts appropriately, known as semi-synthetic medium, such as peeled potato 200g, glucose 20g, potassium dihydrogen phosphate 1.5g, magnesium sulphate 0.75g; Vitamin B110mg, agar 18g, 1000ml of water.
Culture medium in addition to meet the medicinal fungi In addition to meeting the nutritional conditions of medicinal fungi, attention should also be paid to adjusting the appropriate acidity and alkalinity. Generally, the commonly used culture medium has two kinds: solid and liquid.
(C) sterilization
In order to isolate the required medicinal fungi and carry out pure culture, the vessels and culture medium used must be strictly sterilized to kill the microorganisms.
1. Dry heat sterilization
Mainly in the oven. Suitable for glassware such as straws, Petri dishes, test tubes, triangular bottles, etc., before sterilization vessels must be wrapped in paper, the temperature rises slowly, the requirements of the control of 160 ° C, keep 1 hour can be. After sterilization until the temperature drops to below 60 ℃ before removing the items, so as to avoid a sudden drop in temperature caused by the explosion of the vessel.
2. Moist heat sterilization
Because the penetration of moist heat is stronger than the penetration of dry heat, so the medium containing moisture is used in this way. Generally liquid and agar media are sterilized with as little as 15 pounds of pressure for half an hour, while sawdust media are sterilized for one times longer.
3. Soil Sterilization
It is done by steaming in a steamer (or regular pot). This is generally used in the absence of autoclaving conditions or in the case of autoclaving damage to the culture medium. Temperature can only reach about 100 ℃, in order to achieve the purpose of complete sterilization, generally three intermittent sterilization, each round gas after keeping 1 hour, and then placed at room temperature for a day, the next day and the third day and then each steam.
4. Drug sterilization
Commonly used are 70% alcohol, 0.1% mercuric water, 10% bleach, 40% formaldehyde solution and new cleaner.
In addition, there are flame sterilization and ultraviolet sterilization.
(D) inoculation and cultivation
The air is full of stray microorganisms, which is harmful to the pure cultivation of medicinal fungi, inoculation must be carried out under sterile conditions.
1. Oblique inoculation
Inoculation, the test tube to be inoculated with the oblique medium and the original species side by side in the left hand, so that the middle finger clamped between the two test tubes, the slanting side of the face up for easy viewing, the right hand to take the inoculation needle, dipped in alcohol in the flame burning for a few moments (the inoculation needle into the part of the test tube to be burned to the test tube), the right hand with the pinky, ring finger and the palm of the hand to pull out the test tube plug, to the flame slightly roasted! The mouth of the tube for a week, so that the mouth of the tube may be contaminated with stray bacteria were burned to death, then the inoculation needle through the flame into the tube slightly cooled pick a little mycelium together with the medium, quickly transferred to the test tube to be inoculated, placed in the center of the slant, in order to facilitate the extension of the growth of mycelium to the surrounding area, pay attention to do not make the species of bacteria stained by the wall of the tube, and then plugged the cotton plug, which completes the first tube of inoculation procedures. Each tube inoculation are as above. Generally each original strain tube can be expanded with 20 new tubes, placed in a constant temperature box or constant temperature room culture.
2. Liquid inoculation
It is the strain from the slant medium transferred to the liquid medium, and then put into the appropriate conditions of oscillation culture. Inoculation method is basically the same as before, clumsy strain tube can be placed in the strain rack, according to a certain angle screw screw, triangular bottle slightly upward slanting to avoid the base liquid outflow, will take the inoculation of the strains of small shovel into the liquid medium, with the back of the shovel in the wall of the bottle close to the culture liquid crushed bacteria and put into the liquid, the same stuffed with cotton plugs and cauterized inoculation tools. Each tube of strain can be connected to 2-3 bottles of culture liquid.
If the strain is liquid, commonly used sterile pipette inoculation, inoculation amount of 10%, after inoculation put in the constant temperature culture room shaking bed shaking culture.
Three, outdoor work of the general tools and operations
Outdoor cultivation refers to the section of wood cultivation, the general use of tools such as axes, saws, drills, scrapers and plastic film. Segmented wood cultivation to grasp the depth of the cut, slightly up to the xylem is appropriate, favorable growth of bacteria, cut or perforated need to be inoculated immediately after inoculation to ensure the quality of inoculation.