After the culture solution is prepared, it is placed in a 500-mL Erlenmeyer flask with a volume of 100 mL and processed with 0 to 15 small glass beads. After adding tampons, the kraft paper is sealed and sterilized at a pressure of 1.5 kg/cm2. 30 Minutes, remove and cool to below 30 °C, access a piece of about 2 square centimeter bevel strain, stand still culture at 23 °C ~ 25 °C for 48 hours, then set on a reciprocating shaker shake culture, oscillation frequency is 80 ~ 100 Times/minutes, amplitude 6cm to 10cm. If using a rotary shaker, the oscillation frequency is 200 to 220 rpm. Shaker room temperature control at 24 °C ~ 25 °C, culture time varies with bacteria, usually about 7 days. The standard for the end of culture is: the culture fluid is clear and transparent, a large number of small mycelium balls are suspended in the liquid, and a unique flavor of various mushrooms is accompanied.
First, the inspection method of liquid bacteria species Liquid bacteria can be tested using sensory tests and sampling tests combined method.
1. Sensory examinations can be checked using the "see, spin, sniff" steps.
Look: Look at the sample while standing on the table. A look at the color and transparency of the broth, the normal fermentation broth was yellow or brown, clear and transparent, mycelium color varies with the species, the color becomes darker after aging; turbid liquids that are mixed with bacteria are cloudy and opaque. Second, look at mycelium morphology and size, the same size of the normal mycelium, was spherical, flake, floc or rod-like, thick mycelium, clear lines; and after the bacteria, mycelium slender, unclear outline. Third, look at the ratio of supernatant and sediment. The greater the proportion of mycelium is, the better, and the proportion of better liquid bacteria in the bottle can reach 80%. Fourthly, to see if the pH indicator is discolored, add methyl red or compound indicator to the culture solution. After 3 to 5 days of color change, it indicates that the pH of the culture solution reaches 4.0 or so, which is the fermentation point; if it is discolored within 24 hours, This indicates that the acidity of the culture solution drastically changed due to the rapid growth of the bacteria. If there is a gray stripe attached to the wall of the culture liquid and the air at the junction of the culture liquid and the air, this indicates yeast contamination, which is called a yeast line.
Rotation: The sample vial is gently rotated to observe the characteristics of its mycelia. The high viscosity of sputum indicates that the strains have good performance; the thin ones indicate that there are few bacteria spheroids and should not be used. The mycelial levitation force is good, and it is not precipitated when placed for 5 minutes, indicating that the strain has strong growth ability; conversely, if the mycelium is easily precipitated, it indicates that the hyphae have aged or died. Looking at the mycelium state again, the size is different, and the burr is obvious, indicating that the oxygen supply is insufficient; if the ball is shrinking and smooth, or the mycelium is slender and self-dissolving, it indicates contamination of the bacteria.
Sniff: After rotating the sample, open the bottle cap to smell the odor. Well-trained, high-quality liquid strains all have aromatic odors, while culture fluids that contend with bacteria emit odors such as acid, sweet, mildew, and odor.
2. Sampling test may use liquid bacteria for weighing and viscosity testing; growth force measurement and fruiting test; chemical examination, including pH, sugar content and oxygen content; microscopic examination, including cell division status observation, ordinary Dyeing and special staining.
Second, the use of liquid strains Liquid strains can be used as the original species can also be used for cultivation.
1. Take a 100ml for the original species. Veterinary syringe, remove the needle tip, change a stainless steel pipe with an inner diameter of 1mm to 2mm and a length of 100mm to 120mm to make a strain inoculum. Before use, wash the inoculator and wrap it with gauze, sterilize it by autoclave, and then inoculate liquid culture strains after cooling.
After sterilizing the seed bottle of the strain to be inserted, the tampon should first be removed under aseptic conditions, and the sterile film should be used to wrap the bottle. When inoculating, insert the needle into the film on the bottle, the inoculum volume per bottle is 10 mL to 15 mL. Care should be taken to keep the liquid bacteria evenly distributed on the surface of the culture medium. Immediately after pulling out the needle, seal the needle hole with tape and put it upright. Room for cultivation on the bed frame.
2. As a cultivar or direct cultivation of liquid strains When used for cultivation, bottle inoculation volume per bottle is 10mL ~ 15mL; clinker bag planted inoculum volume per bag, pouch 10mL ~ 15mL, large bags of 20mL ~ 30mL; open bed planted, inoculation volume per square meter of 500mL ~ 1000mL, do not need to inoculate the syringe, can be directly sprinkled on the material surface, or sowing.
First, the inspection method of liquid bacteria species Liquid bacteria can be tested using sensory tests and sampling tests combined method.
1. Sensory examinations can be checked using the "see, spin, sniff" steps.
Look: Look at the sample while standing on the table. A look at the color and transparency of the broth, the normal fermentation broth was yellow or brown, clear and transparent, mycelium color varies with the species, the color becomes darker after aging; turbid liquids that are mixed with bacteria are cloudy and opaque. Second, look at mycelium morphology and size, the same size of the normal mycelium, was spherical, flake, floc or rod-like, thick mycelium, clear lines; and after the bacteria, mycelium slender, unclear outline. Third, look at the ratio of supernatant and sediment. The greater the proportion of mycelium is, the better, and the proportion of better liquid bacteria in the bottle can reach 80%. Fourthly, to see if the pH indicator is discolored, add methyl red or compound indicator to the culture solution. After 3 to 5 days of color change, it indicates that the pH of the culture solution reaches 4.0 or so, which is the fermentation point; if it is discolored within 24 hours, This indicates that the acidity of the culture solution drastically changed due to the rapid growth of the bacteria. If there is a gray stripe attached to the wall of the culture liquid and the air at the junction of the culture liquid and the air, this indicates yeast contamination, which is called a yeast line.
Rotation: The sample vial is gently rotated to observe the characteristics of its mycelia. The high viscosity of sputum indicates that the strains have good performance; the thin ones indicate that there are few bacteria spheroids and should not be used. The mycelial levitation force is good, and it is not precipitated when placed for 5 minutes, indicating that the strain has strong growth ability; conversely, if the mycelium is easily precipitated, it indicates that the hyphae have aged or died. Looking at the mycelium state again, the size is different, and the burr is obvious, indicating that the oxygen supply is insufficient; if the ball is shrinking and smooth, or the mycelium is slender and self-dissolving, it indicates contamination of the bacteria.
Sniff: After rotating the sample, open the bottle cap to smell the odor. Well-trained, high-quality liquid strains all have aromatic odors, while culture fluids that contend with bacteria emit odors such as acid, sweet, mildew, and odor.
2. Sampling test may use liquid bacteria for weighing and viscosity testing; growth force measurement and fruiting test; chemical examination, including pH, sugar content and oxygen content; microscopic examination, including cell division status observation, ordinary Dyeing and special staining.
Second, the use of liquid strains Liquid strains can be used as the original species can also be used for cultivation.
1. Take a 100ml for the original species. Veterinary syringe, remove the needle tip, change a stainless steel pipe with an inner diameter of 1mm to 2mm and a length of 100mm to 120mm to make a strain inoculum. Before use, wash the inoculator and wrap it with gauze, sterilize it by autoclave, and then inoculate liquid culture strains after cooling.
After sterilizing the seed bottle of the strain to be inserted, the tampon should first be removed under aseptic conditions, and the sterile film should be used to wrap the bottle. When inoculating, insert the needle into the film on the bottle, the inoculum volume per bottle is 10 mL to 15 mL. Care should be taken to keep the liquid bacteria evenly distributed on the surface of the culture medium. Immediately after pulling out the needle, seal the needle hole with tape and put it upright. Room for cultivation on the bed frame.
2. As a cultivar or direct cultivation of liquid strains When used for cultivation, bottle inoculation volume per bottle is 10mL ~ 15mL; clinker bag planted inoculum volume per bag, pouch 10mL ~ 15mL, large bags of 20mL ~ 30mL; open bed planted, inoculation volume per square meter of 500mL ~ 1000mL, do not need to inoculate the syringe, can be directly sprinkled on the material surface, or sowing.
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