CN110618056B - Method for measuring biomass of fungal hyphae - Google Patents
Method for measuring biomass of fungal hyphae Download PDFInfo
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- CN110618056B CN110618056B CN201910935958.2A CN201910935958A CN110618056B CN 110618056 B CN110618056 B CN 110618056B CN 201910935958 A CN201910935958 A CN 201910935958A CN 110618056 B CN110618056 B CN 110618056B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder
Abstract
The invention belongs to the technical field of hypha biomass measuring methods, and particularly relates to a method for measuring fungal hypha biomass, which comprises the following steps: (1) and (3) glass slide treatment: sticking a medium paper sheet soaked by the base solution on a potato substrate sheet, sticking the potato substrate sheet on a circular glass slide, placing the potato substrate sheet in a culture dish, filling the base solution into the culture dish until the medium paper sheet is submerged, covering the culture dish with a cover, and sterilizing at high pressure; (2) and (3) inoculation element treatment: sterilizing the inoculated cells under high pressure, cooling to room temperature, and soaking with pure fungus culture solution; (3) inoculating and culturing; (4) and (3) detection: culturing for several days, separating the medium paper sheet with the hyphae from the potato substrate sheet by using a pair of tweezers, dehydrating, drying and weighing; the method for measuring the hypha biomass has the characteristics of simple and convenient operation, accurate measurement, low cost, simple calculation mode, strong repeatability, wide measurement range and small deviation of results.
Description
Technical Field
The invention belongs to the technical field of hypha biomass measuring methods, and particularly relates to a method for measuring fungal hypha biomass.
Background
Biomass is a basic parameter of the growth of all microorganisms, and its determination is the basis for determining the growth kinetics of the microbial culture process and for further study of the metabolic regulation of microorganisms. Direct determination of biomass is difficult due to the mixed nature of most microorganisms with their growth substrates.
Currently, there are two main types of assay methods in common use: (1) judging according to the diameter of the bacterial colony, but having poor accuracy and only playing a role in qualitative judgment; (2) liquid culture, filtration, drying and weighing are adopted for judgment, and although the accuracy is high, the experiment is complicated.
In addition, for example, patent No. CN201110236660.6 discloses an online in situ monitoring method for solid state fermentation fungal biomass. The method is characterized in that: carrying out fermentation test outside the reactor by using a production strain, shooting thalli-matrix images at different fermentation stages, obtaining a fractal dimension through digital image processing, simultaneously determining corresponding thalli biomass by using a chemical method, and calculating a functional relation between the fractal dimension and the thalli biomass; and shooting a thallus growth image in the solid-state fermentation process by using a built-in camera in the reactor, calculating the fractal dimension of the thallus growth image, and obtaining corresponding biomass according to the fractal dimension-biomass function relation. The invention can realize the on-line monitoring of the biomass of the solid fermentation fungi and promote the automatic control of the solid fermentation. However, the method is complex in calculation mode and high in resolution requirement of used equipment.
Also, for example, patent No. cn201310305536.x discloses a method for detecting live cell biomass of phanerochaete chrysosporium under heavy metal stress, which comprises the following steps: (1) culturing the bacterial balls: adding phanerochaete chrysosporium into a liquid culture medium for constant-temperature culture, and then adding a heavy metal solution for stress treatment; (2) and (3) color development reaction: adding the Phanerochaete chrysosporium strain balls obtained after the heavy metal stress into a sterile thiazole blue solution, carrying out color reaction for 0.5-4 h at 30-50 ℃, and then quickly adding a hydrochloric acid solution to stop the reaction to obtain a mixed solution after the reaction; (3) and (3) centrifugal treatment: centrifuging the mixed solution, and removing supernatant to obtain lower-layer precipitate; (4) and (3) extraction detection: adding an extracting agent into the precipitate for extraction, measuring the absorbance of the obtained extract liquor, and calculating the living cell biomass of phanerochaete chrysosporium under the stress of heavy metals; however, the detection method has strict requirements on the instrument, and the maintenance and cleaning cost of the instrument is high.
Therefore, the research of a detection method with high accuracy, simplified steps and low cost has important significance for the detection of the fungal hypha biomass.
Disclosure of Invention
The invention provides a method for measuring the biomass of fungal hyphae to solve the technical problems.
The method is realized by the following technical scheme:
a method for measuring the biomass of fungal hyphae comprises the following steps:
(1) and (3) glass slide treatment: sticking a medium paper sheet soaked by a basic solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, then fixing the circular glass slide on a bracket by inclining the circular glass slide by 20-25 degrees, then placing the circular glass slide in a culture dish, filling the basic solution into the culture dish until the medium paper sheet is submerged by 0.3-0.7cm, covering the culture dish with a cover, and sterilizing the culture dish for 28-35min under high pressure;
(2) and (3) inoculation element treatment: sterilizing the inoculating element under high pressure for 28-35min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
(3) inoculating and culturing: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
(4) and (3) detection: culturing for several days, separating the medium paper sheet with the hyphae from the potato substrate sheet by using a pair of tweezers, dehydrating, drying and weighing the medium paper sheet with the hyphae, and removing the weight of the medium paper sheet to obtain the hyphae biomass.
The basic solution comprises a carbon source, a nitrogen source, inorganic salt and water.
The total concentration of the carbon source, the nitrogen source and the inorganic salt is 25-40 g/L.
The carbon source is any one or a mixture of more of glucose, rhamnose, maltose, soluble starch, citric acid, fatty acid, cellulose and lignin.
The nitrogen source is any one or a mixture of more of fishbone powder, peanut powder, bran, peptone and corn steep liquor.
The medium paper sheet is a paper sheet which is cut into the same area with the circular glass slide by using medium-speed double-circle qualitative filter paper.
Before the base solution is soaked, the medium paper sheet is soaked by starch/vinyl acetate graft copolymer; the preparation method of the starch/vinyl acetate graft copolymer comprises the following steps: weighing 5g of starch, mixing with 200mL of deionized water, heating in the mixing process to prepare gelatinized starch, introducing nitrogen into the system, adding 3g of lauryl sodium sulfate, mixing for 3-5min, dropwise adding 0.25g of potassium persulfate, 0.2g of sodium acetate and 5g of vinyl acetate, reacting for 2-3h at the temperature of 70-80 ℃ after dropwise adding within 45min, adding a polymerization inhibitor to terminate the reaction, and cooling to normal temperature.
The potato substrate sheet is prepared by cleaning and peeling potatoes, and cutting the potatoes into potato chips with the same area as a circular glass slide.
And a mixed layer of diatomite and silicon carbide with the thickness of 50-70 mu m is laid on the potato substrate sheet.
The mass ratio of the diatomite to the silicon carbide mixed layer is as follows: silicon carbide ═ (1.3-2.3): 1.
the inoculation unit is to use a puncher to make filter paper into a circular sheet, and the circular sheet is arranged in a test tube and is plugged with a rubber plug.
The pure fungus culture solution comprises the following components in parts by weight: 3.5 to 4.8 parts of corn flour, 2.3 to 3.1 parts of wheat bran powder, 1.2 to 2.2 parts of urea, 0.8 to 1.6 parts of ammonium sulfate, 0.3 to 0.9 part of vitamin C, 0.1 to 0.7 part of vitamin K, 0.2 to 0.6 part of iron phosphate, 0.1 to 0.5 part of zinc phosphate, 8 to 10 parts of water and 4.5 to 6.5 parts of beer yeast fermentation liquor.
Has the advantages that:
we have demonstrated through a number of experiments: the method for measuring the hypha biomass has the characteristics of simple and convenient operation, accurate measurement, low cost, simple calculation mode, strong repeatability, wide measurement range and small result deviation, and provides a convenient and efficient method for analyzing the hypha biomass under different culture conditions in a laboratory.
The invention has the advantages of less instruments and equipment, simple and convenient operation, no use of high-precision instruments such as spectrometers and the like in the detection process, capability of detecting the biomass of various fungal hyphae, wide application range and high practicability.
According to the invention, the diatomite and silicon carbide mixed layer is laid on the potato culture medium, so that the effects of heat insulation and shading are achieved, and further the reduction of the measured value caused by the serious influence of external conditions such as temperature and illumination on the growth of hyphae and the components of the culture solution is avoided.
According to the invention, the starch/vinyl acetate graft copolymer is used for soaking the medium paper, so that the medium paper is easy to adsorb a basic solution, hypha culture is facilitated, the breaking resistance of the medium paper is improved, the medium paper is weighed more accurately, the measurement accuracy is effectively improved, and the medium paper is recycled.
According to the invention, the fungus culture solution is optimized, and particularly, the vitamin C, the vitamin K, the iron phosphate and the zinc phosphate are added into the culture solution, so that the rapid growth of hyphae is promoted, the culture time is shortened, the test can be carried out after about 2 days of culture, and the method is also suitable for the culture of various fungi.
Drawings
FIG. 1 is a top view of a culture dish according to example 1;
FIG. 2 is a side view of the culture dish of example 2.
Wherein, 1-culture dish, 2-glass slide and 3-bracket.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Control groups 1-4
The control groups 1-4 respectively provide a detection method for hypha biomass of basidiomycetes lactarius deliciosus fungus, aspergillus niger fungus, trichoderma virens fungus and phanerochaete chrysosporium fungus, which specifically comprises the following steps:
preparation before detection is as follows:
firstly, preparation of an inoculation element: making filter paper into a round piece with the diameter of 0.3cm by using a puncher, and plugging the round piece with a rubber plug in a test tube;
a culture dish: the inner diameter (diameter) is 14cm, and the dish depth is 4.5 cm;
③ round glass slide: the diameter is 6.4 cm;
fixing the circular glass slide bracket: the height of the equilateral 4cm triangular support is 4 cm;
the potato substrate sheet comprises: cleaning and peeling the potatoes, and cutting the potatoes into potato chips with the same area and thickness of 0.2cm as the circular glass slide;
sixthly, paper medium: cutting the glass slide into a piece of paper with the same area as the circular glass slide by using medium-speed double-circle qualitative filter paper;
seventh, base solution: the total substance concentration of the carbon source, the nitrogen source and the inorganic salt is 30 g/L; the carbon source is glucose; the nitrogen source is corn steep liquor.
The detection method comprises the following steps:
s1 slide treatment: sticking a medium paper sheet soaked by a base solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, fixing the circular glass slide on a bracket by inclining the circular glass slide by 20 degrees, then placing the circular glass slide in a culture dish (as shown in figures 1 and 2), filling the base solution into the culture dish until the medium paper sheet is submerged by 0.5cm, covering the culture dish with a cover, and sterilizing the culture dish for 30min under high pressure;
s2 inoculation element treatment: sterilizing the inoculating element under high pressure for 30min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
s3 inoculation and culture: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
s4: culturing for 2 days, separating the medium paper sheets with the hyphae from the potato substrate sheets by using tweezers, dehydrating, drying and weighing the medium paper sheets with the hyphae, removing the weight of the medium paper sheets to obtain the mass of the hyphae, and averaging three medium paper sheets to obtain the biomass of fungal hyphae growth under the condition of a basic solution;
the pure fungus culture solution comprises the following components in parts by weight: 4.8 parts of corn flour, 3.1 parts of wheat bran powder, 2.2 parts of urea, 1.6 parts of ammonium sulfate, 0.9 part of vitamin C, 0.7 part of vitamin K, 0.6 part of iron phosphate, 0.5 part of zinc phosphate, 10 parts of water and 6.5 parts of beer yeast fermentation liquor.
Example 1-example 7 the following preparations were made prior to testing:
firstly, preparation of an inoculation element: making filter paper into a round piece with the diameter of 0.3cm by using a puncher, and plugging the round piece with a rubber plug in a test tube;
a culture dish: the inner diameter (diameter) is 14cm, and the dish depth is 4.5 cm;
③ round glass slide: the diameter is 6.4 cm;
fixing the circular glass slide bracket: the height of the equilateral 4cm triangular support is 4 cm;
the potato substrate sheet comprises: cleaning and peeling potatoes, cutting the potatoes into potato chips with the same area as a circular glass slide and the thickness of 0.2cm, and paving a mixture layer of diatomite and silicon carbide with the thickness of 50-70 mu m on a potato substrate sheet; the mass ratio of the diatomite to the silicon carbide mixed layer is diatomite: silicon carbide ═ (1.3-2.3): 1;
sixthly, paper medium: shearing the medium-speed double-circle qualitative filter paper into paper sheets with the same area as the circular glass slide, and soaking the medium paper sheets by using starch/vinyl acetate graft copolymer; the preparation method of the starch/vinyl acetate graft copolymer comprises the following steps: weighing 5g of starch, mixing with 200mL of deionized water, heating in the mixing process to prepare gelatinized starch, introducing nitrogen into the system, adding 3g of lauryl sodium sulfate, mixing for 4min, dropwise adding 0.25g of potassium persulfate, 0.2g of sodium acetate and 5g of vinyl acetate, reacting for 2h at the temperature of 70-80 ℃ after dropwise adding within 45min, adding a polymerization inhibitor to terminate the reaction, and cooling to normal temperature;
seventh, base solution: the total substance concentration of the carbon source, the nitrogen source and the inorganic salt is 30 g/L; the carbon source is glucose; the nitrogen source is corn steep liquor.
Example 1
The embodiment provides a method for detecting hypha biomass of basidiomycete lactarius deliciosus fungus, which comprises the following steps:
s1 slide treatment: sticking a medium paper sheet soaked by a base solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, fixing the circular glass slide on a bracket by inclining the circular glass slide by 20 degrees, then placing the circular glass slide in a culture dish (as shown in figures 1 and 2), filling the base solution into the culture dish until the medium paper sheet is submerged by 0.5cm, covering the culture dish with a cover, and sterilizing the culture dish for 30min under high pressure;
s2 inoculation element treatment: sterilizing the inoculating element under high pressure for 30min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
s3 inoculation and culture: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
s4: culturing for 2 days, separating the medium paper sheets with the hyphae from the potato substrate sheets by using tweezers, dehydrating, drying and weighing the medium paper sheets with the hyphae, removing the weight of the medium paper sheets to obtain the mass of the hyphae, and averaging three medium paper sheets to obtain the biomass of fungal hyphae growth under the condition of a basic solution;
the pure fungus culture solution comprises the following components in parts by weight: 4.8 parts of corn flour, 3.1 parts of wheat bran powder, 2.2 parts of urea, 1.6 parts of ammonium sulfate, 0.9 part of vitamin C, 0.7 part of vitamin K, 0.6 part of iron phosphate, 0.5 part of zinc phosphate, 10 parts of water and 6.5 parts of beer yeast fermentation liquor.
Example 2
The embodiment provides a method for detecting hypha biomass of basidiomycete lactarius deliciosus fungus, which comprises the following steps:
s1 slide treatment: sticking a medium paper sheet soaked by a base solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, fixing the circular glass slide on a bracket by inclining 20 degrees, then placing the bracket in a culture dish, filling the base solution into the culture dish until the medium paper sheet is submerged for 0.5cm, covering the culture dish, and sterilizing for 30min under high pressure;
s2 inoculation element treatment: sterilizing the inoculating element under high pressure for 30min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
s3 inoculation and culture: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
s4: culturing for 2 days, separating the medium paper sheets with the hyphae from the potato substrate sheets by using tweezers, dehydrating, drying and weighing the medium paper sheets with the hyphae, removing the weight of the medium paper sheets to obtain the mass of the hyphae, and averaging three medium paper sheets to obtain the biomass of fungal hyphae growth under the condition of a basic solution;
the pure fungus culture solution comprises the following components in parts by weight: 3.5 parts of corn flour, 2.3 parts of wheat bran powder, 1.2 parts of urea, 0.8 part of ammonium sulfate, 0.3 part of vitamin C, 0.1 part of vitamin K, 0.2 part of iron phosphate, 0.1 part of zinc phosphate, 8 parts of water and 4.5 parts of beer yeast fermentation liquor.
Example 3
The embodiment provides a method for detecting hypha biomass of basidiomycete lactarius deliciosus fungus, which comprises the following steps:
s1 slide treatment: sticking a medium paper sheet soaked by a base solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, fixing the circular glass slide on a bracket by inclining 20 degrees, then placing the bracket in a culture dish, filling the base solution into the culture dish until the medium paper sheet is submerged for 0.5cm, covering the culture dish, and sterilizing for 30min under high pressure;
s2 inoculation element treatment: sterilizing the inoculating element under high pressure for 30min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
s3 inoculation and culture: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
s4: culturing for 2 days, separating the medium paper sheets with the hyphae from the potato substrate sheets by using tweezers, dehydrating, drying and weighing the medium paper sheets with the hyphae, removing the weight of the medium paper sheets to obtain the mass of the hyphae, and averaging three medium paper sheets to obtain the biomass of fungal hyphae growth under the condition of a basic solution;
the pure fungus culture solution comprises the following components in parts by weight: 4 parts of corn flour, 2.8 parts of wheat bran powder, 1.7 parts of urea, 1.2 parts of ammonium sulfate, 0.6 part of vitamin C, 0.4 part of vitamin K, 0.4 part of iron phosphate, 0.3 part of zinc phosphate, 9 parts of water and 5.5 parts of beer yeast fermentation liquor.
Example 4
The embodiment provides a method for detecting hypha biomass of aspergillus niger fungus, which comprises the following steps:
s1 slide treatment: sticking a medium paper sheet soaked by a base solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, fixing the circular glass slide on a bracket by inclining 20 degrees, then placing the bracket in a culture dish, filling the base solution into the culture dish until the medium paper sheet is submerged for 0.5cm, covering the culture dish, and sterilizing for 30min under high pressure;
s2 inoculation element treatment: sterilizing the inoculating element under high pressure for 30min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
s3 inoculation and culture: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
s4: culturing for 2 days, separating the medium paper sheets with the hyphae from the potato substrate sheets by using tweezers, dehydrating, drying and weighing the medium paper sheets with the hyphae, removing the weight of the medium paper sheets to obtain the mass of the hyphae, and averaging three medium paper sheets to obtain the biomass of fungal hyphae growth under the condition of a basic solution;
the pure fungus culture solution comprises the following components in parts by weight: 4 parts of corn flour, 2.8 parts of wheat bran powder, 1.7 parts of urea, 1.2 parts of ammonium sulfate, 0.6 part of vitamin C, 0.4 part of vitamin K, 0.4 part of iron phosphate, 0.3 part of zinc phosphate, 9 parts of water and 5.5 parts of beer yeast fermentation liquor.
Example 5
The embodiment provides a hypha biomass detection method of trichoderma virens, which comprises the following steps:
s1 slide treatment: sticking a medium paper sheet soaked by a base solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, fixing the circular glass slide on a bracket by inclining 20 degrees, then placing the bracket in a culture dish, filling the base solution into the culture dish until the medium paper sheet is submerged for 0.5cm, covering the culture dish, and sterilizing for 30min under high pressure;
s2 inoculation element treatment: sterilizing the inoculating element under high pressure for 30min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
s3 inoculation and culture: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
s4: culturing for 2 days, separating the medium paper sheets with the hyphae from the potato substrate sheets by using tweezers, dehydrating, drying and weighing the medium paper sheets with the hyphae, removing the weight of the medium paper sheets to obtain the mass of the hyphae, and averaging three medium paper sheets to obtain the biomass of fungal hyphae growth under the condition of a basic solution;
the pure fungus culture solution comprises the following components in parts by weight: 4 parts of corn flour, 2.8 parts of wheat bran powder, 1.7 parts of urea, 1.2 parts of ammonium sulfate, 0.6 part of vitamin C, 0.4 part of vitamin K, 0.4 part of iron phosphate, 0.3 part of zinc phosphate, 9 parts of water and 5.5 parts of beer yeast fermentation liquor.
Example 6
The embodiment provides a hypha biomass detection method of phanerochaete chrysosporium fungus, which comprises the following steps:
s1 slide treatment: sticking a medium paper sheet soaked by a base solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, fixing the circular glass slide on a bracket by inclining 20 degrees, then placing the bracket in a culture dish, filling the base solution into the culture dish until the medium paper sheet is submerged for 0.5cm, covering the culture dish, and sterilizing for 30min under high pressure;
s2 inoculation element treatment: sterilizing the inoculating element under high pressure for 30min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
s3 inoculation and culture: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
s4: culturing for 2 days, separating the medium paper sheets with the hyphae from the potato substrate sheets by using tweezers, dehydrating, drying and weighing the medium paper sheets with the hyphae, removing the weight of the medium paper sheets to obtain the mass of the hyphae, and averaging three medium paper sheets to obtain the biomass of fungal hyphae growth under the condition of a basic solution;
the pure fungus culture solution comprises the following components in parts by weight: 4 parts of corn flour, 2.8 parts of wheat bran powder, 1.7 parts of urea, 1.2 parts of ammonium sulfate, 0.6 part of vitamin C, 0.4 part of vitamin K, 0.4 part of iron phosphate, 0.3 part of zinc phosphate, 9 parts of water and 5.5 parts of beer yeast fermentation liquor.
Comparative example 1
The difference from example 3 is that: the pure fungus culture solution comprises the following components in parts by weight: 4 parts of corn flour, 2.8 parts of wheat bran powder, 1.7 parts of urea, 1.2 parts of ammonium sulfate, 0.4 part of vitamin K, 0.4 part of iron phosphate, 0.3 part of zinc phosphate, 9 parts of water and 5.5 parts of beer yeast fermentation liquor.
Comparative example 2
The difference from example 3 is that: the pure fungus culture solution comprises the following components in parts by weight: 4 parts of corn flour, 2.8 parts of wheat bran powder, 1.7 parts of urea, 1.2 parts of ammonium sulfate, 0.6 part of vitamin C, 0.4 part of iron phosphate, 0.3 part of zinc phosphate, 9 parts of water and 5.5 parts of beer yeast fermentation liquor.
Comparative example 3
The difference from example 3 is that: the pure fungus culture solution comprises the following components in parts by weight: 4 parts of corn flour, 2.8 parts of wheat bran powder, 1.7 parts of urea, 1.2 parts of ammonium sulfate, 0.6 part of vitamin C, 0.4 part of vitamin K, 0.4 part of iron phosphate, 9 parts of water and 5.5 parts of beer yeast fermentation liquor.
The rates of change in the hyphal biomass after 2 days of culture in examples 1 to 3 and comparative examples 1 to 3 were as shown in Table 1, as compared with control 1:
in example 4, the hyphal biomass increased 65% after 2 days of culture, compared with control 2.
In example 5, the hyphal biomass increased 65% after 2 days of culture, compared with control 3.
In example 6, the hyphal biomass increased 72% after 2 days of culture, compared with control 4.
The examples, comparative examples and control groups are shown in Table 2, wherein the growth of hyphae (observed by a 10-fold microscope) is observed after 2 days and 5 days of culture:
TABLE 2
Claims (8)
1. A method for measuring the biomass of fungal hyphae is characterized by comprising the following steps:
(1) and (3) glass slide treatment: sticking a medium paper sheet soaked by a basic solution on a potato substrate sheet, then sticking the medium paper sheet on a circular glass slide together to enable the medium paper sheet to face upwards, then fixing the circular glass slide on a bracket by inclining the circular glass slide by 20-25 degrees, then placing the circular glass slide in a culture dish, filling the basic solution into the culture dish until the medium paper sheet is submerged by 0.3-0.7cm, covering the culture dish with a cover, and sterilizing the culture dish for 28-35min under high pressure;
(2) and (3) inoculation element treatment: sterilizing the inoculating element under high pressure for 28-35min, cooling to room temperature under aseptic condition, and soaking the inoculating element in pure fungus culture solution;
(3) inoculating and culturing: selecting an inoculation element by using an inoculation needle, inoculating the inoculation element on a medium paper sheet, covering a cover, and performing standing culture in a constant temperature and humidity environment;
(4) and (3) detection: culturing for several days, separating the medium paper sheet with the hyphae from the potato substrate sheet by using a pair of tweezers, dehydrating, drying and weighing the medium paper sheet with the hyphae, and removing the weight of the medium paper sheet to obtain hyphae biomass;
the basic solution comprises a carbon source, a nitrogen source, inorganic salt and water;
the medium paper sheet is a paper sheet which is cut into the same area with the circular glass slide by using medium-speed double-circle qualitative filter paper.
2. The method for measuring fungal hyphal biomass according to claim 1, wherein the total concentration of the carbon source, the nitrogen source and the inorganic salt is 25 to 40 g/L.
3. The method for measuring the biomass of fungal hyphae according to claim 1, wherein the carbon source is any one or a mixture of more of glucose, rhamnose, maltose, soluble starch, citric acid, fatty acid, cellulose and lignin.
4. The method for measuring the biomass of fungal hyphae according to claim 1, wherein the nitrogen source is any one or more of fish bone meal, peanut meal, bran, peptone and corn steep liquor.
5. The method for measuring fungal hyphal biomass according to claim 1, wherein the potato substrate is potato chips that are obtained by washing and peeling potatoes and cutting the potato chips into pieces having the same area as a circular slide.
6. The method for measuring the fungal hypha biomass according to claim 1 or 5, wherein a mixed layer of diatomite and silicon carbide with the thickness of 50-70 μm is laid on the potato substrate sheet; the mass ratio of the diatomite to the silicon carbide mixed layer is as follows: silicon carbide ═ (1.3-2.3): 1.
7. the method for measuring the biomass of fungal hyphae according to claim 1, wherein the inoculation unit is a round piece of filter paper made by a puncher and is arranged in a test tube, and the tube opening is plugged by a rubber plug.
8. The method for determining the biomass of fungal hyphae according to claim 1, wherein the pure fungal culture solution comprises the following components in parts by weight: 3.5 to 4.8 parts of corn flour, 2.3 to 3.1 parts of wheat bran powder, 1.2 to 2.2 parts of urea, 0.8 to 1.6 parts of ammonium sulfate, 0.3 to 0.9 part of vitamin C, 0.1 to 0.7 part of vitamin K, 0.2 to 0.6 part of iron phosphate, 0.1 to 0.5 part of zinc phosphate, 8 to 10 parts of water and 4.5 to 6.5 parts of beer yeast fermentation liquor.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519642A (en) * | 2009-04-08 | 2009-09-02 | 中国农业科学院特产研究所 | Special efficient culture medium for necrotic fusobacterium and preparation method thereof |
| CN108715813A (en) * | 2018-05-25 | 2018-10-30 | 福建农林大学 | A method of collecting pure mycelia |
| CN110129407A (en) * | 2019-05-30 | 2019-08-16 | 红河学院 | The separation of corn stover efficient degradation fungi and identification method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519642A (en) * | 2009-04-08 | 2009-09-02 | 中国农业科学院特产研究所 | Special efficient culture medium for necrotic fusobacterium and preparation method thereof |
| CN108715813A (en) * | 2018-05-25 | 2018-10-30 | 福建农林大学 | A method of collecting pure mycelia |
| CN110129407A (en) * | 2019-05-30 | 2019-08-16 | 红河学院 | The separation of corn stover efficient degradation fungi and identification method |
Non-Patent Citations (3)
| Title |
|---|
| 不同碳氮源对竹荪液体培养菌丝生物量影响;闫永亮 等;《食用菌》;20061231;第26页 * |
| 元蘑液体菌种的优化培养;李延雷 等;《安徽农业科学》;20161231;第44卷(第26期);第1-3页 * |
| 菌落直径法和菌丝干重法在优化真姬菇菌种固体斜面培养条件的比较;孙磊 等;《中国食用菌》;20151231;第34卷(第03期);第29页 * |
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