CN105693330A - High-transparency PDA (potato dextrose agar) culture medium preparing method - Google Patents
High-transparency PDA (potato dextrose agar) culture medium preparing method Download PDFInfo
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- CN105693330A CN105693330A CN201610081117.6A CN201610081117A CN105693330A CN 105693330 A CN105693330 A CN 105693330A CN 201610081117 A CN201610081117 A CN 201610081117A CN 105693330 A CN105693330 A CN 105693330A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
Abstract
The invention belongs to the field of PDA (potato dextrose agar) culture medium preparing methods and discloses a high-transparency PDA culture medium preparing method.The method includes steps: A, potato dicing; B, stewing, to be more specific, stewing over strong fire and then slow fire to make potato dices thoroughly cooked but not mushy; C, filtering; D, material adding, to be more specific, adding 20g of glucose, 0.8-1.4g of monopotassium phosphate and 1.2-1.7g of magnesium sulfate into filtrate obtained at the step C, well mixing, and adding water to a constant volume of 1000mL; E, pH value regulation, to be more specific, using a hydrochloric acid and a sodium hydroxide solution for regulating the pH value to 6.5-7 to obtain a liquid culture medium, distributing 19-23g of high-purity agar powder into a plurality of mixing containers, then distributing the liquid culture medium into the mixing containers, and well mixing; F, transferring to flat-plate petri dishes; G, dehumidifying, namely putting the petri dishes over flame of an alcohol lamp to remove moistures; H, sealing and sterile testing.The high-transparency PDA culture medium preparing method has the advantages that raw materials can be proportionally mixed precisely and quickly, and system errors and operation errors are avoided; moistures in the petri dishes can be well controlled to avoid breeding of contaminating microorganisms.
Description
Technical field
The present invention relates to the manufacture method field of PDA culture medium, particularly relate to the manufacture method of a kind of high grade of transparency PDA culture medium。
Background technology
Field is cultivated at fungi microbe, people commonly use potato dextrose agar, existing common practice is: first clean peeling, weigh 200g Rhizoma Solani tuber osi again to be cut into small pieces, add water well-done, with eight layers of filtered through gauze, heating, need to add 1-10g agar according to actual experiment again, continue heated and stirred mixing, after agar has dissolved, add glucose, stir, slightly supply moisture again to 1000 milliliters after cooling, subpackage test tube or conical flask, jump a queue, wrap up, take out test tube pendulum inclined-plane after sterilizing about 20 minutes or shake up, storing standby after cooling。This preparation method has following defect: one is cause operating error, add glucose after first adding agar powder, after agar powder contact water, become pasty state, easily agglomerating, mix pot irregular, easy to stick, cause other to add compositions to be clung by agar group, it is difficult to be mixed even, and mix and also to shift to the containers such as triangular flask afterwards, the culture fluid viscosity having added agar is big, adhere to above reinforcing bar pot, funnel, Glass rod and spoon, not only time-consuming but also affect proportioning degree of accuracy, operating error can be caused；Two is cause systematic error。After the Rhizoma Solani tuber osi liquid constant volume prepared, adding adjuvant and agar powder continues heated and boiled and makes agar melt completely, so also have many moisture to evaporate in heating process, in the PDA caused, the change of each constituent concentration, does not reach standard volume, causes systematic error；The PDA culture medium reduced transparency that three are formed into is low, is unfavorable for follow-up inoculation operation。
Summary of the invention
It is an object of the invention to provide the manufacture method of a kind of high grade of transparency PDA culture medium, can precisely, proportioning rapidly, it is to avoid systematic error and operating error。
The present invention proposes manufacture method of a kind of high grade of transparency PDA culture medium and preparation method thereof, comprises the following steps:
A, potato cutting: Rhizoma Solani tuber osi 200g is cleaned, the square fourth block removing the peel, go eye, trimming length to be 4-6mm；
B, boil block: place in hot water by Rhizoma Solani tuber osi fourth block, first boil 5 minutes to softening with big fire, then change slow fire into and boil 10-14 minute, make potato nutritional fully distribute to water, make Rhizoma Solani tuber osi fourth thoroughly well cooked but not mushy simultaneously；
C, filtration: use 6-8 layer filtered through gauze 2-3 time, retain filtrate；
D, reinforced: glucose 20g, potassium dihydrogen phosphate 0.8-1.4g, magnesium sulfate 1.2g-1.7g to be added above-mentioned filtrate, stirs evenly, be settled to 1000mL with water；
E, tune pH value: use hydrochloric acid solution and sodium hydroxide solution to regulate pH value to 6.5-7, obtain fluid medium；First by high-purity agar powder 19-23g subpackage to several allotment containers, then fluid medium is dispensed in described several allotment container, mixing；
F, it is down flat plate: having added the culture medium high temperature sterilize of agar powder, and after cooling, aseptically be dispensed in sterilized slat chain conveyor ware, the bottom of each culture dish is level, and the thickness of each culture dish culture medium is 3.0 ± 0.5mm；
G, dry: cool down culture dish further, ware is covered and on alcohol burner flame, steam is dried；
H, seal and carry out Sterility testing: with ventilative sealed membrane, under aseptic conditions culture dish is sealed up mouth, install in aseptic polypropylene plastics pocket, be put into inside the constant incubator of 28 DEG C, within 2-4 days, do Sterility testing, obtain solid medium after integral asepsis after testing。
Preferably, described " F, be down flat plate " step refers to culture medium at 123 DEG C, high pressure 0.122MPa sterilizing 26min, temperature be down to 50 DEG C again subpackage to sterilized slat chain conveyor wares。
In described " D, reinforced " step, add food grade salts 8-14g。
The invention has the beneficial effects as follows: one is to obtain the PDA culture medium that transparency is high, in " boiling block " step, first boil again slow fire in short-term to boil with big fire, make Rhizoma Solani tuber osi fourth thoroughly well cooked but not mushy, such boil existing potato nutritional in liquid, limpid again, transparent, and good fluidity is easy to follow-up reinforced, adopt high-purity agar powder, because highly purified agar has the advantages of impure few, moisture is up to standard, miscellaneous bacteria quantity does not exceed standard, considerably less containing harmful metal ion, solidifiability is strong, the culture medium definition allotted, transparency is high, culture medium color will not variable color, few containing heavy metal substance, it is substantially less than national standard, mycelial growth will not be produced toxic action, therefore the transparent shape of culture medium made。Two is " D, reinforced " step can be accurate, proportioning rapidly, first add glucose, potassium dihydrogen phosphate, magnesium sulfate enters Rhizoma Solani tuber osi filtrate, after stirring evenly, this three is lyotrope, quickly it is dissolved in water, finally high-purity agar powder is first weighed and pour allotment container afterwards into, such as triangular flask, pour the above-mentioned PDA nutritional solution without agar prepared again into, and shake triangular flask, agar powder is made to be evenly distributed in nutritional solution, what adopt is the high-purity agar powder that quality is high, so only need to quickly rock, vibration triangular flask is just good at being mixed thoroughly by high-purity agar powder soon, save the operating time, improve distributing precision, glucose in prior art, agar powder adds simultaneously, and whipping process also needs with the little fire heating of heating kettle, present invention advantage compared with prior art: (1) is more convenient for cleaning glass drying oven and reinforcing bar pot, culture fluid without agar does not stick to reinforcing bar pot, funnel, above Glass rod and spoon；(2) change of each constituent concentration in the PDA that systematic error causes is reduced, as being in the past after the Rhizoma Solani tuber osi liquid constant volume prepared, add adjuvant and agar powder continues heated and boiled and makes agar melt completely, many moisture are so also had to evaporate in heating process, do not reach standard volume, cause systematic error；(3) operating error is reduced: after first adding agar powder, add glucose, pasty state is become after agar powder contact water, easily agglomerating, mix pot irregular, easy to stick, cause other to add compositions to be clung by agar group, it is difficult to be mixed even, and mix and also to shift to the containers such as triangular flask afterwards, the culture fluid viscosity having added agar is big, adheres to above reinforcing bar pot, funnel, Glass rod and spoon。Three is interpolation food grade salts in PDA culture medium, at vegetative stage, owing to containing the metal ions such as sodium, magnesium, calcium in Sal, it is possible to cultivate healthy and strong mycelia；During original female ultralow temperature preservation planted, it is possible to make mycelia absorb the little molecule of glycerol faster, better play a protective role；Original female plant mycelia Restoration stage due to the flow of water of strain block poor, strain block easily absorbs water mycelium germination survival rate height。Four is adopt control bacterium technology of drying, prior art, by adding chloromycetin or oxytetracycline, suppresses the growth of antibacterial, reduces interference, and the present invention adopts increase agar powder consumption, make culture medium partially dry, in " G, dry " step of original creation, ware lid alcohol burner flame is thoroughly dried, sterilized, control the moisture of subenvironment in each culture dish, play and control the effect that mycete grows, more more environmentally-friendly than addition chloromycetin or oxytetracycline, it is beneficial to the growth of follow-up parent edible fungus kind。Five be add potassium dihydrogen phosphate, the effect of magnesium sulfate is to be provided that female demand planted in growth course necessary trace element, better cultivates the mycelium of robust growth。Six is the effect adopting high pressure short-term sterilization method both to play sterilizing, maintains again the height solidification degree of agar powder。
In a word, the present invention can fast, save time, proportioning is made to precisely the PDA culture medium of the high grade of transparency, the culture medium high grade of transparency, can be easier to find very faint living contaminants in educating bacterium process, more clearly observe mycelia whether sturdy and neat and make a variation situation, seeing more clearly when cutting the operations such as Tip Splitting, operation is more accurate, and culture medium is pure, transparent, without miscellaneous bacteria, culture medium thickness is uniform, surfacing, and ware lid upper interior does not have steam, and makes more convenient。
Detailed description of the invention
Embodiment one the present embodiment proposes the manufacture method of a kind of high grade of transparency PDA culture medium, comprises the following steps:
A, potato cutting: Rhizoma Solani tuber osi 200g is cleaned, the square fourth block removing the peel, go eye, trimming length to be 4-6mm；
B, boil block: place in hot water by Rhizoma Solani tuber osi fourth block, first boil 5 minutes to softening with big fire, then change slow fire into and boil 10-14 minute, make potato nutritional fully distribute to water, make Rhizoma Solani tuber osi fourth thoroughly well cooked but not mushy simultaneously；
C, filtration: use 6-8 layer filtered through gauze 2-3 time, retain filtrate；
D, reinforced: by glucose 20g, potassium dihydrogen phosphate 0.9g, magnesium sulfate 1.3g, food grade salts 8g, to add above-mentioned filtrate, stir evenly, be settled to 1000mL with water；
E, tune pH value: use hydrochloric acid solution and sodium hydroxide solution to regulate pH value to 6.5-7, obtain fluid medium；Being installed to by high-purity agar powder 21g average mark in the triangular flask of 5 250 milliliters, before subpackage, namely the bottom of each triangular flask adds high-purity agar powder 4.2g, is then installed to by fluid medium average mark in described several allotment container, mixing；
F, it is down flat plate: the culture medium of agar powder will have been added at 123 DEG C, high pressure 0.122MPa sterilizing 26min, temperature is down to 50 DEG C, and subpackage is to sterilized slat chain conveyor ware again, and the bottom of each culture dish is level, and the thickness of each culture dish culture medium is 3.0 ± 0.5mm；
G, dry: cool down culture dish further, ware is covered and on alcohol burner flame, steam is dried；
H, seal and carry out Sterility testing: with ventilative sealed membrane, under aseptic conditions culture dish is sealed up mouth, install in aseptic polypropylene plastics pocket, be put into inside the constant incubator of 28 DEG C, within 2-4 days, do Sterility testing, obtain solid medium after integral asepsis after testing。
Embodiment two
The present embodiment proposes the manufacture method of a kind of high grade of transparency PDA culture medium, comprises the following steps:
A, potato cutting: Rhizoma Solani tuber osi 200g is cleaned, the square fourth block removing the peel, go eye, trimming length to be 4-6mm；
B, boil block: place in hot water by Rhizoma Solani tuber osi fourth block, first boil 5 minutes to softening with big fire, then change slow fire into and boil 10-14 minute, make potato nutritional fully distribute to water, make Rhizoma Solani tuber osi fourth thoroughly well cooked but not mushy simultaneously；
C, filtration: use 6-8 layer filtered through gauze 2-3 time, retain filtrate；
D, reinforced: by glucose 20g, potassium dihydrogen phosphate 1.0g, magnesium sulfate 1.4g, food grade salts 10g, to add above-mentioned filtrate, stir evenly, be settled to 1000mL with water；
E, tune pH value: use hydrochloric acid solution and sodium hydroxide solution to regulate pH value to 6.5-7, obtain fluid medium；Being installed to by high-purity agar powder 22g average mark in the triangular flask of 5 250 milliliters, before subpackage, namely the bottom of each triangular flask adds high-purity agar powder 4.4g, is then installed to by fluid medium average mark in described several allotment container, mixing；
F, it is down flat plate: the culture medium of agar powder will have been added at 123 DEG C, high pressure 0.122MPa sterilizing 26min, temperature is down to 50 DEG C, and subpackage is to sterilized slat chain conveyor ware again, and the bottom of each culture dish is level, and the thickness of each culture dish culture medium is 3.0 ± 0.5mm；
G, dry: cool down culture dish further, ware is covered and on alcohol burner flame, steam is dried；
H, seal and carry out Sterility testing: with ventilative sealed membrane, under aseptic conditions culture dish is sealed up mouth, install in aseptic polypropylene plastics pocket, be put into inside the constant incubator of 28 DEG C, within 2-4 days, do Sterility testing, obtain solid medium after integral asepsis after testing。
Embodiment three
The present embodiment proposes the manufacture method of a kind of high grade of transparency PDA culture medium, comprises the following steps:
A, potato cutting: Rhizoma Solani tuber osi 200g is cleaned, the square fourth block removing the peel, go eye, trimming length to be 4-6mm；
B, boil block: place in hot water by Rhizoma Solani tuber osi fourth block, first boil 5 minutes to softening with big fire, then change slow fire into and boil 10-14 minute, make potato nutritional fully distribute to water, make Rhizoma Solani tuber osi fourth thoroughly well cooked but not mushy simultaneously；
C, filtration: use 6-8 layer filtered through gauze 2-3 time, retain filtrate；
D, reinforced: by glucose 20g, potassium dihydrogen phosphate 1.2g, magnesium sulfate 1.6g, food grade salts 12g, to add above-mentioned filtrate, stir evenly, be settled to 1000mL with water；
E, tune pH value: use hydrochloric acid solution and sodium hydroxide solution to regulate pH value to 6.5-7, obtain fluid medium；Being installed to by high-purity agar powder 23g average mark in the triangular flask of 5 250 milliliters, before subpackage, namely the bottom of each triangular flask adds high-purity agar powder 4.6g, then pours in triangular flask by fluid medium average weight, rocks triangular flask and is mixed by agar powder；
F, it is down flat plate: the culture medium of agar powder will have been added at 123 DEG C, high pressure 0.122MPa sterilizing 26min, temperature is down to 50 DEG C, and subpackage is to sterilized slat chain conveyor ware again, and the bottom of each culture dish is level, and the thickness of each culture dish culture medium is 3.0 ± 0.5mm；
G, dry: cool down culture dish further, ware is covered and on alcohol burner flame, steam is dried；
H, seal and carry out Sterility testing: with ventilative sealed membrane, under aseptic conditions culture dish is sealed up mouth, install in aseptic polypropylene plastics pocket, be put into inside the constant incubator of 28 DEG C, within 2-4 days, do Sterility testing, obtain solid medium after integral asepsis after testing。
The foregoing is only the preferred embodiments of the present invention; not thereby the scope of the claims of the present invention is limited; every equivalent structure transformation utilizing description of the present invention to make, or directly or indirectly it is used in other relevant technical fields, all in like manner include in the scope of patent protection of the present invention。
Claims (3)
1. the manufacture method of a high grade of transparency PDA culture medium, it is characterised in that comprise the following steps:
A, potato cutting: Rhizoma Solani tuber osi 200g is cleaned, the square fourth block removing the peel, go eye, trimming length to be 4-6mm；
B, boil block: place in hot water by Rhizoma Solani tuber osi fourth block, first boil 5 minutes to softening with big fire, then change slow fire into and boil 10-14 minute, make potato nutritional fully distribute to water, make Rhizoma Solani tuber osi fourth thoroughly well cooked but not mushy simultaneously；
C, filtration: use 6-8 layer filtered through gauze 2-3 time, retain filtrate；
D, reinforced: glucose 20g, potassium dihydrogen phosphate 0.8-1.4g, magnesium sulfate 1.2g-1.7g to be added above-mentioned filtrate, stirs evenly, be settled to 1000mL with water；
E, tune pH value: use hydrochloric acid solution and sodium hydroxide solution to regulate pH value to 6.5-7, obtain fluid medium；First by high-purity agar powder 19-23g subpackage to several allotment containers, then fluid medium is dispensed in described several allotment container, mixing；
F, it is down flat plate: having added the culture medium high temperature sterilize of agar powder, and after cooling, aseptically be dispensed in sterilized slat chain conveyor ware, the bottom of each culture dish is level, and the thickness of each culture dish culture medium is 3.0 ± 0.5mm；
G, dry: cool down culture dish further, ware is covered and on alcohol burner flame, steam is dried；
H, seal and carry out Sterility testing: with ventilative sealed membrane, under aseptic conditions culture dish is sealed up mouth, install in aseptic polypropylene plastics pocket, be put into inside the constant incubator of 28 DEG C, within 2-4 days, do Sterility testing, obtain solid medium after integral asepsis after testing。
2. the manufacture method of high grade of transparency PDA culture medium according to claim 1, it is characterized in that, described " F, be down flat plate " step refers to culture medium at 123 DEG C, high pressure 0.122MPa sterilizing 26min, temperature be down to 50 DEG C again subpackage to sterilized slat chain conveyor wares。
3. the manufacture method of high grade of transparency PDA culture medium according to claim 1, it is characterised in that in described " D, reinforced " step, add food grade salts 8-14g。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114794145A (en) * | 2022-05-30 | 2022-07-29 | 广东省农业科学院果树研究所 | Potassium bicarbonate bacteriostatic agent for inhibiting banana wilt |
| CN114794110A (en) * | 2022-05-30 | 2022-07-29 | 广东省农业科学院果树研究所 | Potassium sorbate bacteriostatic agent for inhibiting banana wilt |
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| CN114794145A (en) * | 2022-05-30 | 2022-07-29 | 广东省农业科学院果树研究所 | Potassium bicarbonate bacteriostatic agent for inhibiting banana wilt |
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