CN105132289A - Method for efficiently isolating tricholoma matsutake fungus - Google Patents

Abstract

The invention provides a method for efficiently isolating tricholoma matsutake fungus. According to the method, spore isolation of tricholoma matsutake is utilized, specifically, tissue blocks containing lamella and context of tricholoma matsutake are clamped, and are adhered on the right middle on the inner surface of a culture dish cover with the lamella facing downwards, and the ripened lamella sprays a large number of spores which fall on a culture medium to germinate to form pure mycelia. With the adoption of the method and the culture medium for isolating the tricholoma matsutake fungus, the success rate achieves 90% or above, the operation is simple and feasible, and the amount of the needed materials is small; the tissue blocks and the culture medium have no direct contact, so that mixed fungi contamination is avoided, and the contamination rate is also greatly reduced; with the adoption of the culture medium for culturing tricholoma matsutake spores, the germination rate is high, the mycelia are sturdy, the growth speed is high, and obvious aerial mycelia are formed. With the adoption of the method, the problems that at present, tricholoma matsutake is isolated by lamella, the success rate is not ideal, contamination is easily caused, the growth of the mycelia on the culture medium is extremely slow, and hardly forms amplification rapidly, and the like, are solved.

Description

A kind of method of high efficiency separation matsutake bacterial classification

Technical field

The present invention relates to a kind of method of high efficiency separation matsutake bacterial classification.

Background technology

Matsutake (Tricholomamatsutake), also known as Trichotoma matsutake, is famous edible and medicinal fungi, has very high pharmaceutical use and multiple biological activity.The effects such as modern medicine study shows that matsutake has antitumor, antibacterial, antiviral, anti-diabetic, anti-inflammatory, treatment cardiovascular diseases; Simultaneously again because it has good radiation-proof healthcare function, extensively pursue by people in the world, there is high economic worth.

Matsutake growth conditions is special, needs the fibrous root belonged to Pinus, oak in certain circumstances to produce symbiotic relationship, forms mycorhiza, could grow sporophore further.The production of matsutake can only take semi-artificial mode, with the mycelia of artificial culture host's root induced synthesis bacterium pool under field conditions (factors) or bacterium colony, finally develops into sporophore.Therefore be separated obtains and cultivate the pure mycelium of matsutake to the production of matsutake, study tool and be of great significance.The method of the separation tricholoma matsutake mycelium the best of generally acknowledging at present is the lamella position utilizing sporophore, but this separation method still also exists some problems, as utilized lamella Success rate of virus isolation, still undesirable, easy pollution, the mycelia speed of growth on substratum is extremely slow, is difficult to amplification rapidly etc.

Summary of the invention

For above-mentioned the deficiencies in the prior art, the present invention is to provide a kind of method of high efficiency separation matsutake bacterial classification, adopt present method to make mycelium germination fast, growing way is strong.

The technology used in the present invention is as follows: a kind of method of high efficiency separation matsutake bacterial classification, as follows: select outward appearance typical case when selecting Tricholoma matsutake (lto et lmai) Singer sporophore, be of moderate size, bacterial context is plump, color normally and the sporophore of not yet parachute-opening, Tricholoma matsutake (lto et lmai) Singer sporophore mass concentration 75% alcohol swab is cleaned 2-3 time, puts into sterilized Bechtop; Tricholoma matsutake (lto et lmai) Singer sporophore is broken into two with one's hands along cap and stem intersection, then cap surface epidermis is torn together with velum, expose aseptic bacterial context and lamella part; Cap edge is clamped, gripping 1-2cm with aseptic nipper ³the tissue block of size, the tissue block of gripping not only comprises the bacterial context on upper strata but also comprises the lamella part of lower floor; Now plate culture medium temperature is at about 60 DEG C, ot-yet-hardened, dips do not solidify substratum on a small quantity by the bacterial context part of tissue block, lamella part does not contact with substratum, tissue block is sticked at culture dish interior surface middle, lamella part is downward, tissue block and substratum distance 0.6cm; The sealing that closes the lid is preserved, and under 25 DEG C of dark conditions, just put cultivation, the bacterial context part of tissue block supplies nutrition to lamella, makes lamella ripe and sprays spore, and ripe spore is sprayed on substratum sprouts into pure mycelium.

The present invention also has following technical characteristic:

Described culture medium prescription is:

Phellinus igniarius mycelium 6d fermented liquid 200ml/L, potato 200g/L, glucose 20g/L, agar 14g/L, retinol1 .5mg/L, vitamins B ₁2.5mg/L and vitamins B ₂2.5mg/L;

Described phellinus igniarius mycelium 6d fermented liquid: access Phellinus inclined-plane kind in 300mlPD liquid nutrient medium, inclined-plane kind substratum is PDA, and 25 DEG C of lucifuges cultivate 7d, 25 DEG C of shaking culture, 160r/min, after cultivating 6d, filters and obtains fermented liquid; Phellinus bacterial classification derives from China typical culture collection center (CCTCC) preserving number: Phellinus DL101CCTCCM2011137.

The present invention has following technical characterstic:

1, use the present invention to be separated the success ratio of matsutake bacterial classification up to more than 90%, and operation is simple, material requested is few;

2, spore separation uses the ripe spore of matsutake, possesses the hereditary property of parents, and vitality is strong, and the strain quality cultivated is good, is better than general separate tissue;

3, in this separation method, tissue block does not directly contact with substratum, avoids some living contaminantses, greatly reduces pollution rate.

4, use the culture medium culturing spores from mushroom in the present invention, germination rate is high, and mycelia is sturdy, fast growth, forms obvious aerial hyphae.

Embodiment

The present invention will be further described in citing below:

Embodiment 1

One, culture medium prescription:

Phellinus igniarius mycelium 6d fermented liquid 200ml/L, PDA: potato 200g/L, glucose 20g/L, agar 14g/L, retinol1 .5mg/L, vitamins B ₁2.5mg/L, vitamins B ₂2.5mg/L.

Concrete operation method, 1L substratum is example: peeling potatoes, takes 200g, is cut into about 1cm ³fritter, add water 500ml, boil rear maintenance boiling 20min, filter to get filtrate.Filtrate is mixed with 200ml phellinus liteus fermented liquid, adds retinol1 .5mg, vitamins B ₁2.5mg, vitamins B ₂2.5mg, heated and stirred is to dissolving completely, and constant volume is to 1000ml.Be dispensed in triangular flask, high pressure steam sterilization 121 DEG C, 0.105MPa, 20min.

Phellinus igniarius mycelium 6d fermented liquid: (PD medium component: potato 200g/L in 300mlPD liquid nutrient medium; glucose 20g/L) (inclined-plane kind substratum is PDA to access a test tube Phellinus inclined-plane kind; 25 DEG C of lucifuges cultivate 7d); 25 DEG C of shaking culture; 160r/min; after cultivating 6d, filter and obtain fermented liquid.Phellinus bacterial classification derives from China typical culture collection center (CCTCC) preserving number: Phellinus DL101CCTCCM2011137.

Two, plate is down flat:

After substratum and culture dish (d=90mm) are used high-pressure sterilizing pot 121 DEG C of sterilizing 20min, put into Bechtop, after uv sterilisation 30min, be down flat plate, operate near spirit lamp flame, each culture dish substratum of falling 25-30ml, uses when substratum ot-yet-hardened.

Embodiment 2

A method for high efficiency separation matsutake bacterial classification is as follows:

Step one: plant mushroom and choose and surface sterilization

Outward appearance typical case should be selected when selecting Tricholoma matsutake (lto et lmai) Singer sporophore, be of moderate size, bacterial context is plump, color normally and the sporophore of not yet parachute-opening.Tricholoma matsutake (lto et lmai) Singer sporophore 75% alcohol swab is cleaned 2-3 time, puts into sterilized Bechtop.

Step 2: stripping and slicing is inoculated

Tricholoma matsutake (lto et lmai) Singer sporophore is broken into two with one's hands along cap and stem intersection, then cap surface epidermis is torn together with velum, expose aseptic bacterial context and lamella part.Cap edge is clamped, gripping 1-2cm with aseptic nipper ³the tissue block of size, the tissue block of gripping not only comprises the bacterial context on upper strata but also comprises the lamella part of lower floor.Now plate culture medium temperature is at about 60 DEG C, ot-yet-hardened.Dip by tissue block bacterial context part and do not solidify substratum (lamella part can not contact with substratum) on a small quantity, tissue block is sticked at culture dish interior surface middle, lamella part is downward, tissue block and substratum distance about 0.6cm.The sealing that closes the lid is preserved.

Step 3: culture purified

Under 25 DEG C of dark conditions, just put cultivation, the bacterial context part of tissue block can supply nutrition to lamella, makes lamella ripe and sprays spore.Ripe spore is sprayed on substratum can sprout into pure mycelium.

Claims (2)

1. a method for high efficiency separation matsutake bacterial classification, is characterized in that, method is as follows:

Select outward appearance typical case when selecting Tricholoma matsutake (lto et lmai) Singer sporophore, be of moderate size, bacterial context is plump, color normally and the sporophore of not yet parachute-opening, Tricholoma matsutake (lto et lmai) Singer sporophore mass concentration 75% alcohol swab is cleaned 2-3 time, puts into sterilized Bechtop; Tricholoma matsutake (lto et lmai) Singer sporophore is broken into two with one's hands along cap and stem intersection, then cap surface epidermis is torn together with velum, expose aseptic bacterial context and lamella part; Cap edge is clamped, gripping 1-2cm with aseptic nipper ³the tissue block of size, the tissue block of gripping not only comprises the bacterial context on upper strata but also comprises the lamella part of lower floor; Now plate culture medium temperature is at about 60 DEG C, ot-yet-hardened, dips do not solidify substratum on a small quantity by the bacterial context part of tissue block, lamella part does not contact with substratum, tissue block is sticked at culture dish interior surface middle, lamella part is downward, tissue block and substratum distance 0.6cm; The sealing that closes the lid is preserved, and under 25 DEG C of dark conditions, just put cultivation, the bacterial context part of tissue block supplies nutrition to lamella, makes lamella ripe and sprays spore, and ripe spore is sprayed on substratum sprouts into pure mycelium.

2. the method for a kind of high efficiency separation matsutake bacterial classification as claimed in claim 1, it is characterized in that, described culture medium prescription is:

Phellinus igniarius mycelium 6d fermented liquid 200ml/L, potato 200g/L, glucose 20g/L, agar 14g/L, retinol1 .5mg/L, vitamins B ₁2.5mg/L and vitamins B ₂2.5mg/L;

Described phellinus igniarius mycelium 6d fermented liquid: access Phellinus inclined-plane kind in 300mlPD liquid nutrient medium, inclined-plane kind substratum is PDA, and 25 DEG C of lucifuges cultivate 7d, 25 DEG C of shaking culture, 160r/min, after cultivating 6d, filters and obtains fermented liquid; Phellinus bacterial classification derives from China typical culture collection center (CCTCC) preserving number: Phellinus DL101CCTCCM2011137.