A small method for the separation and culture of strains.

In nature, edible bacteria do not exist alone, but with many bacteria, radioactive bacteria, mold and so on live together.
the so-called species separation, is to these and edible bacteria living together with the separation of the hybrid bacteria, through culture, to obtain pure excellent strains.
, primary and cultivated species of the bacteria species.
breeding program can be referenced.
(a) the separation of the mother species of culture edible bacteria can be divided into spore separation method, tissue separation method and the separation of the base mycelium.
1. Spore separation method spore separation method is a method of germination into mycelium and culture of bacteria using sexual spores or asynsional spores of edible bacteria.
the bacteria life force is strong, but there are differences between individual spores, and the phenomenon of natural differentiation is more serious, large variation, need to be tested by mushrooms in order to be applied in production.
(l) monosporidium separation method: is a method of taking only one spore at a time or per test tube, allowing it to germinate into mycelium to obtain purebred species.
mushrooms and grass mushrooms with single spore separation of the mycelium, has a strong capacity, can be used to isolate the production of pure bacteria.
used in the production of single spore separation, and the technology is complex, the polysporidium separation method is generally used.
(2) polysporidium separation method: is a way to inoculate many spores on the same culture base and allow them to germinate and mate freely to obtain pure strains of edible bacteria.
Specific operating methods, there are the following: (1) mushroom spore bullet shooting method: choose individual robust, flower-shaped round, no pests and diseases, mushrooms uniform, high yield and stable yield, adaptable eight or nine mature mushrooms, cut off most, the handle with sterile water rinsed several times before using sterilized gauze or degreased cotton, filter paper to dry surface moisture.
in the inoculation box or sterile room, hang the mushroom folds face down with wire under the glass funnel, which covers the petri dish upside down, and the upper hole is stuffed with cotton.
dishes are placed on a gauze-covered enamel plate and placed for 12 to 20 hours, and the spores on the folds are scattered in the petri dish.
a layer of powdered spores (flat mushrooms very lilac, mushrooms, mushrooms are brown, mushrooms, golden needle mushroom spores printed white).
a small amount of spores with an inoculation needle and line the agar outside the test tube or on a petri dish.
spores to germinate, the production of mycobacteria, the selection of spores germination early, long-term good microbiosis for test tube culture.
spores can also be collected using spore collectors.
method is to select the mushrooms, according to the above procedure, gently open the glass bell cover, the mushroom handle is inserted down on the wire frame of the spore harvester, placed in the center of the petri dish.
the glass cover and stuff the bell around the palm with gauze.
and pour a little mercury or sterile water over the gauze.
into a thermostat for culture at about 20 degrees C.
(2) Smearing method on the folds: when separated by sterile operation, choose mature mushrooms, insert directly between the folds with a inoculation needle, gently wipe the spores on the surface of the folds that have not yet empirged, and then line the base.
(3) Hook suspension: Take a few folds of the mature bacteria cap or a small piece of ear (black wood ear, woolen ear, white wood ear), with aseptic stainless steel wire (or wire, cotton wire and other hanging materials) hanging above the culture base inside the triangular bottle, do not come into contact with the culture base or the wall around the bottle.
culture and transfer can be placed at suitable temperature.
(4) Affix: Take a small piece of mature bacterial folds or ear tablets according to sterile operation, and attach them to the test tube wall directly above the tube oblique meth with a dissolved agar culture or Arabic glue, paste, etc.
after 6 to 12 hours of culture, waiting for the spores to fall on the slope, immediately transplant the spores together with part of the agar medium into a new test tube culture.
Spore separation of the mother species, must be further purified and strong, when the mother plant planted for about a week, mycelium covered with slope, choose mycelium robust, vigorous growth without aging, no infection of the mother test tube, and then transfer tube expansion, generally to cultivated species, transfer tube should not be more than 5 times.
isolated from the spores, must pass the mushroom test and be identified as a high-quality strain before it can be used in production.
general bacteria such as mushrooms, mushrooms, anchovies, mushrooms, mushrooms and mushrooms, can be used polysporidium separation method to obtain the mother species.
is now on the separation of silver ear spores outlined below: according to sterile operation to obtain the species of ear, hanging ear triangular bottle after 12 hours of culture, the bottom of the bottle culture base surface has a "spore print."
take out the ear, place 20 degrees C - 25 degrees C temperature box culture 2 to 3 days, the surface of the culture base will appear milky white transparent paste-like small bacteria, that is, silver ear yeast-like spores formed a small amount of silver ear bacteria.
this time to transfer to the test tube sloped meth, to be fully oblique, and then to be transferred to the nutrient-rich, and the surface is relatively dry on the culture.
after 30 days or so, the bacteria grow white mycelium.
Silver ear yeast-like spores, mycelium only mixed with feather-like mycelium subcystic bacteria (scented ash mycelium) culture, by the latter to help decompose wood and some other fibrous substances, provide nutrition, in order to benefit the silver ear spores germination, mycelium planting and the formation of sub-entities.
two pure myceliums are selected when the two myceliums are at the meeting.
Feather-like mycelium to purify breeding, generally to choose a fast-growing, wall-climbing force of the test tube slope or bottle, take the first end of the mycelium, transfer tube transfer, place 25 degrees C - 28 degrees C culture, repeat the transfer tube several times can get good thoroughbred.
characteristics of silver ear mycelium, mycelium growth is slow, and spores are not easy to germinate.
In the mixing of two bacteria, first take 8 to IO days cultured silver ear mycelium slope, according to sterile operation method in this slope from silver ear mycelium about 0.5 cm to access a small feather-like mycelium.
the silver ear mother species is mixed for 1 week at 25 degrees C.
2. Organizational separation culture method uses the internal organization of sub-entities to carry out asym sexless reproduction and obtain the mother species, that is, tissue separation.
the method is simple to operate, mycelium growth and development is fast, variety characteristics are easy to save, especially after hybrid breeding, excellent strains with tissue separation method can stabilize the genetic characteristics.
separation methods are often used.
(l) sub-entity separation: mushrooms to choose a large cover thick, short handle, 89 cents mature excellent varieties.
Cut off the two base parts of the mushrooms, soak in 0.1% liter mercury in a sterile box for a few minutes, then rinse with sterile water and dry or wipe the lid and bacterial handle 2 times with 75% alcohol cotton ball, surface disinfection.
inoculation, as long as the mushrooms are torn apart, at the junction of the bud cover and the handle or the folds of the bacteria, pick a small piece of tissue;
cultured for 3-5 days at a temperature of about 25 degrees C, you can see that white fluffy mycelium is produced on the tissue, and the transfer tube expands to obtain the bacteria.
mushrooms, mushrooms, etc. can be used in this way.
(2) microbial nucleus separation: the sub-entities of bacteria such as sepsis, pig pythroids, and thunder pills are not easy to collect.
common is that it stores nutrient-rich nuclei.
with the micronucleus, the same can be obtained.
method is to wash the surface of the bacteria core, with alcohol or mercury disinfection, cut open the nucleus, take a small piece of intermediate tissue, about the size of soybeans, inoculated in the PDA medium slope, insulation culture.
It should be noted that the nucleus is a storage organ, most of which is polysaccharides, contains only a small amount of mycelium, so pick the tissue block to be bigger, if the tissue block is too small, it is not easy to split the species.
(3) mycelium separation: some sub-entities are not easy to find, there are no bacterial nucleus, can be isolated with mycelium.
such as honey ring bacteria, false honey ring bacteria.
Its operation method is to use alcohol or mercury to gently wipe the black cort vein on the surface of the bacteria 2 to 3 times, and then remove the black outer cort vein (bacteria), pull out the white myelin part;
of mycelium should be paid attention to: because the bacteria is relatively small, the separator is also relatively small, the separation is very easy to contaminate the bacteria, so strict operation.
3. The ethyl mycelium separation culture method uses the substation of edible fungi as the separation material to obtain a method of pure bacteria species, called the base mycelium separation method.
method of separation is appropriate only in certain seasons, and is a child entity that dies in the twilight and is not easy to harvest.
of the inner-base separation method and the tissue separation method is that the mycelium in dried mushrooms or earwoods is often dormant.
sometimes does not return to growth immediately after vaccination.
therefore, it is necessary to keep it for a longer period of time (about 1 month) to determine whether mycelium can survive.
the separation method of the inner mycelium can be divided into the separation of mycelium (i.e., mushroom or ear wood separation method) and the separation method of mycelium in soil.
(1) in the wood of the mycelium separation method: is the mushroom wood or ear wood separation method, in order to reduce the infection of bacteria, mushroom (ear) wood before separation, must be sterile treatment.
The surface of the mushroom (ear) water can be gently burned with an alcohol lamp flame to burn the mold spores, or another 0.1% of the mercury water soaked in spores for a few minutes, and then rinsed with sterile water and then dried with sterile filter paper.
should be taken when cutting the inoculation block.
inoculation block must be cut within the distribution of the mycelium.
so, the type of slow growth of mycelium should be shallow;
at the same time.
should also determine the range of mycelium distribution according to the type of mushroom bacteria, wood texture, mushroom (ear) wood weight, the length of development time.
then cut with a sharp knife.
should be as small as possible to reduce the chance of miscellaneous infection and to raise the purity of the bacteria.
the inoculation block to the medium, it should be placed suitable for the growth of mycelium in a greenhouse or temperature box of 22 to 26 degrees C culture, so that mycelium can resume growth.
(2) soil mycelium separation: many types of edible bacteria, many native edible bacteria;
tissue separation is not easy to succeed, using the method of pure species separation of mycelium in soil, called the separation of mycelium in soil.
When the separation of mycelium in soil, it should be noted that because there are a variety of soil microorganisms living around the soil mycelium, so the separation must avoid the interference of these microorganisms as far as possible, as far as possible to remove the tip of clean mycelium, without debris mycelium inoculation, repeatedly rinse with sterile water, in the medium to add some inhibitory bacteria growth drugs, such as 40 micrograms / liter of mycocin or gin.
if infected bacteria are found, the mycelium on the edge of the microselith can be picked out and inoculated into a wood chip medium.
because bacteria do not have the ability to break down gluten, they are not easy to expand in wood chip mediums;
the bacteria grow out of the infected area, it can be expanded and purified.
(3) sub-solid base part away: from the bottle planting, bag planting or queen bed cultivation of sub-solid base part of the method of new mycelium, called sub-solid base part away, is now to bag-planted silver ear as an example: from the ear early, ear rate away, pest-free cultivation room;
after 7 to 10 days of culture, the body of the to be treated can be recovered when the diameter of the child body is 4 to 5 cm, as a separate mother.
then sift through the ideal one, cut off the silver ear body with a sharp knife, and place it overnight in a refrigerator or hostile container at 0C to kill the pests in the bottle.
then scrub the earbuds and impurities outside the bag with 75% alcohol or mercury, along with inoculation tools, inoculation cultures, etc. into the sterile room.
sterilized, the old root of the bag is about 15 mm thick with an inoculation knife and cultured.
When the white mycelium is exposed at the mouth of the bag, pick a half-grain white bacterial colon with the inoculation needle, quickly move into the center of the mother test tube media, gently remove the inoculation needle, plug the cotton plug.
in order to obtain more mother species, a vaccination should have 100-200 test tubes, in order to choose from.
, it should be moved into a thermostat or greenhouse at 22 to 24 degrees C in time for culture.
because there is more moisture in the culture, mycelium recovers faster than earwood.
2-3 days after the separation, the edge of the separation can be seen white mycelium.
observe at least twice a day for purification.
observation and purification methods are the same as the separation of earwoods.
10-15 days after warm culture, when the inoculation block kinks appear red and yellow water beads, the original species can be expanded.
(ii) the inoculated cultured mother species of the original and cultivated species is obtained, in order to meet the needs of the production of the strains.
the mother species with high purity should be selected to further expand into the original species.
original culture base generally uses cottonseed shell, wood chips, grass and other culture base.
the original culturebest in a bottle or bag can be sterilized, it can be fed into the sterilized inoculation box, and the culture be cooled to less than 30 degrees C in the bottle can be inoculated in accordance with sterile operating procedures.
(bags) into the culture room, to be frequently checked, as soon as the discovery of hybrid contamination, immediately remove.
cultured into the original species, mycelium body must be strong and powerful, close to the wall of the bottle without dry shrinkage, pure color, with a certain fragrance, strong life force, expanded into cultivated species when eating fast.
cultivated species is to further expand the original species into a three-stage species, it and the original species of vaccination and cultivation of the same.
mushrooms, mushrooms, mushrooms, monkey head, wood ear, silver ear cultivation species with sawn wood, cotton skin as a culture base.
mushrooms use manure grass as a culture.
cultivated species requires that the material blocks are not dehydrated and shrink.
mycelium body strong and powerful, pure color, has a clear fragrance, no aging phenomenon, no hybrid pollution, some species licensed to have a small number of original base, access to cultivation materials, bacteria fast, good growth, strong life force.
the original species in the cultivation of species, the original bottle mouth of a layer of mycelium should be dug out without.
(iii) The simple cultivation of liquid species at present, the production of bacteria by liquid deep fermentation method, with large production volume, short cycle, neat age, low cost, easy inoculation and so on, is to achieve the goal of factory production of edible bacteria.
the liquid breeding process is now briefly described for reference.
In short, the pure and excellent strains, into the liquid culture (solid culture without coagulants), so that mycelium reproduction to form a large number of small bacteria, and then the culture of this culture into wood chips or cottonseed skin, into the bacteria, culture.