What is tissue separation, and briefly describes the operation process of tissue separation

Tissue segregation, also known as asexual segregation, is a simple method to isolate and cultivate pure hyphae using the tissue blocks of fruiting bodies under suitable medium and growth conditions. This isolation method is easy to operate, and the offspring can maintain the excellent properties of the original strain, and it is not easy to mutate. This is usually done as follows:

1) Mushroom disinfectionPut the preferred standard mushrooms in the inoculation box, scrub and disinfect the surface of the mushrooms with 75% alcohol, and blot them dry with sterile gauze, and place them in the sterilized petri dish for later use. The selection of mushrooms and the disinfection of utensils are the same as before. (2) Cut the mushrooms apart, and cut a small piece of the mushroom at the junction of the cap and the stipe or the fold of the fungus, and use the sterilized inoculation knife to make the inoculation block.

It is then slitted into small slices for later use. In order to reduce the chance of carrying miscellaneous bacteria, try to take small pieces of tissue when cutting them to obtain bacteria with high purity, and the small pieces are easy to survive. Matsutake tissue isolation is better to pick small pieces of fungus folds with an inoculation needle.

3) Inoculation and culture use an inoculation needle to pick a small inoculation block, carefully access the ** of the oblique tube medium, and place it in the incubation room (box) of about 25 for culture. Pay attention to aseptic operation when inoculation, and do not be careless. After 5 to 7 days of culture, when white hyphae grow on the tissue inoculum block and spread to the medium, robust and excellent mycelium should be selected, purified and selected, and another tube should be cultivated into a generation of mother species.

The method of isolating a strain from a certain part of the fruiting body is called tissue isolation. The tissue isolation method is simple, and the offspring are not easy to mutate, which can maintain the excellent characteristics of the original strain. It is best to use young fruiting bodies or mushroom buds that are in vigorous growth as the separation material, and the tissue at the junction of the cap and the stipe is used for isolation, which is the best effect, for those mushrooms that have inner or outer curtain protection.

In addition, the young and tender folds under the protection of the bacterial curtain are inoculated, and the vitality is more vigorous. For some mycorrhizal fungi, the stipe tissue close to the base is used to survive.

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Sharp separation: Performed with a knife or scissors. When separating with a knife, the blade is used to make a vertical, lightweight, short-distance incision along the tissue gap.

When using scissors, the tip of the scissors is extended into the tissue gap, which should not be too deep, and then the scissors handle is opened, the tissue is separated, and then it is cut off after determining that there are no important blood vessels and nerves. Sharp separation has less tissue damage, fewer postoperative reactions, and faster healing. However, it must be done with anatomy and when discerning tissue structures under direct vision.

The movements should be accurate and delicate. 2.Blunt separation:

Perform with a knife handle, hemostat, stripper or fingers, etc. These instruments or fingers are inserted into the interstitial space and the surrounding tissue is separated with appropriate force. This method is most suitable for the separation of normal muscles, fascia, and benign tumors.

In blunt dissociation, tissue damage is severe, and many inactive tissue cells are often left, so the postoperative tissue response is heavier and healing is slower. In the department with large scars, excessive adhesions, or rich blood vessels and nerves, it should not be used.

It is best to use the limb wheel or mushroom bud of the young fruiting body that is in vigorous growth as the separation material, and the tissue at the junction of the cap and the stipe is used for separation, and the best effect is to use the tissue at the junction of the cap and the stipe.

Precautions: 1) The size of the incision must be appropriate. The incision is too small to be fully revealed; Unnecessary large incisions can damage too much tissue.

2) When making an incision, it must be carried out in layers according to the anatomical level, and care should be taken to keep the incision the same size from the outside to the inside. Both sides of the incorporation should be covered and fixed with sterile towels to avoid bringing bacteria on the surface into the incision during the operation and causing contamination.

3) The incision tissue must be neat, and strive to cut it at one time. The scalpel is perpendicular to the **, muscle, to prevent oblique cuts or multiple cuts in the same plane, causing unnecessary tissue damage.

4) When cutting the deep fascia, in order to prevent damage to the deep blood vessels and nerves, a small incision can be made first, separated and opened with a hemostat, and then cut.

5) When cutting the muscles, it is necessary to separate them with the handle or fingers along the direction of the muscle fibers, and cut off less to reduce the damage and affect the healing.

6) When cutting the peritoneum and pleura, it is necessary to prevent internal organ damage.

7) When cutting the bone tissue, it is necessary to cut and separate the periosteum first, and preserve the healthy part as much as possible to facilitate the healing of the bone tissue.

Tissue isolation, which usually refers to the isolation of hyphae from fruiting bodies.

A quick, easy, and easy way to do it. There are two ways to isolate the tissue of Tremella fungus: one is to isolate the germination hyphae of the fungus from the ear piece; The other is to isolate the hyphae directly from the white hair ball. Strains can be obtained. Here's how to do it:

1) Ear piece separation method.

Pick the seed ears. In the enclosed, non-spurious fungus specially used for the separation and selection of the fruiting body of Tremella fuciformis, the white hairy mass is strong and plump, the fruiting body original base block is formed early, the ear emerges early, the fruiting body is round, the ear piece is thick, the leaf is coarse, and the color is white as the separation material. It is also possible to select fruiting bodies that meet the standard seed ear conditions from the Tremella fuciformis cultivation population as the separation material.

Cut the ears into the bottle. In the inoculation box, cut the fruiting body 1 cm square (the size of a small fingernail) of small ear pieces, rinse them in sterile water or wipe the surface of the ear pieces with alcohol, and then absorb the water with gauze, and then insert the pre-sterilized bottled sawdust medium.

on, let the spores germinate hyphae. Inoculate more than 30 vials at a time for screening.

Spore germination. After the ear piece is inserted, it is placed under 23 24 culture, and observed day by day, and if it is found that it is contaminated by miscellaneous bacteria, it will be eliminated immediately. After the spores are scattered on the medium and germinate into white spores, take out the ear pieces and plug the cotton at the mouth of the bottle.

Generally, it takes 10-15 days from the time the ear piece enters the bottle to the appearance of the spores. When millet grains appear in the original seed lugs.

After the large white hair ball, one of the white and complete grains was hooked with an inoculation needle, and transferred to the sawdust seed culture medium, and continued to be cultivated for 30 35 days at 22 24 times. Among them, the mycelium of the ear friend grows vigorously and secretes melanin.

Uniform, the white hair mass gelatinizes quickly, the primordium is strong, the ears emerge early, and the fruiting body is rounded, and it grows to the thumb or a ping pong ball.

When it is large, it becomes a first-class species of sawdust.

2) Single group separation method.

The single-cluster isolation method is fast and can produce a large number of strains in a short period of time. However, it can only be used once, and if it is used repeatedly, it is easy to cause the mycelium of otophilia to degenerate, affecting the yield. Here's how:

Selection. In the culture room, the strains with traits in line with commercial production were selected and the bacterial age was about 10 days as the separation material. The mycelium on the surface of the medium is required to grow robustly and the white hair mass is successfully established; The mycelium of the inner ear friend in the medium grows vigorously, secretes clear black patterns, no bacterial contamination, and no insect pests.

Transfer. Move the selected stock into the inoculation box, open the tampon on the bottle mouth, carefully aim at the ideal one among the many white hair balls, hook a single white hair ball with an inoculation needle, quickly transfer it to the surface of agar medium or sawdust medium, and transfer 1 grain per test tube or strain bottle. Transfer 40 to 50 bottles or bottles at a time for easy screening.

Cultivate. After inoculation, under 22 24, after 10 to 15 days of culture and observation, wait for the spread of otophilia hyphae to the slope.

When pale yellow water droplets appear around the white hair mass, and then form a broken rice-like primordium, it is a first-order agar species. The white hairy mass inoculated on the medium of the sawdust seed bottle is generally cultured for 30 to 35 days, and when the fruiting body appears to be thumb large, the best is selected to become the first-class species of sawdust.

Tissue isolation is a method of isolating a strain from a part of the fruiting body.

1.The famous black fungus species "Jusan", "Dongmei No. 1", "Xuemei No. 1", "Xuemei No. 3" and "984" are the Dongmei edible fungus factory of the Chaihe Edible Fungi Society of Heilongjiang Province, which has been successfully domesticated and bred through the isolation of wild fungus tissues.

2.Tissue isolation method for shiitake mushrooms.

The fruiting bodies are disinfected with mercury water or 70% alcohol. Rinse with sterile water and wipe off surface moisture with sterile gauze. When separating shiitake mushrooms, use a sterile scalpel to cut a little from the stigium, then break the fruiting body into two by hand, cut a small piece of mushroom meat in cubic centimeters at the junction of the cap and the fungus fold, and transfer it to the inclined medium**.

If the umbrella has been opened, choose the mushroom meat at the junction of the cap and the stipe. When separating the straw mushroom, use a sterile scalpel to cut a little longitudinal bud; Then gently peel off the cap by hand, cut a fine piece of 1 5 mm above the stipe and at the junction of the cap, and connect it in the inclined medium. If the mushroom has been exposed to rain and absorbs more water, the fungus folds should be used as inoculation material.

After tissue isolation, place the outside of the tube in an incubator and culture. After the hyphae germinate around the tissue block and spread to the culture medium, the robust hyphae are selected for tube culture. The tissue isolation method is easy to operate, and it is not easy to bring in miscellaneous bacteria, and it is easy to obtain pure strains.

But for glial fungi such as white fungus and black fungus; Because the content of hyphae in the fruiting body is very small, it is often difficult to succeed if it is isolated and cultured with tissue.