How to inoculate edible mushrooms? A full analysis of the technical key points

Inoculation is one of the key steps in edible mushroom production. If the inoculation technique is not up to standard, it will cause contamination by foreign microorganisms, a low survival rate, and all previous efforts wasted, let alone cultivation. Whether it is strain isolation, subculturing, or propagation, all must be carried out under sterile conditions and in strict accordance with sterile operating procedures. The specific key points are as follows:

1 Personnel quality

1.1 Inoculation personnel must possess a spirit of conscientiousness, meticulousness, self-discipline, and responsibility toward the work.

1.2 Inoculation personnel must firmly establish a sterile mindset, must not harbor any wishful thinking, and must never be negligent or careless.

1.3 Inoculation personnel must have the ability to identify the superiority or inferiority of strains; substandard strains must not be used for production.

2 Environmental disinfection

2.1 For a newly made inoculation box or one that has been idle for more than 15 days, before its first use, both the inside and outside of the box must be thoroughly scrubbed and disinfected with 0.1% mercuric chloride water or 2% Lysol.

2.2 Before reusing an inoculation room and buffer room that have been idle for more than 30 days, the walls must be sprayed and disinfected with 2% Lysol. For a newly renovated or more than 3 months unused inoculation room, before use, the indoor walls must be whitewashed with lime slurry, and the concrete floor must be washed with clean water.

3 Space disinfection

After placing the culture medium to be inoculated into the inoculation box or inoculation room, the space inside the box or room must be disinfected.

3.1 If it is stock culture subculturing or a small amount of mother spawn inoculation, the inoculation box can be used with the inoculation tools placed inside at the same time, and then chemical fumigation or ultraviolet irradiation can be used for space disinfection for 30 minutes.

3.2 If it is large-volume mother spawn or third-level spawn production, place the culture medium into the inoculation room, place the inoculation tools inside at the same time, then use chemical fumigation together with ultraviolet irradiation for space disinfection for about 40 minutes.

3.3 Place work clothes, disinfectant water, washbasins, etc. in the buffer room, and complete all preparatory work in the buffer room before inoculation. Before entering, turn on the ultraviolet lamp for 30 minutes; when leaving the buffer room and entering the inoculation room, the ultraviolet lamp must still be kept on until inoculation is finished.

4 Disinfection of carried items

4.1 Cut off the cotton plug of the mother culture test tube or mother spawn bottle (bag) flush with the tube or bottle mouth; wipe the outer wall of the mother culture test tube with 75% alcohol cotton, and wash the outer wall of the mother spawn bottle (bag) with 0.25% potassium permanganate water, then bring them into the inoculation box (room) together with the inoculation personnel.

4.2 After inoculation personnel have changed clothes and washed their hands in the buffer room and entered the inoculation room, they must not enter and exit freely; they should move around as little as possible and avoid air convection.

5 Sterilization

Use an alcohol flame for necessary sterilization.

5.1 First burn the inoculation tools (hook, spatula, knife, etc.) on the flame of an alcohol lamp, then place them in a clean empty test tube to cool for later use.

5.2 Place the test-tube mother culture or mother spawn bottle on a universal clamp, remove the cotton plug with a twisting motion, pick up the alcohol lamp with the right hand so that the flame touches the tube mouth or bottle mouth, slowly rotate the test tube or mother spawn bottle with the left hand for 3 to 4 turns, then secure the test tube or mother spawn bottle firmly.

6 Inoculation

During mother culture isolation, subculturing, or propagation, the test tube mouth must be kept within 10 cm of the alcohol flame during operation.

6.1 Use the sterilized spare inoculation hook to remove the tip portion of the mother culture slant and the central mother culture block, then put the inoculation hook back into the mother culture test tube.

6.2 Hold the test tube culture medium to be inoculated with the left hand, with the tube mouth tilted slightly downward; with the right hand, use the little finger and ring finger, index finger and middle finger to hold the inoculation hook, hook out a spawn block about 2 mm in diameter from the mother culture test tube, place it in the center of the slant of the culture medium to be inoculated, withdraw the inoculation hook, put it back into the mother culture test tube, briefly singe the cotton plug held in the right hand, insert it into the inoculated test tube with a twisting motion, place it in an appropriate position with the right hand, and continuously complete all test tubes to be inoculated.

Note: ① Each inoculation session must not exceed 2 hours continuously. ② During operation, the tip of the inoculation tool and the spawn block must not touch the tube mouth or other external objects. If the tool tip touches an external object, replace the tool; if the spawn block touches an external object, discard it. ③ General spawn usage: each test-tube mother culture can propagate about 100 first-level spawn tubes; or subculture 6 to 8 bottles of mother spawn.

7 Two special inoculation methods

7.1 When subculturing and propagating Armillaria mellea, carry it out while the culture medium still has condensation water; the diameter of the inoculated spawn block must not be less than 0.5 mm, and it must also carry rhizomorphs. Each test-tube culture medium can be inoculated with 3 mother culture blocks at the same time, and each mother culture block must be pressed with the inoculation tool until it sinks about 2 mm into the culture medium, so that the rhizomorphs can extend into the culture medium as quickly as possible; when inoculating mother spawn, each test tube can inoculate 3 to 4 bottles of mother spawn.

7.2 When subculturing and propagating the yuan mushroom fungus, the diameter of the spawn block must not be less than 0.3 mm. During inoculation, draw a longitudinal line along the middle of the slant from about 1.5 cm from the tail end of the test-tube culture medium to about 2 cm from the tip of the slant, and place the spawn block there; this method can shorten the culture cycle by about 3 days.

8 Safety

8.1 A lit alcohol lamp must not be placed near cotton plugs, plastics, or other flammable materials.

8.2 A lit alcohol lamp must be placed securely; during operation, strictly prevent knocking over the alcohol lamp to avoid causing a fire.

8.3 When using chemical drugs for space disinfection in an inoculation box or inoculation room, calculate the space volume and use them according to the drug instructions; do not arbitrarily increase the dosage, so as to avoid causing chemical damage.

8.4 Ultraviolet light can harm the human body, especially causing electric ophthalmia. Therefore, do not work while the ultraviolet lamp is turned on, and even more so, do not look directly at a ultraviolet lamp that is turned on.