MS培养基的配制与灭菌

MS培养基的配制与灭菌 Preparation and sterilization of MS medium A common medium in plant tissue culture is the MS medium. The preparation of the MS medium comprises the following steps. Preparation and preservation of MS culture medium containing nearly 30 kinds of nutritional components of base liquor, in order to avoid every medium preparation should be carried out on the weighing dozens of ingredients, can be cultured in the medium of various components, according to the original amount of 20 times or 200 times respectively weighing, with concentrated liquid, this concentrated solution is called culture base liquor. In this way, each use, take the total amount of 1/20 (50 mL) or 1/200 (5 mL), dilute with water, into the culture medium. The quantities of various substances required for the preparation of the mother liquid of the culture medium are listed for use in preparation. A large number of elements (mother liquor I) mg / L NH4NO3 33000 KNO3 38000 CaCl2 2H2O 8800 MgSO4 7H2O 7400 KH2PO4 3400 Trace elements (Mother Liquor II) KI 166 H3BO3 1240 MnSO4 4H2O 4460 ZnSO4 7H2O 1720 Na2MoO4 2H2O 50 CuSO4 5H2O 5 CoCl2 6H2O 5 Iron salt (mother liquor III) FeSO4 7H2O 5560 Na2-EDTA 2H2O 7460 Organic ingredients (mother liquor IV) IV A Inositol 20000 IV B Niacin 100 Pyridoxine hydrochloride (vitamin B6) 100 Thiamine hydrochloride (vitamin B1) 100 Glycine 400 The amount of all the nutrients mentioned above was 200 times of concentrated liquid except for mother liquor, which was 20 times concentrated liquid. Each of these mother liquids shall be individually separated into 1 L stock solution. The preparation method of mother liquor, liquor and liquor are: II IV weighing several components each in the mother liquor after respectively with distilled water to dissolve a small amount, and then they are immiscible, the final volume to 1 L. The preparation method is: Liquor III will be called FeSO4 7H2O and Na2-EDTA 2H2O respectively in 450 mL distilled water, heating and stirring to dissolve them, then the two solutions are mixed, and the pH was adjusted to 5. 5 and final volume to 1 L, stored in brown glass bottle. After all the mother liquor has been prepared, they are stored in glass bottles, labeled with mother's numbers, preparation times, dates and so on. They are kept in the refrigerator. MS medium adding 2 4-, two chlorophenoxyacetic acid (2, 4-D) and NAA (NAA), 6 (6BA) and benzyladenine and other plant growth regulating substances, and respectively with mother liquor (0. 1 mg/mL). The preparation method is as follows: each of the 3 substances is respectively 10 mg, and 2, 4-D and NAA are pre dissolved with small amount (1 mL) anhydrous ethanol, and the 6BA is dissolved with a small amount (1 mL) of a substance with a concentration of 0.1 mol/L of NaOH solution, The dissolution process requires water bath heating, finally to volume 100 mL, the concentration of 0. 1 mg/mL mother liquor. Preparation of liquid or using a pipette to remove the required amount from a variety of mother liquor respectively: mother liquor was 50 mL, II, III and IV liquor A and IV B 5 mL. Take 2, 4 D 5 mL, NAA 1 mL, and all kinds of liquor together into a beaker. Should pay attention to preparation of medium: in the use of advance preparation of mother liquor, should take all kinds of liquor in the amount before, gently shake the bottle containing mother liquor, if found a bottle of precipitation, suspended or microbial contamination should be immediately eliminated, the mother liquor, re preparation; using a pipette or the amount of training base liquor before, must be taken by the volume of the cylinder or pipette liquor Guan Runxi 2 times; the amount of liquor, the liquor will be all the best according to the amount of the order written on paper, Volume 1, crossed 1, in order to avoid mistakes. Melt agar with coarse balance were weighed, steamed sugar agar 9 g 30 g 1000 mL into the enamel cup, then add 750 mL of distilled water, with electric heating, heating and stirring with a glass rod until translucent liquid. Then add the prepared mixture to the boiling agar, and finally add the distilled water to 1000 mL, stirring evenly. It should be noted that in the process of heating the agar and preparing the medium, the operator must not leave, otherwise the boiling agar spill will need to be re weighed and prepared. In addition, if there is no enamel cups available instead of beaker. But we should pay attention to the outer surface of the bottom of the beaker cannot touch water, otherwise when heating the solution beaker easily cracked, overflow, scald. PH with the dropper absorb amount of substance concentration of NaOH solution was 1 mol/L, each medium drops into the melt, while drops while stirring, and at any time with the pH test precision (5. 4~7. 0) measuring the pH of the medium, until the pH of the medium was 5. 8 (medium the pH must be strictly controlled in 5. 8). Medium should be packed separately when heated. When loading, the medium is poured into the beaker and the medium in the beaker is poured into a conical flask (50 mL or 100 mL). Be careful not to leave the culture medium to the mouth of the bottle and the wall of the bottle. The amount of the medium in a conical flask is about 1/5 to 1/4 of the capacity of a conical flask. Each 1000 mL medium can be divided into 25~30 bottles. After the medium is packed, the bottle mouth should be sealed in time. 2 pieces of parchment paper (each about the size of 9 cm * 9 cm) sandwiched between 1 thin layers of kraft paper cover the bottle, and binding. Finally, label the outer cone of the conical flask. Autoclaving of high-pressure sterilization medium involves the following steps. First, put the flask. A conical flask containing a culture medium is placed in a small metal basket and placed in a autoclave. If there is no small metal basket, it can be separated by a glass plate between the two conical bottles. Second place other items that need to be sterilized. Other items also need sterilization in high pressure steam sterilization pot, such as conical flask, filled with distilled water with a screw top glass bottle, beaker, jar (the above items are to be wrapped in kraft paper, kraft paper coated with sealing) Petri dishes, scissors, tweezers, scalpel, filter paper and pencil etc.. Third, sterilization. To be sterilized items stacked after cover. At 98 kPa and 121.3 DEG C, 20 min was sterilized. After sterilization, remove the conical flask and allow the medium to cool and solidify naturally. Better place 1 d before using.