Medium Preparation

• Composition of MS medium• Discussion of ingredients and what they are for• Use of prepackaged mixes or stock solutions• How stocks are used to make medium• Preparation of stocks

Murashige-Skoog Medium Composition

Ingredient mg/L

NH4NO3 1650

KNO3 1900

CaCl2•2H2O 440

MgSO4•7H2O 370

KH2PO4 170

H3BO3 6.2

MnSO4•H2O 16.9

ZnSO4•H2O 8.6

KI 0.83

NaMoO4•2 H2O 0.25

Murashige-Skoog Medium Composition

Ingredient mg/L

CuSO4•5H2O 0.025

CoCl2•6H2O 0.025

FeSO4•7H2O 27.8

Na2EDTA•2H2O 37.3

myo-inositol 100.0

nicotinic acid 0.5

thiamine•HCl 0.1

pyridoxine•HCl 0.5

glycine 2.0

sucrose 30000

agar 8000

Medium ingredients

• Inorganics– macronutrient salts– micronutrient salts

• Organics– sugar (sucrose)– "vitamins"– plant growth regulators (PGRs)– undefined ingredients– agar and solidifying agents

Prepackaged mixes vs. medium from stocks

• Prepackaged mixes offer convenience• Stock solutions have several benefits

– the assurance that you know precisely what is in the medium

– the potential to modify the medium; e.g., to adjust the PGR concentrations and ratios

– the assurance that all of the chemicals are in solution

Use of prepackaged mixes

• Most packaged mixes come with directions• There are often many variations of the most

commonly used media• The most widely used come with salts and

PGRs for 1 L, which are dissolved in water• Sucrose is added, pH checked and adjusted if

necessary

Use of prepackaged mixes (contin)

• Solidifying agent is added• Medium is autoclaved and allowed to cool

Use of stock solutions

• For making a liter of MS medium, start with 700 to 800 ml DI H2O in a beaker

• Place a stir bar in the beaker, and stir while adding 30 g sucrose

• Add inorganic and organic stock solutions:– Nitrate stock (50x) 20 ml– Sulfate stock (100x) 10 ml– Halide stock (100x) 10 ml

Use of stock solutions (contin)

• Add inorganic and organic stocks:– P•B•Mo (100x) 10 ml

– Na2FeEDTA (200x) 5 ml

– myo-inositol (100x) 10 ml– vitamins (1000x) 1 ml– PGRs (usu. 1 mg/ml) 0-5 ml

• Bring volume to near 1000 ml with DI water

Use of stock solutions (contin)

• Adjust the pH to 5.7 (most species)• Bring the volume of the medium to 1 L using a

graduated cylinder• Add 8 grams agar• Place beaker in the microwave to melt the agar• While medium is still warm (above 40° C), pour

about 35 ml into each baby-food jar• Loosely cap jars and autoclave 15 min at 121° C

Preparation and use of stock solutions

• Stocks are usually made as 50x, 100x, 200x or 1000x the medium concentration

• For example, for a nitrate stock at 50x, the medium recipe calls for 1650 mg/L of NH4NO3, you would make a stock at a conc. of 50(1650) = 82,500 mg/L or 82.5 g/L

• It may be more convenient to make a smaller volume of stock solution, say 200 ml of nitrate stock

Preparation and use of stock solutions (contin)

• How would that change the mass (no. grams) of NH4NO3 added to the stock?

• Remember, you always want the correct conc. of each ingredient in the medium

• One way to keep track of this is with the formula VsCs = VmCm, where:

– Vs = volume of the stock solution

– Cs = concentration of the stock solution

Preparation and use of stock solutions (contin)

– Vm = volume of the culture medium

– Cm = concentration of the culture medium

• This formula also comes in handy in calculating the volume of stock to add:– if the conc. of the stock is 82.5 g NH4NO3 and you are

making 1 liter of medium, the stock volume is x(82.5 g) = (1000 ml)(1650 mg/L), and solving for x, the stock volume is (?)

– does your answer agree with the volume listed on slide 8?

Making PGR stocks

• A useful concentration for PGRs is 1 mg/ml (1000 mg/L), then, e.g., 2 mg/L BA can easily be made up by dispensing 2 ml of a 1 mg/ml BA stock

• What if you were making only 250 ml of medium?

Making PGR stocks (contin)

• For making PGR stocks, keep in mind that these go into aqueous solution with difficulty

• One trick is to recall that most PGRs are weakly acidic or basic; e.g., cytokinins are weak bases, auxins are weak acids

• Using sl. excess of molar equivalents of the opposing compound allows most PGRs to be completely dissolved

Making PGR stocks (contin)

• For example, for making 1 mg/ml stock of BA, dissolve 100 mg BA in 98.6 ml DI water and 1.4 ml 1 N HCl

• Solution is stirred until all granules go into solution, which may take several hours

• For auxins, use 1 N NaOH or KOH in place of the acid

Considerations in medium prep

• Stock solutions should be stored at 4° C to retard bacterial and fungal growth

• Inorganic stocks can be sterilized; when dispensing from sterile stocks, use sterile pipets

• Some organic compounds must be filter-sterilized– Vitamins and some PGRs can be autoclaved– Exceptions: IAA, GA, ABA– Antibiotics must be filter-sterilized

Considerations in medium prep (contin)

• Water of crystallization– check your reagent against the medium recipe; a

molecule of ZnSO4 and ZnSO4●7H20 weigh different amounts

– using molecular weights, calculate the amount that is proportional to the compound cited in the recipe

• Recommended shelf life of media and stocks– for medium and stocks, about 2 months– for medium with antibiotics, use ASAP– for stable PGRs, up to 6 months