LB agar-plates: (1 L): (rich media, Culture of aerobic bacterial species on solid media)
1. Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized
water, 15 g/L agar before autoclaving
2. Adjust the pH of the medium to 7.0 using 1N NaOH and bring volume up to 1 liter.
3. Autoclave on liquid cycle for 20 min at 15 psi. Allow solution to cool to 55°C, and add
antibiotic if needed (50μg / mL of Amp or Kan).
4. After autoclaving, cool to approx. 55°C, add antibiotic (if needed), and pour into
petridishes.
5. Let harden, then invert and store at +4°C in the dark.
10% Tryptic Soy Agar (1L): (a universal dilute rich medium that supports the growth of a wide variety of microorganisms…both gram-positive & gram-negative bacteria)
• 15 g Tryptone
• 5 g Soytone – enzymatic digest of soybean meal
• 5 g Sodium Chloride
• 15 g Agar
Autoclave as above, cool and pour in plates.
MacConkey Agar (selects for Gram-negatives and identifies lactose fermenting bacteria, bile tolerant)
• 10g lactose
• 20g peptone
• 5g bile salts
• “pinch” or small amount of 1mg/mL Bromcresol purple or Crystal violet solution.
• 15g agar
• 1L distilled water
M9 minimal media Agar (oligotrophic media for enriching slow-growing heterotrophs)
Reagents/Materials:
1. Erlenmeyer flask of volume at 2X for amount of media to be made (i.e. 1L flask to make
500mL)
2. M9 minimal salts solution (5X concentrate)
◦ To 800mL of distilled/deionized water add:
◦ 64g sodium phosphate, penta-hydrate — Na2HPO4-7H2O
◦ 15g potassium phosphate (dibasic) — KH2PO4
◦ 2.5g table salt — NaCl
◦ 5.0g ammonium chloride — NH4Cl
◦ Stir until dissolved.
◦ Adjust volume to 1000mL (1L).
◦ Sterilze by autoclaving or filter sterilization if autoclave is not available.
3. Distilled water
4. 1M solution of magnesium sulfate (MgSO4).
5. 20% solution wt/wt of glucose (20g gulcose in 80g of water). Add powdered glucose
(also called “dextrose”) to water solution slowly while mixing, else the glucose will
clump and not easily dissolve. Filter sterilize when finished – do not autoclave as the heat
will carmelize the sugar.
6. 1M solution of calcium chloride (CaCl2)
7. To make solid media, include agar base and sterile Petrie plates.
Method
• To prepare 1L of media, add:
• 200mL 5X M9 salts solution
• to ~800mL of distilled water
• Add 15g of agar media if agar plates are to be poured.
After autoclaving, swirl to mix evenly and “temper” at room temperature until you can place
your hand on the flask for 2 second (an adult should do this!), then add the following:
• 2mL of 1M MgSO4 solution
• 0.1mL of 1M CaCl2 solution
• 20mL of 20% glucose
Swirl to mix evenly.
Liquid media should be distributed to sterile bottles or tubes with a pipette and pipettor. Small
bottles can hold 250mL of media for use when needed, for instance, or aliquots of 5mL or 10mL
can be transferred to sterile, capped test tubes.
If plates are to be poured, ~25mL of media should be added per Petrie dish and allowed to
solidify at room temperature. Poured/solid plates can be stacked and replaced in the original
plastic sleeve in which they came. Label the sleeve with the type of media and date poured.