Nutrient Agar and Nutrient Broth
Nutrient agar and nutrient broth are widely used for cultivation and maintenance of less fastidious bacteria.
Peptic digest of animal tissue – 5.0 gm
Beef extract – 1.5 gm
Yeast extract – 1.5 gm
Sodium chloride – 5.0 gm
Distilled water – 1000 mL
pH – 7.4 ± 0.2
i. Weigh and dissolve each ingredient except agar in 500 mL of distilled water.
ii. Make the volume to 1000 mL with distilled water. Set the pH to 7.0. This is nutrient broth.
iii. Autoclave the nutrient broth at 15 psi for 15- 20 minutes and dispense in sterile test tubes aseptically.
iv. For nutrient agar, add 15 gm of agar in 1000 mL of nutrient broth. Heat the mixture to dissolve agar.
v Dispense the mixture into flasks and autoclave.
vi. After autoclaving, cool the nutrient agar medium to 45 °C and dispense it aseptically into sterile Petri plates and tubes to make agar plates, slants, and deep tubes.
vii. Allow to solidify. For making slants, keep the tubes in an inclined position for agar solidification.
viii. Inoculate nutrient agar plates, nutrient agar tubes and nutrient broth with bacterial culture and incubate for 24 hours at 37 °C.
• Luria-Bertani (LB), Miller broth & agar
LB media is used for coliform especially E.coli cultivation. Its composition is as follows-
Tryptone – 10 gm
Yeast extract – 5 gm
Sodium chloride – 10 gm
Distilled water – 1000 ml
pH – 7.5 ± 0.2
i. For 1 liter of Miller LB broth, weigh tryptone, yeast extract and sodium chloride in 2-liter conical flask/beaker.
ii. Add 800 ml distilled water and mix them.
iii. The solution will appear translucent with yellowish tint with undissolved media ingredients.
iv. Dissolve all ingredients completely by heating up to a boil with stirring.
v. Bring down the medium’s temperature to room temperature. Adjust the pH 7.5 with 5N NaOH (~0.2 ml).
vi. Adjust the volume to 1000 ml with distilled water. Mix it again.
vii. Transfer the medium to autoclavable bottle or cover the conical flask with a cotton plug and aluminum foil.
viii. Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C).
ix. For LB agar, add 15 g to 1000ml of LB broth. Heat the mixture to dissolve agar, and then autoclave.
x. Allow to solidify. For making slants, keep the tubes in an inclined position for agar solidification.
xi. Inoculate nutrient agar plates, nutrient agar tubes and nutrient broth with bacterial culture and incubate for 24 hours at 37 °C.