NZC broth is a rich microbial culture medium. It can be prepared by dissolving 10 g NZ Amine A, 5 g NaCl, 2 g MgCl2·6H2O and 5 ml of 20% Casamino Acids in water to a final volume of 1000 ml (Elbing & Brent, 2019).
Reagents and Solutions
NZ Amine A
Sodium Chloride (NaCl, Molecular Weight: 58.44)
Magnesium chloride hexahydrate (MgCl2.6H2O, Molecular Weight: 203.30)
20% Casamino Acids (sterile)
Deionized / Milli-Q water
Equipment and disposables
Hot plate / Magnetic stirrer with heating device
1% NZ Amine A
0.5% (85.56 mM) Sodium Chloride (NaCl, Molecular Weight: 58.44)
0.2% (9.84 mM) Magnesium chloride hexahydrate (MgCl2.6H2O, Molecular Weight: 203.30)
0.1% Casamino Acids
Preparation of 1000 ml NZC broth
Step 1: To prepare 1000 ml of NZC broth, weigh out 10 g NZ Amine A, 5 g NaCl, and 2 g MgCl2·6H2O in a 2-liter Erlenmeyer flask. Add 800 ml deionized/Milli-Q water. Mix it using a magnetic stirrer.
One can use manual shaking using a glass pipette to mix all ingredients. Mixing using a Magnetic stirrer is an option. The magnetic stirrer makes the dissolving process easy and convenient.
◊ Do not dissolve in 1000 ml of deionized / Milli-Q water. It is always a good practice to dissolve all media ingredients and then adjust the volume to the desired volume with the solvent. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.
◊ Make sure no lumps remain after making the suspension.
Step 2: Dissolve all ingredients completely by heating to boiling while stirring.
◊ Once all the ingredients of the medium dissolve and the medium appears transparent, stop the heating. Don’t unnecessarily heat the medium.
◊ Make sure to dissolve any residual powder sticking to the glass.
Step 3: Adjust the volume to 995 ml with deionized / distilled water. Mix it again.
Step 4: Transfer the medium to autoclavable bottles or cover the Erlenmeyer flask with cotton plug and aluminum foil.
Step 5: Sterilize the solution by autoclaving [20 minutes at 15 lb/in2 (psi)] from 121-124°C on liquid cycle).
Elbing & Brent, 2019 recommend autoclaving for 30 minutes at 15 lb/in2 (1.05 kg/cm2).
Step 6: Let the medium cool down to room temperature. Add 5 ml of 20% Casamino Acids under aseptic conditions.
Antibiotics and nutritional supplements can also be added. Many supplements, like antibiotics, are degraded at high temperatures, so make sure that the medium temperature is below 40°C.
After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain sterility.
The solution can be stored at 2 – 8 °C.
Bacterial growth medium
|Follow the table to prepare NZC broth of various volumes.|
|Reagents / Volume||100 ml||500 ml||1,000 ml||10,000 ml|
|NZ Amine A||1 gm||5 gm||10 gm||100 gm|
|Sodium chloride||0.5 g||2.5 g||5 g||50 g|
|MgCl2·6H2O||0.2 g||1 g||2 g||20 g|
|20% Casamino Acids||0.5 ml||2.5 ml||5 ml||50 ml|
|Water||Adjust the final volume to 100 ml||Adjust the final volume to 500 ml||Adjust the final volume to 1000 ml||Adjust the final volume to 10000 ml|