Preparation of NZC Broth

OVERVIEW

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).

REQUIREMENTS

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
Measuring cylinder
Erlenmeyer flask
Hot plate / Magnetic stirrer with heating device

Composition
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

Objective: 
Preparation of 1000 ml NZC broth

PREPARATION

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. 

Tip:
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.

Precautions:
◊ 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.

Precautions:
◊ 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).

Note:
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. 

Tip
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. 

Precaution
After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain sterility.

Storage
The solution can be stored at 2 – 8 °C.

Applications
Bacterial growth medium

Follow the table to prepare NZC broth of various volumes.
Reagents / Volume100 ml500 ml1,000 ml10,000 ml
NZ Amine A1 gm5 gm10 gm100 gm
Sodium chloride0.5 g2.5 g5 g50 g
MgCl2·6H2O0.2 g1 g2 g20 g
20% Casamino Acids0.5 ml2.5 ml5 ml50 ml
WaterAdjust the final volume to 100 mlAdjust the final volume to 500 mlAdjust the final volume to 1000 mlAdjust the final volume to 10000 ml

REFERENCES

Elbing & Brent, 2019. Recipes and Tools for Culture of Escherichia coli. Curr Protoc Mol Biol. 125(1), e83. PMID-30412361; Full-Text Links: wiley, PMC

Was this post helpful?

Author: admin

Leave a Reply

Your email address will not be published.