Overview
- H Medium is a moderately rich medium. It contains tryptone and sodium chloride (NaCl).
- Tryptone provides basic nutrients and growth factors, thus supporting growth.
- NaCl maintains a proper isotonic environment of the medium.
REQUIREMENTS
Reagents and solutions
Tryptone
Sodium chloride
5N NaOH (for pH adjustment)
Deionized / Milli-Q water
Equipment and disposables
Measuring cylinder
Erlenmeyer flask / Beaker
Hot plate / Magnetic stirrer with heating device
Composition
1% Tryptone
0.8% Sodium chloride
pH : 7.0 ± 0.2
Objective:
Preparation of 1000 ml H Medium
PROCEDURE
Step 1: To prepare 1000 ml of H medium, weigh out 10 g tryptone and 8 g NaCl in a 2-liter Erlenmeyer flask/beaker. Add 800 ml deionized/Milli-Q water. Mix it using a magnetic stirrer. The solution will appear translucent yellowish with undissolved media ingredients.
Tip:
One can use manual shaking using a glass pipette to mix all ingredients. Mixing using a Magnetic stirrer is an option. 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 first and then adjust the volume to the desired volume with solvent. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.
◊ Make sure no lumps are remaining after making the suspension of medium components.
Step 2: Dissolve all ingredients completely by heating to boiling while stirring.
Note:
Medium with completely dissolved ingredients will appear as a transparent yellowish solution.
Precautions:
◊ Once all the ingredients are dissolved 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: Cool down the medium to room temperature. Adjust the pH 7.0 with 5N NaOH (~0.2 ml).
Note:
The H Medium is not very highly buffered. The pH of the medium drops, as the growing culture reaches near the saturation phase.
Precaution:
Since pH is dependent on temperature, It is always a good practice to adjust the medium pH only after the solution has cooled to 25°C (room temperature).
Step 4: Adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.
Step 5: Transfer the medium to autoclavable bottles or insert the cotton plug and cover the mouth Erlenmeyer flask with aluminum foil.
Tip:
One can make small aliquots of the medium if needed (e.g., 5 ml aliquots in a 50 ml conical flask).
Step 6: Sterilize the solution by autoclaving for 20 minutes at 15 lb/in2 (1.05 kg/cm2) on liquid cycle.
Precautions:
◊ Antibiotics and nutritional supplements should be added only after the solution has cooled to 40°C or below. Many supplements, like antibiotics, are degraded at high temperatures.
◊ After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain sterility.
Storage
The solution can be stored at room temperature for a few days. Keep the medium at 2 – 8 °C for longer storage.
Applications
Bacterial growth medium
Follow the table to prepare H Medium of various volumes. | ||||
Reagents / Volume | 100 ml | 500 ml | 1,000 ml | 10,000 ml |
Tryptone | 1 gm | 5 g | 10 g | 100 g |
Sodium chloride | 2 gm | 4 g | 8 g | 80 g |
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 |