- 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.
Reagents and solutions
5N NaOH (for pH adjustment)
Deionized / Milli-Q water
Equipment and disposables
Erlenmeyer flask / Beaker
Hot plate / Magnetic stirrer with heating device
0.8% Sodium chloride
pH : 7.0 ± 0.2
Preparation of 1000 ml H Medium
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.
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.
◊ 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.
Medium with completely dissolved ingredients will appear as a transparent yellowish solution.
◊ 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).
The H Medium is not very highly buffered. The pH of the medium drops, as the growing culture reaches near the saturation phase.
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.
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.
◊ 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.
The solution can be stored at room temperature for a few days. Keep the medium at 2 – 8 °C for longer storage.
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|