Buffer & media preparation
Make buffers, stock solutions and growth media at a defined concentration and pH.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Why buffers hold their pH
A buffer is a weak acid and its conjugate base (or a weak base and its conjugate acid) in solution together. Added acid is soaked up by the base form and added base by the acid form, so the pH barely moves.
Choose the buffer
Pick a buffer whose pKa is close to the pH you need: phosphate 7.2, HEPES 7.5, Tris 8.1, acetate 4.8.
Weigh
Grams = molarity × volume (L) × molecular weight. Check which salt or hydrate the bottle contains.
Adjust, then make up
Dissolve in most of the water, set the pH with a calibrated meter, then make up to the final volume.
2Temperature and Tris
Some buffers change pKa with temperature. Tris drops about 0.028 pH units per °C: a Tris buffer set to pH 7.5 at room temperature is about pH 8.1 in a cold room.
3Stocks and sterility
Labs keep concentrated stocks (1 M Tris, 5 M NaCl, 0.5 M EDTA) and dilute them into working buffers with C1V1 = C2V2. Media for cells or bacteria must also be sterile: heat-stable solutions are autoclaved (121 °C, 15–20 min), while heat-labile components such as antibiotics, glucose, vitamins or serum are filter-sterilised through 0.22 µm membranes and added after the medium cools.
Key terms
Practise these as flashcards →The pH at which a weak acid is half dissociated; the centre of its buffering range.
How much acid or base a buffer absorbs per unit pH change; highest at pH = pKa and proportional to concentration.
Moles of solute per litre of solution (M = mol/L).
Dilution equation: stock concentration × volume taken = final concentration × final volume.
Setting a pH meter with two standards that bracket the pH you'll measure.
Pressurised steam at 121 °C that kills bacteria, spores and viruses.
When to use it
- Every experiment: almost every reaction, wash and medium depends on a solution at the right concentration and pH.
- Making working buffers from validated stocks to keep batches consistent.
- Preparing sterile media and supplements for cell or bacterial culture.
Compared with alternatives
The pipetting skills behind accurate stock dilutions.
Autoclaving and filtering in more depth.
Choosing and preparing cell-culture media.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Rinse the electrode, calibrate with two or three fresh standard buffers that bracket your target (e.g. pH 7.00 and 10.01 for Tris), at the temperature you'll measure. Check the slope (95–105%) and store the electrode wet in storage solution.