Filtration & sterilization
A disc membrane in a plastic housing on the end of a syringe, for millilitres to tens of millilitres: 13 mm gives about 0.8 cm² of membrane, 25 mm about 3.9 cm² and 33 mm about 5.5 cm². Pick the pore size for what has to be removed, the chemistry for what must not stick, and the diameter for the volume, and stop pushing if it goes hard.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Syringe filters
A disc membrane in a plastic housing on the end of a syringe, for millilitres to tens of millilitres: 13 mm gives about 0.8 cm² of membrane, 25 mm about 3.9 cm² and 33 mm about 5.5 cm². Pick the pore size for what has to be removed, the chemistry for what must not stick, and the diameter for the volume, and stop pushing if it goes hard.
How it differs from the standard method
- 0.22 µm to sterilise, 0.45 µm only to clarify, 0.1 µm where mycoplasma matter. The housing is printed with the rating; read it rather than assuming.
- PES or cellulose acetate for protein; nylon binds one to two orders of magnitude more and is for solvents and mobile phases.
- Flush the first millilitre to waste: it carries wetting agents and extractables, and it is the fraction the membrane strips hardest.
- For a dilute protein, use the smallest filter that will pass the volume, since binding scales with area, and consider blocking the membrane with a carrier protein first.
- A 25 mm, 0.22 µm filter passes roughly 100 mL of clean buffer, far less of anything cloudy. If the plunger stiffens, fit a fresh filter: forcing it splits the membrane and the filtrate is then not sterile.
- Check the membrane is compatible with the solvent: cellulose acetate dissolves in DMSO, where PTFE and nylon survive it.
1Two ways to make something sterile
There are only two routine options at the bench. Moist heat kills: saturated steam at 121 °C denatures everything, including bacterial spores, given enough time.
Sterilising grade
Defined by a bacterial challenge, not by a pore picture: a membrane is sterilising grade if it retains at least 10⁷ Brevundimonas diminuta per cm². 0.45 µm clarifies; it does not sterilise.
Equivalent minutes
Lethality measured in minutes at 121.1 °C. Every 10 °C multiplies the rate by ten, so F₀ adds up the whole cycle, and it has to be measured at the centre of the load.
What heat destroys
Antibiotics, serum, glutamine, vitamins, growth factors, enzymes and most sugars in the presence of peptides. These are filtered and added to the cooled medium.
2What a membrane does and does not remove
Pore size is a rating, not a sieve aperture: a membrane is a tortuous structure with a pore size distribution, and its rating comes from a challenge test. A 0.22 µm membrane is validated against Brevundimonas diminuta, an organism of about 0.3 µm, and gives seven logs of reduction or better.
3Area, pressure and clogging
A membrane will pass a certain volume before the pores plug, roughly in proportion to its area: tens of millilitres per square centimetre for a clean buffer, a few for serum-containing medium, and under a millilitre for a cell lysate. As the fouling layer builds, the flow at a given pressure falls towards zero, so the honest way to size a filtration is volume per unit area rather than time.
4Moist heat: the load, not the chamber
An autoclave's gauge reports the chamber. What matters is the centre of the largest item in it, which lags behind by minutes, about five for a 500 mL bottle, a quarter of an hour for a 2 L one, longer for a packed bag.
Key terms
Practise these as flashcards →A membrane validated to retain at least 10⁷ Brevundimonas diminuta per cm² of membrane, in practice a 0.2 or 0.22 µm rating.
Accumulated lethality of a moist-heat cycle, in equivalent minutes at 121.1 °C with z = 10 °C. The reference cycle, 121 °C for 15 min at the load, is F₀ = 15.
Time at a given temperature to reduce a population tenfold. For Geobacillus stearothermophilus spores, D₁₂₁ is about 1.5 min.
The temperature change that alters the D-value tenfold; 10 °C for moist heat, which is what makes 131 °C ten times as lethal as 121 °C.
A membrane chemistry such as PES or cellulose acetate that adsorbs only a few micrograms of protein per cm², as against 100 µg/cm² or more for nylon.
The volume a membrane will pass before the flux falls to nothing, quoted per unit area. It is the number that decides whether a unit is big enough.
A coarse layer above the sterilising membrane that retains aggregates and lipid, raising throughput without changing the retention rating.
A vial of Geobacillus stearothermophilus spores placed inside the load and incubated afterwards: the definitive test that a cycle worked, available two days later.
A strip placed inside the load whose end point corresponds to a time-at-temperature equivalent, so it reports on the load rather than the chamber.
When to use it
- Making up media, buffers and supplements for cell or bacterial culture, where a single contaminated batch costs weeks of work.
- Preparing anything heat-labile (antibiotics, serum, glutamine, growth factors) which has to be filtered because autoclaving destroys it.
- Clarifying a sample before a column or an instrument, where a 0.45 µm filter is used to remove particles rather than organisms.
- Decontaminating cultures, plates and liquid waste before disposal, which is an autoclave cycle in its own right and usually a long one.
Compared with alternatives
Making the solution in the first place; come here once the question is how to make that solution sterile without wrecking it.
A sterile reagent only stays sterile if it is handled aseptically; prefer that page when the contamination appears after the bottle was opened.
Which supplements a medium needs and what they are for; this page covers how each of them has to be sterilised.
Use it instead to clarify a lysate or remove cells and debris, which spares a sterilising membrane that would otherwise plug immediately.
Choose it when the aim is to change the solvent around a protein rather than to remove organisms from it.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A disc membrane in a plastic housing on the end of a syringe, for millilitres to tens of millilitres: 13 mm gives about 0.8 cm² of membrane, 25 mm about 3.9 cm² and 33 mm about 5.5 cm². Pick the pore size for what has to be removed, the chemistry for what must not stick, and the diameter for the volume, and stop pushing if it goes hard.
- 0.22 µm to sterilise, 0.45 µm only to clarify, 0.1 µm where mycoplasma matter. The housing is printed with the rating; read it rather than assuming.
- PES or cellulose acetate for protein; nylon binds one to two orders of magnitude more and is for solvents and mobile phases.
- Flush the first millilitre to waste: it carries wetting agents and extractables, and it is the fraction the membrane strips hardest.
- For a dilute protein, use the smallest filter that will pass the volume, since binding scales with area, and consider blocking the membrane with a carrier protein first.
- A 25 mm, 0.22 µm filter passes roughly 100 mL of clean buffer, far less of anything cloudy. If the plunger stiffens, fit a fresh filter: forcing it splits the membrane and the filtrate is then not sterile.
- Check the membrane is compatible with the solvent: cellulose acetate dissolves in DMSO, where PTFE and nylon survive it.