0.1 µm vs 0.2 µm Filtration: Which for Cell Culture?

Sterile filtration is the standard way to sterilise heat-sensitive liquids such as serum, media, buffers and reagents. The choice of pore size, 0.2 µm or 0.1 µm, determines what the filter retains, how fast it runs and how much it costs. This guide explains the difference and when each pore size is the right choice.

Short answer

0.2 µm (also labelled 0.22 µm) is the standard sterilising-grade filter. It retains bacteria and is sufficient for most media, buffers and reagents.
0.1 µm additionally retains mycoplasma more reliably. It is the preferred pore size for serum such as FBS and for any material where mycoplasma contamination is a particular risk.
Neither reliably removes viruses, endotoxin or prions.

What a 0.2 µm filter retains

A sterilising-grade 0.2 µm filter is defined by a bacterial challenge test: it must retain at least 10⁷ colony-forming units of Brevundimonas diminuta per cm² of filter area (ASTM F838). B. diminuta is one of the smallest bacteria, so a filter that retains it retains practically all bacteria and fungi.

Why mycoplasma can pass 0.2 µm

Mycoplasma are very small bacteria (about 0.15–0.3 µm) and, unlike other bacteria, have no cell wall. They can deform and squeeze through the pores of a 0.2 µm membrane, especially at high pressure or high load.

Filters rated for mycoplasma retention have pore sizes of around 0.1 µm. They are typically validated with Acholeplasma laidlawii.

Mycoplasma are one of the most common contaminants in cell culture. They usually cause no turbidity and stay undetected for long periods. Serum is a historic source of mycoplasma, which is why 0.1 µm filtration is standard for FBS. SeamlessBio FBS, e.g. FBS Standard, is sterile-filtered through 0.1 µm.

What neither filter removes

Contaminant	Typical size	0.2 µm	0.1 µm	Effective control
Bacteria, fungi	> 0.3 µm	retained	retained	Sterile filtration
Mycoplasma	0.15–0.3 µm, deformable	not reliably	retained	0.1 µm filtration, testing
Viruses	approx. 0.02–0.3 µm	no	no	Sourcing, testing, gamma irradiation, virus filters (~20 nm)
Endotoxin	molecular (LPS)	no	no	Raw material selection, endotoxin testing, e.g. FBS Low Endotoxin
Prions	molecular	no	no	Origin, TSE/BSE documentation

Comparison

	0.2 µm	0.1 µm
Bacterial retention	sterilising grade	sterilising grade
Mycoplasma retention	not reliable	reliable (with validated filters)
Flow rate	higher	lower; more filter area needed
Clogging with viscous or particulate liquids	lower	higher; prefiltration usually needed
Cost per litre	lower	higher
Typical use	Media, buffers, supplements, reagents, most diagnostic reagents	Serum (FBS, animal and human sera), mycoplasma-critical materials, particle-free solutions

Which pore size for which application?

Cell culture media and buffers: 0.2 µm is usually sufficient, provided the raw materials are of controlled quality.
Serum (FBS, other animal sera, human serum): 0.1 µm is preferred because of the mycoplasma risk. Many animal sera are offered sterile-filtered at 0.2 µm. For mycoplasma-sensitive applications, ask for the filtration grade.
GMP and ATMP manufacturing: Pore size and filter validation are defined in the process risk assessment. Mycoplasma-retentive filters are often specified for critical raw materials.
Diagnostics (IVD):
Reagents and buffers are generally filtered through 0.2 µm.
For particle-sensitive methods (flow cytometry, light scattering, nanoparticle analysis), 0.1 µm can reduce background.
Supplements with large molecules or colloids (e.g. lipid concentrates): test whether 0.1 µm retains the active ingredient or clogs.

Protocol: Sterile-filtering serum or medium in the lab

Choose the membrane. For serum and protein solutions, use a low protein-binding membrane such as polyethersulfone (PES) or PVDF. Nylon binds more protein and is less suitable for serum.
Choose the format.
- Up to approx. 50 mL: syringe filters
- 100 mL–1 L: vacuum bottle-top filters
- Larger volumes: capsule filters with a pump. These can also fill directly into single-use bags or bag assemblies.
Prefilter viscous or turbid liquids. Serum, especially after thawing or heat inactivation, should be passed through a 0.45 µm or glass-fibre prefilter first. This protects the final filter from clogging.
Filter aseptically in a biosafety cabinet. Use moderate pressure or vacuum. High pressure increases the risk of mycoplasma passing through and can damage the membrane.
Do not let the filter run dry and do not exceed the filter’s rated capacity. If the flow drops sharply, change the filter instead of increasing the pressure.
Integrity test (GMP). In regulated manufacturing, the filter integrity is tested before and after use, e.g. with a bubble point or diffusion test.
Aliquot and label the filtrate (pore size, date, lot) and store according to the material.

FAQ

Is 0.22 µm the same as 0.2 µm?
In practice, yes. Both terms describe the same class of sterilising-grade filters; the label depends on the manufacturer.

Does a 0.2 µm filter remove mycoplasma?
Not reliably. Mycoplasma have no cell wall and can deform through 0.2 µm pores. For mycoplasma retention, use 0.1 µm filters.

Does 0.1 µm filtration remove viruses?
No. Most viruses are smaller than 0.1 µm. Viral safety comes from sourcing, testing, gamma irradiation or specific virus filters.

Why is FBS filtered through 0.1 µm?
Serum is a known historical source of mycoplasma contamination. 0.1 µm filtration retains mycoplasma that could pass a 0.2 µm filter.

Does filtration remove endotoxin?
No. Endotoxins are molecules (lipopolysaccharides) that pass both pore sizes. Low endotoxin levels come from raw material quality and are confirmed by testing.

Should I re-filter commercial FBS before use?
Normally not. Commercial FBS is supplied sterile-filtered. Re-filtering only makes sense after manipulation (e.g. opening under non-sterile conditions or adding supplements), and it can reduce some proteins.

Sterile-filtered sera from SeamlessBio

SeamlessBio sera are supplied sterile-filtered, each lot with CoA:
– Fetal Bovine Serum in 0.1 µm sterile-filtered grades
– Human Serum
– other animal sera

Prices and availability on request – request a quote.

0.1 µm vs 0.2 µm Sterile Filtration: Which Should You Use for Cell Culture and Diagnostics?