Microbial Count of Pharmaceutical Water by Membrane Filtration
Filters a measured volume of water through a 0.45 µm membrane, places the membrane on R2A agar and counts colonies after incubation, giving cfu per mL (purified water) or cfu per 100 mL (WFI). Results are compared with alert and action levels and trended.
- Membrane filtration onto R2A agar
- Filtration unit
- LAF
- Incubator
- Autoclave
- Colony counter
Reference: Typical approach based on USP <1231> Water for Pharmaceutical Purposes. Action and alert levels are set by each site; always follow your approved water monitoring SOP.
Why this method works
Bacteria in pharmaceutical water are usually few in number and adapted to low nutrient levels. Filtering a large volume concentrates them on a membrane, which lowers the limit of detection. A low-nutrient medium such as R2A agar, with a longer incubation at a moderate temperature, recovers these stressed waterborne organisms better than rich media. Each colony on the membrane is one cfu from the volume filtered.
Instruments used
Reagents & standards
| Reagent | Grade / specification |
|---|---|
| R2A agar | Low-nutrient medium for waterborne bacteria; growth promotion tested batch |
| Sterile 0.45 µm membranes (47 mm, gridded) | Mixed cellulose esters or as validated |
| Sterile diluent / rinse fluid | e.g. peptone water, for pre-wetting and rinsing the funnel |
| Sterile sample bottles | With sodium thiosulfate if the water may contain chlorine (potable water) |
Preparing the sample
- 01
Sample
Flush the user point, then collect aseptically into a sterile bottle.
- 02
Transport
Test within the defined time, refrigerated if the delay is longer than allowed.
- 03
Filter
Filter the defined volume through a 0.45 µm membrane.
- 04
Plate
Place the membrane on R2A agar without air bubbles.
- 05
Incubate
Incubate at the validated temperature and time.
- 06
Count
Count colonies and calculate cfu/mL or cfu/100 mL.
Procedure
- 1Collect samples as the water is used in production (same hose, same flush) so the result represents the water actually used.
- 2Test samples promptly; if a delay is unavoidable, keep them at 2–8 °C and test within the time validated in your SOP (often not more than 24 hours).
- 3In the LAF, assemble a sterile funnel with a 0.45 µm membrane on the manifold and run a negative control (sterile diluent) first.
- 4Shake the sample and filter the defined volume: purified water typically 1–100 mL (USP <1231> suggests at least 1 mL); WFI typically 100–300 mL (at least 100 mL).
- 5Rinse the funnel walls with sterile diluent and filter.
- 6Transfer the membrane to an R2A agar plate with sterile forceps, rolling it on to avoid air bubbles.
- 7Incubate, typically at 30–35 °C for at least 5 days, or another validated time and temperature (see the current <1231>).
Standard preparation
- 1Growth promotion of each R2A batch with suitable organisms (e.g. a reference strain plus a site water isolate), not more than 100 cfu.
- 2Negative control: sterile diluent filtered through the same system.
Notes for analysts
- The action levels above are typical values cited in USP <1231>; your site's levels are in the approved water monitoring SOP.
- If no colonies grow, report as less than 1 cfu per volume filtered (e.g. < 1 cfu/100 mL).
- Trend results per user point; a slow rise can show biofilm before an action level is exceeded.
- Chemical tests (conductivity and TOC) are done on the same water system but are separate methods.
Method parameters (typical)
| Membrane | 0.45 µm, 47 mm |
|---|---|
| Medium | R2A agar (or another validated medium) |
| Volume — purified water | Typically 1–100 mL (at least 1 mL) |
| Volume — WFI | Typically 100–300 mL (at least 100 mL) |
| Incubation | Typically 30–35 °C for at least 5 days, or as validated |
| Typical action level — purified water | 100 cfu/mL |
| Typical action level — WFI | 10 cfu/100 mL |
| Countable range on a membrane | Typically 20–80 cfu per membrane for best accuracy, as per SOP |
System suitability & sequence
| Parameter | Limit |
|---|---|
| Negative control | No growth |
| Growth promotion | R2A batch passes before use |
| Sample holding | Tested within the validated time and temperature |
Typical sequence
- 1Negative control (sterile diluent)
- 2Samples in order from cleanest (WFI) to least clean (PW), changing funnels between samples
- 3Duplicate membranes per sample if the SOP requires
Calculation with a worked example
Where
- Mean colonies
- Average colonies on the membranes for one sample
- Volume filtered
- Volume of water filtered through each membrane, in mL
- Dilution factor
- 1 for undiluted water
Given
- Purified water, user point PW-07
- 10 mL filtered on each of two membranes; 24 and 20 colonies
- WFI, user point WFI-03
- 200 mL filtered; 3 colonies
- Typical action levels
- PW 100 cfu/mL; WFI 10 cfu/100 mL
Working
- PW mean = (24 + 20) ÷ 2 = 22 colonies
- PW count = 22 ÷ 10 × 1 = 2.2 cfu/mL
- WFI count = 3 ÷ 200 = 0.015 cfu/mL
- WFI count per 100 mL = 0.015 × 100 = 1.5 cfu/100 mL
PW-07 = 2.2 cfu/mL (below 100 cfu/mL); WFI-03 = 1.5 cfu/100 mL (below 10 cfu/100 mL). Both below the typical action levels; compare also with site alert levels and trend.
Try it with your numbersWhat result passes
- Purified water: typical action level 100 cfu/mL (USP <1231>); sites usually set lower alert levels from historical data.
- WFI: typical action level 10 cfu/100 mL (USP <1231>).
- Results above the alert level prompt closer review; results above the action level require investigation and corrective action. These levels are process control values, not product release specifications, unless your specification says otherwise.
- Isolates from WFI or from excursions are identified, and objectionable organisms are assessed even at low counts.
Validation parameters (ICH Q2(R2))
| Parameter | Typical requirement |
|---|---|
| Medium and incubation | Recovery of waterborne isolates on the chosen medium and conditions compared with alternatives |
| Sample holding time | Count stable over the allowed holding time and temperature |
| Membrane | No inhibition of test organisms by the membrane type |
Run this method in LimsPulse: specifications and STPs under version control, results calculated and checked against limits, and a signed Certificate of Analysis.