How to sample for microbial process control data

Where regular sampling is required as part of your operation, it’s important the exact same process is used by all personnel to ensure consistency. We often get asked the best wat to carry out microbial sampling and we have put together the below guidance to explain.

In the case of process control sampling, the water of importance is the water within the system – in other words the water behind the valves. This water could pick up substantial microbial contaminants as it exits through the valve and perhaps also from a hose attached to it before it enters a sample container. Every effort must be made to avoid or prevent this additional microbial contribution to the systemic bacteria that are intended to be collected in that sample.

Routine, well-defined practices should be instituted to avoid or prevent this additional sample contamination from occurring. The sampling procedure described below should avoid any substantial microbial contribution from the outlet during sampling.

  1. Assure that valves used for microbial sampling are of a sanitary design (i.e. don’t retain water downstream of the sealing surfaces and all upstream surfaces can be reached by sanitizing agents when in the closed position)
  2. Sanitise the external and the internal surfaces of the valve, particularly if lumen is narrow (1/4 inch or less) regardless of orientation, and susceptible to water retention by surface tension phenomena. Be sure to thoroughly saturate all surfaces with alcohol and allow adequate contact time (at least 30 seconds to 1 minute). It may be necessary with small lumen valves to inject alcohol directly into the downstream side of the valve using a long cannula and syringe.
  3. Aseptically attach a sterile or recently sanitized hose (and gasket) to the outlet to be able to direct the water flow and minimize the splashing/spraying of water from a bare outlet.
  4. Direct the end of the hose to a suitably-sized* flush bucked or drain and open the valve sufficient to achieve at least an 8ft/sec flow velocity through the widest part of the valve/hose assembly. For convenience and consistency between samples, specify a fully open valve for at least 30 seconds.

*Never reduce the flush rate to accommodate a convenient bucket capacity – adjust the bucket capacity to the needed flush rate. Also assure that the drain or flush collection funnel have the capacity to accommodate the needed flushed water flow rate. Do not compromise this flow rate since it provides the needed shear forces to remove the fragile portion of the biofilm that may be colonizing the valve and flow path of the water.

  1. After this initial 30 second full-force flush that sloughs off biofilm down to about 40-50 microns thick, throttle back the flow rate to a manageable sampling flow rate and allow an additional 30 seconds of flush time. This lower flow rate now moves the boundary layer and turbulence to far above the surface of the remaining biofilm allowing very little of it to slough off into the sample container.
  2. Aseptically collect the water sample and close the valve. Please note that after the valve has been opened for pre-sampling flushing, never close and reopen the valve to resume the sampling procedure. Just leave it open until after the sample is collected. Closing the valve after flushing – but before sample collection – could slough additional biofilm organisms off the sealing surfaces within the valve and release them into the samples water.
  3. Detach the hose and gasket and set them aside to be re-sanitised and/or properly stored for full drain ability and drying.
  4. Inject alcohol all the way up into the downstream valve lumen again with a cannula and alcohol-filled syringe to displace any retained water within this downstream portion of this valve.
  5. If the valve is in an environmentally compromised location, cover the outlet side with a porous Tyvek or equivalent cover to reduce interim environmental contamination prior to the next use.

If personnel compliance with the procedure is enforced, the vast majority of sampling variability will be eliminated. We hope this has proved useful. If you have any more questions, please get in touch with us on 0113 232 0005 or

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