Appendix D: Infection Prevention and Control Guideline for Flexible Gastrointestinal Endoscopy and Flexible Bronchoscopy – Bioburden test method

APPENDIX D – Bioburden Test Method

Sample collection usually requires two people. It may be feasible to have a staff person from Infection Prevention and Control work with a staff person in the endoscope reprocessing area to collect the samples. If Infection Prevention and Control staff are not available, reprocessing personnel can perform the sampling, however, care must be taken to ensure aseptic technique during sample collection.

1. Sample Collection

Staff should wear appropriate personal protective equipment (gloves, gowns, face shields).


  1. Sterile tubing connector suitable to attach to the umbilical suction barb (or other channels if tested)
  2. Sterile syringes; one for each channel to be sampled (30 cc for suction/biopsy channel and air/water channel, 20 cc for other smaller channels)
  3. Sterile container for collecting the sample (sterile urine specimen containers work well)
  4. Sterile reverse osmosis (RO) water (adequate total volume to collect samples from each channel; e.g., for a colonoscope ~ 50 mls, for a bronchoscope ~ 30 mls, for a duodenoscope ~ 50 mls will be needed). Ideally separate 20 mls aliquots of sterile RO water should be used for each channel sampled (this ensures that the risk of contaminating the sterile RO water as a result of aspirating several samples from the same container is reduced).

Ensure the specimen collection container is properly labelled. It will be necessary to ensure that the suction valve and biopsy port are closed so that the fluid used to collect the sample is flushed through the suction/biopsy channel (the air/water valve needs to be closed to collect the sample from the air/water channel). Use sterile RO water to collect the sample from the channel. This ensures that the optimal sample will be collected as RO water is very “active” and will “strip” surface material more efficiently than buffer or both media. In addition, it will not have to be cleaned away and as such will not interfere with subsequent use of the scope on patients.


  1. Aspirate 10 mls of sterile RO water into a sterile 30cc syringe.
  2. Attach this syringe via a piece of sterile tubing (e.g., manufacturer’s recommended connector) to the suction/biopsy barb of the umbilical end and flush 10 mls of sterile RO water through the channel.
  3. Collect the channel sample from the distal end of the endoscope by holding the end of the insertion tube in a sterile plastic container (urine specimen container can be used).
  4. Use a syringe of air to flush out any residual fluid sample from the channel.

The sterile tubing used for sample collection should be packaged and steam sterilized prior to next usage. The air/water channel can be sampled in a similar fashion by attaching a sterile piece of tubing (e.g., manufacturer’s recommended connector) to the air/water barb on the umbilical end and flushing 10 mls of sterile RO water through the air/water channel. For other smaller channels that may be present on some flexible endoscopes (e.g., elevator wire channel and auxiliary water channel) the same process can be performed using 3 mls of sterile RO water instead of 10mls, injected using a sterile 10cc syringe.

Once the channel sample(s) have been collected, and adequately labelled, they should be sent immediately to the microbiology laboratory for culture. If transit time is anticipated to be more than 30 minutes, the sample(s) should be held on ice or refrigerated until cultured (samples should not be held in the refrigerator longer than 24 hours prior to culture). This ensures that microbial growth does not occur during storage.

2. Bacterial Culture


  1. Mix the sample well (e.g., vortex mixer)
  2. Inoculate 0.1 mls of the sample onto a blood agar (BA) plate and a Sabaroud agar (SA) plate.
  3. Spread the inoculum over the entire surface of the media to allow quantitation of any colonies that grow.
  4. Incubate the BA plate aerobically at 35°C for 48hrs (the BA plate will detect a wide range of organisms that might come from humans and/or the environment)
  5. Incubate the SA plate aerobically at 30°C for 5 days (the SA plate at the lower temperature will detect the presence of Methylobacter species, which is commonly found in potable water).

If no growth is detected after 5 days of incubation the sample should be reported as having no detectable organisms. If growth of organisms is detected, the number of colonies should be counted and the colony forming units (cfu)/ml determined (cfu/ml = total number of colonies on the entire plate/0.1 mls (e.g., If 10 colonies are detected, the cfu/ml = 10/0.1 = 100 cfu/ml).

3. Interpretation

How to interpret the presence of any detectable bioburden is controversial. One published interpretation criterion is that endoscope channels would be expected to have bioburden levels that are no worse than 200 cfu/ml, which represents the cut-off of viable microorganisms for dialysis water and is within the 500cfu/ml or less cut-off (heterotrophic plate count, excluding coliforms) accepted by Canadian guidelines for potable waterFootnote (93).  If there are 20 or more colonies on the plate (e.g., 20 colonies per 0.1 mls = 200 cfu/ml), this is considered unacceptable and would be reported as < 200 cfu/ml. If 1 - 19 colonies are detected, this is considered acceptable, as a few organisms may be present due to the collection method. This result would be reported as < 200 cfu/ml. If no growth is detected the result would be reported as “no growth”.

4. Actions for Unacceptable Bioburden Levels

If the level of bioburden is ≥200 cfu/ml, then the scope should be removed from use and re-tested. If the second testing shows no problem, then the scope can be returned to use (i.e., the elevated bioburden level was an isolated situation that is not due to an ongoing process problem). If the bioburden level is ≥200 cfu/ml, on the second evaluation, then infection prevention and control needs to be contacted to identify what the problem might be and recommend solutions. At this stage testing of all scopes may be necessary to determine how wide-spread the problem is. Items to be assessed (not an exhaustive list) when unacceptable bioburden levels are repeatedly detected include:

  • Was there residual moisture in the channels during storage (review the procedure for alcohol rinsing and forced air drying prior to storage)? This is the most common reason for sporadic unacceptable bioburden levels.
  • Was the cleaning being done properly (review cleaning process to ensure fresh detergent was prepared for each scope)?
  • Is the high level disinfectant still effective (review daily minimum effective concentration (MEC) testing results)?
  • Was the final rinse post-high level disinfection (HLD) contaminated (check filter integrity if using an AER, take samples of the final rinse water from the distal side of the filters to determine bioburden levels)?
  • Is there a problem with the high level disinfectant?  To evaluate whether this is a problem, bioburden testing should be performed (sample collected as described previously) immediately after HLD to assess if the disinfection stage is ineffective.

If HLD problems are identified, Infection Prevention and Control should review the charts of patients on whom this scope had been used since the time of the last testing to evaluate if there have been any potential patient-related problems. If potential patient infections are identified, an assessment should be performed to determine the need for further outbreak investigation and/or patient notification.

The data from bioburden testing and any follow-up investigations should be reviewed with Infection Prevention and Control at the time of the occurrence.

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