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6.4 Microbiological Testing

Appropriate bacteriological surveillance of endoscopes and AFERs has proved one of the most difficult and controversial areas of infection control in endoscopy. It is therefore appropriate to state the principles involved together with the details of sample acquisition, processing and interpretation.


Microbiological monitoring of endoscopes should be viewed as an indirect marker of adequacy and completeness of the cleaning and disinfection process. It is a marker of rigorous adherence to the recommended protocol and also as a measure of the structural integrity of the instrument.

Numerous studies document the transmission of infection by contaminated duodenoscopes during ERCP. In many of these outbreaks the endoscopy units involved were unaware of the instrument contamination and the serious clinical infections being caused. The outbreaks were frequently overlooked for prolonged periods and only came to light as a result of investigation of a series of infections with similar or unusual organisms.

Microbiological contamination

Contamination of endoscopes may occur if:

Reprocessing deficiencies

This may occur during:

Endoscope damage

It is not possible to adequately inspect the internal channels of endoscopes. Cracking, splitting, fissuring, joint disruption, and channel wall holes can all be the source of bacterial contamination within the endoscope which can be difficult or impossible to detect by routine inspection and testing. Bacteriological surveillance of endoscopes is frequently the only means of detecting these problems at this time.


Close interaction with a specialist clinical microbiologist where possible will provide optimum outcomes. Because of differential risks of infection transmission, recommendations which are themselves empiric, vary with both the proposed use of endoscopes and with the method of disinfection:

  1. Automated flexible endoscope reprocessors (AFER's) should be monitored every 4 weeks.
  2. Duodenoscopes, bronchoscopes and endoscopic ultrasound instruments should be monitored very 4 weeks.
  3. All other gastrointestinal endoscopes should be monitored every 3 months.
  4. Endoscopes that have been reprocessed through a sterilisation cycle and stored in a wrapped state should be monitored every 3 months.
  5. The water used for manual rinsing of endoscopes should be monitored every 4 weeks if a filter bank is not in use or every 3 months where rinse water is filtered to 0.2u.
  6. Endoscopes on loan are to be tested within 72 hours of receipt of the instrument. The loan instrument should then be retested according to the routine schedule for the type of endoscope if it remains on loan for that period of time.
  7. Further microbiological screening may be undertaken in consultation with a Clinical Microbiologist if:

          - there is clinical suspicion of cross infection related to endoscopy

          - positive surveillance cultures occur

          - alterations are made to the plumbing of the endoscopy reprocessing area

          - new reprocessing protocols are introduced in the unit

          - new models of equipment (endoscope or AFER) are used

          - as a means of quality check for new staff responsible for endoscope reprocessing


Routine microbiological surveillance for viruses is not recommended because:


Bacterial cultures should be directed to the detection of:


Instruments should be sampled after usual processing and following storage of at least 12 hours to allow detection of micro-organisms arising from a biofilm. Endoscopes that have undergone sterilisation and been stored in a wrapped state should be removed from the packaging and tested at the interval indicated above.

Method of Sampling - Endoscopes

  1. 10 mL of sterile water or normal saline is withdrawn from a freshly opened container using a sterile cannula/needle and syringe and put into a sterile specimen container.
  2. 10 mL of sterile water is flushed into each of the channels to be brush sampled. Any fluid that emerges from the distal tip is collected into the sterile specimen container. Attention should be paid to keeping the tip of the endoscope from touching the container so as to avoid contamination.
  3. A sterilised or single-use endoscope brush is passed down the biopsy channel, withdrawn and swirled in the container containing the sterile water. This procedure should also be performed on air and water channels of endoscopes designed with brushable channels. The brush will need to be handled using sterile gloves; sterile gowns are optional. Reusable endoscope brushes should be cleaned and sterilised by steam under pressure or low temperature sterilisation prior to sampling.
  4. Using a sterile syringe, aliquots of sterile water are flushed through each of the air and water channels, suction channel and the forceps elevator and jet channels where applicable. Flushing should be performed from the connection points in the light guide plug and flow to the distal tip. The volume of fluid required is different for each endoscope and will vary from 5 to 50 ml. Fluid should be flushed until it emerges from the distal tip. Air is then syringed through to empty the remaining fluid from each of the channels. The total rinse fluid is collected in a sterile specimen container.
  5. The samples should be pooled in a single container that is labelled and sent with a request form detailing the following:
    - type of scope sampled and serial number
    - name of person to whom the report should be sent
    - test requested
  6. In the event of a persistently positive surveillance culture from an endoscope, the individual channels may need to be sampled and the rinse fluid placed into separate collection containers.
  7. Antegrade sampling may need to be supported by retrograde sampling in selected instances; e.g suspicion of clinical transmission, irregular positive cultures, AFER contamination, 'pseudo infections' associated with bronchoscopy. Retrograde sampling is obtained by using the suction button of the endoscope to suction back the fluid used for flushing, to the proximal channel opening.

Method of Sampling - AFERs

Early detection of machine contamination is best effected by a concentration process. The exact method of sample collection for AFERs will vary depending upon the design of the individual machine.

Connect a sterile sealed bacteria-retentive 0.2µ or 0.45µ filter to the outlet of the machine where it normally attaches to the endoscope and cycle at least 200 ml of fluid through the filter in the rinse cycle mode. When completed, the filter should be placed into a specimen container and forwarded to the laboratory. There the disc can then be removed and plated directly.

Method of Sampling - Water for Manual Rinsing or the Water Supply to an AFER

It is likely that a concentration process will also best effect detection of rinse-water micro-organisms. Following wiping of the tip of the water faucet with 70% alcohol and allowing it to air dry, run 50 ml through the faucet and discard. Then using aseptic handling techniques, collect a 400 ml sample of water in a sterile container and send to the laboratory where a filtration process will concentrate the sample.

Note: Micro-organisms (especially Pseudomonas spp.) can multiply in fluids. Any delay, such as samples being collected in the late afternoon and not processed until the following day, may lead to erroneous results. Therefore it is essential that the sample is promptly processed after collection. If there is likely to be any delay the sample should be refrigerated.

Laboratory Procedure

  1. Centrifuge the collected sample for 15 minutes at approximately 3000 rpm, then decant to 1 ml and resuspend.
  2. Inoculate 0.1 ml sample onto each of two blood agar plates.
  3. Incubate one plate at 35°C and the other at 28°C for 7 days under aerobic conditions. Plates will need to be checked at 48 hours to identify rapidly growing bacteria and attention paid to ensure the plates do not dry out.
  4. Perform semi-quantification of bacterial growth, e.g no growth, 1 to 10 colonies, 10 to 100 colonies, > 100 colonies.
  5. Identify any micro-organisms isolated as far as necessary to allow interpretation as detailed below. Sensitivities are not routinely required.
  6. If there is any growth of micro-organisms the unit that sent the samples should be notified that working day.
  7. Place microfilter discs used to sample the final rinse water of an automated reprocessor directly onto a blood agar plate and incubate aerobically for 2 days at 35°C then 28°C for 3 days.


Each endoscopy unit in conjunction with a Clinical Microbiologist must set its own threshold for the initiation of action if cultures are positive. The following flowcharts reprinted from the New Zealand Standard SNZ HB 8149:2001 will guide decision making.

 Click on image to open: Response to positive bronchoscope cultures  Click on image to open: Response to positive duodenoscope cultures  Click on image to open: Response to positive gastroscope or colonoscope cultures

Some examples are given below:

  1. Low numbers of skin micro-organisms, such as Staphylococcus epidermidis, are most likely to represent collection process contamination rather than a significant problem with the disinfection or cleaning process. The most appropriate initial response is to review the sample processing technique to reduce the chance of contamination e.g. use sterile long-sleeved gown and sterile brush.
  2. A growth of Pseudomonas spp. or other non-fermentative gram-negative bacilli from a duodenoscope, bronchoscope or an AFER that processes duodenoscopes or bronchoscopes would be cause for serious and immediate concern. This is a high-risk clinical situation and the immediate responses should include removing the AFER and endoscope from service, careful culturing of the AFER to see if it is the source of contamination, careful inspection of the endoscope for defects and repeated cultures after manual reprocessing to see if contamination persists. Clinical follow up of patients recently undergoing ERCP or bronchoscopy procedures with that endoscope would also be indicated.
  3. Significant numbers of enteric micro-organisms, such as E. coli or enterococci being repeatedly recovered from one instrument only. This suggests that there is a mechanical defect in that instrument and it requires careful inspection with replacement of the channels if no other defect can be identified.
  4. Significant numbers of enteric micro-organisms, such as E. coli or enterococci, being recovered from a variety of instruments within the unit. This suggests inadequate reprocessing, most likely defects in the manual cleaning program. Much less likely would be a problem in an AFER, (e.g worn valves, serious biofilm accumulation). The appropropriate response here would be a detailed review of all staff members' cleaning and disinfection techniques, if necessary by an independent assessor.
  5. Culture of Mycobacterium tuberculosis from a flexible bronchoscope. This is a serious problem. Responses would include removal of the bronchoscope from service, mechanical review of the instrument by the manufacturer, review of any AFER used including detailed cultures and clinical surveillance of patients recently bronchoscoped with that instrument.
  6. Growth of Mycobacterium chelonae from a bronchoscope. It is almost certain that this will prove to be due to a contaminated AFER that needs to be taken out of service and decontaminated.
  7. ANY isolation of Salmonella or Shigella should cause concern.

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Last Updated: 29 August 2011
Last Reviewed: 29 August 2011