Modified CDC Duodenoscope Culture Method

Modified CDC Duodenoscope Culture Method

http://www.cdc.gov/hai/settings/lab/lab-duodenoscope-culture-method.htmL

  1. Sterile brush used to sample the distal end: Vortex the sample for 2 minutes in 10 second bursts and aseptically, remove the brush using sterile forceps from the PBST solution. Samples are transferred to blood and MaConkey agar plates, see further Membrane Filtration, step 5.
  2. Transfer the fluid samples (instrument channel flush, channel-opening brush fluid) to 50-cc conical tubes
  3. Consider including an internal process positive control, which may provide insight on whether the culture protocol was conducted appropriately and if remaining residual disinfectant, if any, may have had an impact on organism viability and detection.
    1. Aliquot 5 mL of the instrument channel flush sample to a sterile conical tube. Reserve the remaining 45 mL for further processing as described below (see Step 4).
    2. Inoculate the 5 mL instrument channel flush sample with a gram-positive (e.g. Staphylococcus aureus) and/or a gram-negative (e.g. Escherichia coli) at a low inoculum (e.g. 100 CFU)
    3. Process the control sample as described in the following steps for the chosen method. Take precautions to not cross-contaminate the positive control with the duodenoscope samples.
  4. Choose either I. Centrifugation or II. Membrane Filtration to concentrate the sample:
    1. Centrifugation
      1. Concentrate by centrifugation on a benchtop centrifuge equipped for high volume suspensions (range: 3,500 - 5,000 x g for 10 - 15 min)
      2. Remove supernatant without disrupting the pellet to a final volume of 1 mL. If needed, add PBST to a final volume of 1 mL and re-suspend.
      3. Vortex the sample for 10 sec
      4. Pipet the following on to blood agar and MacConkey agar plates.
      5. Continue with Step 5 in II. Membrane Filtration
    2. Membrane Filtration
      1. Set up membrane filtration equipment in a laboratory (e.g. sidearm filtering flask, vacuum, filter housings, gridded filters, sterile forceps, etc.)
      2. The total volume needed to assay samples is 20 mL at a minimum.
      3. Filter the samples, making sure to rinse the filter housing liberally with a sterile buffered solution after each sample
      4. Place the gridded filter using sterile forceps, grid side up, on the agar plate; taking care to place the filter completely flat and removing any air bubbles or creases in the filter
      5. Incubate at 35°C to 37°C for 48 h.
      6. Check and record growth at 18 to 24 h and 48 h.
        1. Count and record number of colonies from plates
        2. Calculate CFU/mL from the blood agar plates and account for the volume of the sample filtered to determine the total CFU/sampled duodenoscope (20 mL sample).
      7. Streak suspect colonies for isolation
      8. Work up pure isolates for characterization of “low- concern” bacteria, which represent flora from skin and the environment, and species identification of “high-concern” bacteria.
        1. “Low-concern” bacteria include, but are not limited to, coagulase-negative staphylococci, micrococci, diptheroids, Bacillus spp. and other gram-positive rods
        2. “High-concern” bacteria include, but are not limited to, Staphylococcus aureus, Enterococcus spp., Streptococcus sp. viridians group, Pseudomonas aeruginosa, Klebsiella spp., Salmonella spp., Shigella spp. and other enteric gram-negative bacilli.