$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Prepare MIC Panel Dilutions
- Prepare a stock solution no higher than 1,600 μg/mL of dalbavancin in neat (pure) dimethyl sulfoxide (DMSO) in sterile glass or plastic tubes, and use on the same day of preparation or store at -20 to -60 °C or below for future use in a non-defrosting freezer. Take into consideration the potency of dalbavancin as provided on the documentation received with the powder when weighing the powder.
- Dilute the stock dilution similarly to the scheme with neat DMSO in sterile glass or plastic tubes. Use one pipet for measuring diluent and another pipet for adding the initial dalbavancin stock to the first tube. For each subsequent dalbavancin stock concentration use a new pipet.
- Prepare 100X final MIC panel concentration dilutions (intermediate concentrations) with neat DMSO. Combine appropriate volumes of source and DMSO to achieve desired intermediate concentration (volumes to be used will depend on the number of MIC panels to be made).
- Prepare 0.004% P80: Prepare a fresh working stock solution of 2% P80 by adding 0.1 mL P80 to 4.9 mL dH₂O. Sterilize by passing through a 0.22 μm filter and use the solution on the same day of preparation. Prepare 0.004% P80 diluent by making a 1:500 dilution of 2% P80 (e.g., 0.3 mL of 2% P80 to 149.7 mL of CAMHB).
- Further, dilute the intermediate concentrations 1:100 in cation-adjusted Mueller Hinton broth (CAMHB) supplemented with 0.004% (v/v) polysorbate-80 (P-80) and/or LHB (for streptococci) added at double the final concentration because the addition of inoculum will result in a 1:2 dilution.
2. Prepare MIC Panels
- Dispense 50 μL of each dalbavancin solution prepared into appropriate wells of the MIC panel and include media only in one well (growth control well). A multi-channel pipette with sterile tips can be used for this step.
- Use panels immediately or seal with plastic film, place them in plastic bags, and immediately place in a non-defrosting freezer at ≤ -20 °C (preferably at ≤ -60 °C) until needed. If frozen panels are used, remove seals and place individual panels on the lab bench for 15–30 min (until well contents are thawed) before proceeding to panel inoculation.
3. Inoculate MIC Panels, Perform Purity, and Set Up Colony Count
- Select several well-isolated colonies from an 18–24 hr blood agar or other non-selective agar plate. Touch the top of each colony with a sterile loop or swab and transfer to 1–5 mL CAMHB or saline until turbidity is equivalent to a 0.5 McFarland standard. Assess turbidity by visual comparison to the 0.5 McFarland or with a photometric device.
- Within 15–30 min of preparation, dilute the inoculum 1:100 in CAMHB (100 μL into 10 mL CAMHB). For most bacteria tested against dalbavancin, with the exception of S. pneumoniae, this dilution will provide a final well concentration of 5 × 10⁵ CFU/mL (acceptable range is 2–8 × 10⁵ CFU/mL). For S. pneumoniae, bacterial concentration based on a comparison of turbidity to a 0.5 McFarland is typically considerably less. Therefore, dilute the inoculum 1:25 (400 μL into 10 mL CAMHB + 10% LHB).
- Within 15 min after inoculum preparation, transfer 50 μL of the final inoculum to each well (with the exception of the sterility control well) of the MIC panel prepared. A multi-channel pipette using sterile tips can be used for this step.
- Perform a purity check by transferring and spreading a 1–10 μL aliquot from the positive growth control well using a sterile loop to a non-selective agar (e.g., trypticase soy agar with 5% sheep blood).
- Set up colony count by removing 10 μL from the growth control well with a single-channel pipette and sterile tip and transferring to 10 mL of saline (1:1,000 dilution). Mix and transfer 100 μL with a single-channel pipette and sterile tip to a suitable non-selective agar medium (e.g., trypticase soy agar with 5% sheep blood) and spread over the entire agar surface with a sterile loop, repeating twice in different directions to assure even distribution of the inoculum (1:10 dilution).
4. Incubate MIC Panels, Colony Count, and Purity Plates
- Seal each MIC panel or stack of no more than four panels in a plastic bag with plastic tape or with a tight-fitting plastic cover before incubating. Alternatively, place an empty MIC panel on the top of the stack of no more than 4 MIC panels, place a damp paper towel in a plastic container, place MIC panels in the plastic container, and close the container securely with a lid. Incubate MIC panels in an ambient air incubator at 35 °C ± 2 °C for 16–20 hr (staphylococci and enterococci) and 20–24 hr (streptococci) within 30 minutes of inoculation. Incubate the colony count and purity plates under the same conditions, except incubate streptococci in a 5% CO₂ incubator.
5. Read the MIC and Colony Count Plates; Check Purity Plate
- Read the MIC as the lowest concentration that completely inhibits bacterial growth in the wells as detected by the unaided eye.
- Count colonies on the colony count plate. Multiply each colony by the dilution factor (1:10,000) (e.g., 50 colonies are equivalent to 5 × 10⁵ CFU/mL). An acceptable range is 20–80 colonies (2–8 × 10⁵ CFU/mL) and is used as an approximate guideline.
- Check the purity plate. If all colonies are similar to each colonies, then the inoculum can be considered pure. If there are any other colonies present, then there is a potential for a contaminant to be present in the MIC panel, and the test should be repeated.