TITLE: TD test protocol 9/26/19
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tolerance detection test</div><div class = "text-block">Bikash(Adapted from(Gefen et al. 2017), Scientific reports)</div></div>

[STEPS]
?. [TD test: Step 1 -  Day 1]
Preparation of Mueller-Hinton plate (Hudzicki n.d.)
?. [TD test: Step 1 -  Day 1]
Allow an MH agar plate (one for each organism to be tested) to come to room temperature. It is preferable to allow the plates to remain in the plastic sleeve while they warm to minimize condensation.
?. [TD test: Step 1 -  Day 1]
Inoculation of the MH plate
?. [TD test: Step 1 -  Day 1]
If the surface of the agar has visible liquid present, set the plate inverted, ajar on its lid to allow the excess liquid to drain from the agar surface and evaporate. Plates may be placed in a 37°C incubator or in a laminar flow hood at room temperature until dry (usually 10 to 30 minutes).
?. [TD test: Step 1 -  Day 1]
Appropriately label each MH agar plate for each organism to be tested
?. [TD test: Step 1 -  Day 1]
Transfer 2 ml of stationary phase culture into the inoculum tube. Do not transfer any cell debris. Dip a sterile swab into the inoculum tube.
?. [TD test: Step 1 -  Day 1]
Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to remove excess fluid. The swab should not be dripping wet.
?. [TD test: Step 1 -  Day 1]
Inoculate the dried surface of an MH agar plate by streaking the swab three times over the entire agar surface; rotate the plate approximately 60 degrees each time to ensure an even distribution of the inoculum.
?. [TD test: Step 1 -  Day 1]
Placement of antibiotic discs
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Rim the plate with the swab to pick up any excess liquid.
?. [TD test: Step 1 -  Day 1]
Discard the swab into an appropriate container
?. [TD test: Step 1 -  Day 1]
Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the agar plate to dry before proceeding to the next step.
?. [TD test: Step 1 -  Day 1]
Place the appropriate antimicrobial-impregnated disks on the surface of the agar, using either forceps to dispense each antimicrobial disk one at a time
?. [TD test: Step 1 -  Day 1]
Once all disks are in place, replace the lid, invert the plates, and place them in a 35°C air incubator for 16 to 18 hours. When testing Staphylococcus against oxacillin or vancomycin, or Enterococcus against vancomycin, incubate for a full 24 hours before reading.
?. [TD test: Step 1 -  Day 1]
You should avoid placing disks close to the edge of the plate as the zones will not be fully round and can be difficult to measure.
?. [TD test: Step 1 -  Day 1]
Each disk must be pressed down with forceps to ensure complete contact with the agar surface or irregular zone shapes may occur.
?. [TD test: Step 1 -  Day 1]
If the surface of the agar is disrupted in any way (a disk penetrating the surface, visible lines present due to excessive pressure of the swab against the plate during inoculation, etc.) the shape of the zone may be affected.
?. [TD test: Step 2 - Day 2]
Replace the antibiotic disk with a glucose (2 mg) disk after 18 hours and incubate the plate for an additional overnight.
Alternatives for this step:Add the glucose solution directly on the antibiotic disk, instead of replacing it.Use other nutrients instead of glucose.
?. [Step 2 - Day 3]
Result: Note the difference in the zone of inhibition and/or the number of tolerant colonies on the zone of inhibition. Use the imaging system in the photodoc room (if needed talk to HGB lab to operate the machine) to take images of the plates (export images as tif or png file).