Preparation and use of 12-well plates for the rapid detection of terbinafine-resistant dermatophytes
Khalid El Moussaoui
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Abstract
This protocol describes the steps necessary for the preparation and use of screening plates for the rapid detection of terbinafine resistant dermatophytes.
Steps
Terbinafine preparation
Weigh 16.33mg of 5mL of 0h 10m 0s to dissolve. The final concentration of this terbinafine preparation is 3200µg/mL . Do not forget to add a magnetic bar. Cover each flask with glass wool and aluminium foil.
Equipment
| Value | Label |
|---|---|
| Heated magnetic stirrer | NAME |
| AG602 | TYPE |
| LMR | BRAND |
| AG602 | SKU |
| https://www.labomoderne.com | LINK |
Autoclave this flask at 121°C for 0h 30m 0s
Equipment
| Value | Label |
|---|---|
| Autoclave | NAME |
| Tuttnauer | BRAND |
| 3850EL | SKU |
Under hood, transfer 1mL of this preparation into 2 mL eppendorf tubes and store at -80°C for later use.
Equipment
| Value | Label |
|---|---|
| Biological safety cabinet | NAME |
| AC2-4S8-TU | TYPE |
| Esco | BRAND |
| 303110 | SKU |
Medium preparation
Dissolve 30g of 1L of 0h 5m 0s (temperature and mixing speed knob at mid-step)
Equipment
| Value | Label |
|---|---|
| Heated magnetic stirrer | NAME |
| AG602 | TYPE |
| LMR | BRAND |
| AG602 | SKU |
| https://www.labomoderne.com | LINK |
Add 15g of 0h 10m 0s while mixing and heating
Pour 250mL of this medium into 4 erlenmeyer flasks and label each with final terbinafine concentrations (0.2 µg/mL for the first flask, 0.1 µg/mL for the second, 0.05 µg/mL for the third and 0 µg/mL for the last flask). Given the uncertainty of measurement, it is advisable to work from the highest concentration to the lowest. Do not forget to add a magnetic bar. Cover each flask with glass wool and aluminium foil.
Autoclave these 4 flasks at 121°C for 0h 30m 0s
Equipment
| Value | Label |
|---|---|
| Autoclave | NAME |
| Tuttnauer | BRAND |
| 3850EL | SKU |
Plates preparation
For the first flask (0.2 µg/mL) : add 15.6µL of a 3200 µg/mL terbinafine preparation (see section number 1). Let mix while heating (temperature and mixing speed knob at mid-step) for 0h 2m 0s . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
For the second flask (0.1 µg/mL) : add 7.8µL of a 3200 µg/mL terbinafine preparation (see section number 1). Let mix while heating (temperature and mixing speed knob at mid-step) for 0h 2m 0s . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
For the third flask (0.05 µg/mL) : add 3.9µL of a 3200 µg/mL terbinafine preparation (see section number 1). Let mix while heating (temperature and mixing speed knob at mid-step) for 0h 2m 0s . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
For the last flask (0 µg/mL) : does not contain terbinafine.
In a 12-well plate (3 rows and 4 columns), the first well of each row does not contain terbinafine. It serves as a growth control. Using an automated pipettor, add 3mL of the medium that does not contains terbinafine (flask number 4) to all the first wells of the plate.
Equipment
| Value | Label |
|---|---|
| Motorized Pipette Filler | NAME |
| Levo Plus | TYPE |
| DR. LAB | BRAND |
| 7033100100 | SKU |
.
Equipment
| Value | Label |
|---|---|
| Microwell plate | NAME |
| Thermo Scientific | BRAND |
| 130185 | SKU |
The second wells in each row contain a concentration of 0.05 µg/mL of terbinafine. Using an automated pipettor, add 3mL of the medium that contains 0.05µg/mL of terbinafine (flask number 3) to all the second wells of the plate.
Equipment
| Value | Label |
|---|---|
| Motorized Pipette Filler | NAME |
| Levo Plus | TYPE |
| DR. LAB | BRAND |
| 7033100100 | SKU |
Equipment
| Value | Label |
|---|---|
| Microwell plate | NAME |
| Thermo Scientific | BRAND |
| 130185 | SKU |
The third wells in each row contain a concentration of 0.1 µg/mL of terbinafine. Using an automated pipettor, add 3mL of the medium that contains 0.1µg/mL of terbinafine (flask number 2) to all the third wells of the plate.
Equipment
| Value | Label |
|---|---|
| Microwell plate | NAME |
| Thermo Scientific | BRAND |
| 130185 | SKU |
Equipment
| Value | Label |
|---|---|
| Motorized Pipette Filler | NAME |
| Levo Plus | TYPE |
| DR. LAB | BRAND |
| 7033100100 | SKU |
The last wells in each row contain a concentration of 0.2 µg/mL of terbinafine. Using an automated pipettor, add 3mL of the medium that contains 0.2µg/mL of terbinafine (flask number 1) to all the last wells of the plate.
Equipment
| Value | Label |
|---|---|
| Motorized Pipette Filler | NAME |
| Levo Plus | TYPE |
| DR. LAB | BRAND |
| 7033100100 | SKU |
Equipment
| Value | Label |
|---|---|
| Microwell plate | NAME |
| Thermo Scientific | BRAND |
| 130185 | SKU |
Cover the plates with their lids and let the medium solidify at room temperature. Label the plates with the corresponding terbinafine concentrations for each well and store at 4°C for future use.
Use of the plates
Using a sterile swab, prepare a suspension (sterile water) 0.5 McF of the dermatophyte to be studied.
Transfer 25µL of this suspension to each of the 4 wells of the plate.
Annotate the plate with the number of the studied strains and incubate at 30°C for 96h before reading the result.
