TITLE: Preparation and use of 12-well plates for the rapid detection of terbinafine-resistant dermatophytes 
AUTHORS: Khalid  El Moussaoui

[DESCRIPTION]
This protocol describes the steps necessary for the preparation and use of screening plates for the rapid detection of terbinafine resistant dermatophytes.

[GUIDELINES]
1) When you take the medium out of the autoclave, be as quick as possible to add the terbinafine and transfer the medium to the plate wells. Since the medium contains agar, it will solidify fairly quickly.

2) Work in sterile conditions as much as possible. The medium is very easy to contaminate and can, if so, falsify the results.

[STEPS]
SECTION: Medium preparation
4. Dissolve 30 g of   in 1 L of   and let mix on the heated magnetic stirrer for 5 min (temperature and mixing speed knob at mid-step)

 

SECTION: Medium preparation
5. Add 15 g of   and let dissolve for 10 min while mixing and heating

SECTION: Medium preparation
6. Pour 250 mL  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.

SECTION: Medium preparation
7. Autoclave these 4 flasks at 121 °C for 30 min 

 

SECTION: Terbinafine preparation
1. Weigh 16.33 mg of   and add to an erlenmeyer flask containing 5 mL of  . Mix without heating for 10 min to dissolve. The final concentration of this terbinafine preparation is 3200 µg/mL . Do not forget to add a magnetic bar. Cover each flask with glass wool and aluminium foil.
 

SECTION: Terbinafine preparation
2. Autoclave this flask at 121 °C for 30 min 
 

SECTION: Terbinafine preparation
3. Under hood, transfer 1 mL  of this preparation into 2 mL eppendorf tubes and store at -80 °C  for later use.
 

SECTION: Plates preparation
8. For the first flask (0.2 µg/mL) : add 15.6 µL of a 3200 µg/mL terbinafine preparation (see section number 1). Let mix while heating (temperature and mixing speed knob at mid-step) for 2 min . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.

SECTION: Plates preparation
9. For the second flask (0.1 µg/mL) : add 7.8 µL of a 3200 µg/mL terbinafine preparation (see section number 1). Let mix while heating (temperature and mixing speed knob at mid-step) for 2 min . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
SECTION: Plates preparation
10. For the third flask (0.05 µg/mL) : add 3.9 µL of a 3200 µg/mL terbinafine preparation (see section number 1). Let mix while heating (temperature and mixing speed knob at mid-step) for 2 min . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
SECTION: Plates preparation
11. For the last flask (0 µg/mL) : does not contain terbinafine.
SECTION: Plates preparation
12. 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 3 mL  of the medium that does not contains terbinafine (flask number 4) to all the first wells of the plate.
 
.

 

SECTION: Plates preparation
13. The second wells in each row contain a concentration of 0.05 µg/mL of terbinafine. Using an automated pipettor, add 3 mL  of the medium that contains 0.05 µg/mL  of terbinafine (flask number 3) to all the second wells of the plate.

 

 

SECTION: Plates preparation
14. The third wells in each row contain a concentration of 0.1 µg/mL of terbinafine. Using an automated pipettor, add 3 mL  of the medium that contains 0.1 µg/mL  of terbinafine (flask number 2) to all the third wells of the plate.
 

 

SECTION: Plates preparation
15. The last wells in each row contain a concentration of 0.2 µg/mL of terbinafine. Using an automated pipettor, add 3 mL  of the medium that contains 0.2 µg/mL  of terbinafine (flask number 1) to all the last wells of the plate.
 

 

SECTION: Plates preparation
16. 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.

SECTION: Use of the plates
17. Using a sterile swab, prepare a suspension (sterile water) 0.5 McF of the dermatophyte to be studied.
SECTION: Use of the plates
18. Transfer 25 µL of this suspension to each of the 4 wells of the plate.

SECTION: Use of the plates
19. Annotate the plate with the number of the studied strains and incubate at 30°C for 96h before reading the result.