May 20, 2022
Preparation and use of 12-well plates for the rapid detection of terbinafine-resistant dermatophytes
- 1AIDS Reference Laboratory, Department of Clinical Microbiology, University Hospital of Liege, 4000 Liege, Belgium
Protocol Citation: Khalid El Moussaoui 2022. Preparation and use of 12-well plates for the rapid detection of terbinafine-resistant dermatophytes . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6116zvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 11, 2022
Last Modified: May 20, 2022
Protocol Integer ID: 58072
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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.
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.
Protocol materials
Terbinafine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8826
Step 1
Dimethyl sulfoxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4540
Step 1
Sabouraud dextrose brothMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3306
Step 4
MilliQ water
Step 4
Difco granulated agarFisher ScientificCatalog #BD 214530
Step 5
Terbinafine preparation
Terbinafine preparation
13m
13m
Weigh 16.33 mg of Terbinafine hydrochlorideSigma AldrichCatalog #T8826 and add to an erlenmeyer flask containing 5 mL of Dimethyl sulfoxideSigma AldrichCatalog #D4540 . Mix without heating for 00:10:00 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.
Equipment
Heated magnetic stirrer
NAME
AG602
TYPE
LMR
BRAND
AG602
SKU
13m
Autoclave this flask at 121 °C for 00:30:00
Equipment
Autoclave
NAME
Tuttnauer
BRAND
3850EL
SKU
LINK
30m
Under hood, transfer 1 mL of this preparation into 2 mL eppendorf tubes and store at -80 °C for later use.
Equipment
Biological safety cabinet
NAME
AC2-4S8-TU
TYPE
Esco
BRAND
303110
SKU
LINK
5m
Medium preparation
Medium preparation
4h 25m
4h 25m
Dissolve 30 g of Sabouraud dextrose brothSigma AldrichCatalog #S3306 in 1 L of MilliQ waterSigma Aldrich and let mix on the heated magnetic stirrer for 00:05:00 (temperature and mixing speed knob at mid-step)
Equipment
Heated magnetic stirrer
NAME
AG602
TYPE
LMR
BRAND
AG602
SKU
8m
Add 15 g of Difco granulated agarSigma AldrichCatalog #BD 214530 and let dissolve for 00:10:00 while mixing and heating
12m
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.
5m
Autoclave these 4 flasks at 121 °C for 00:30:00
Equipment
Autoclave
NAME
Tuttnauer
BRAND
3850EL
SKU
LINK
4h
Plates preparation
Plates preparation
1h 11m
1h 11m
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 00:02:00 . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
2m
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 00:02:00 . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
2m
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 00:02:00 . Caution : do not leave for more than 2 minutes otherwise the medium will solidify.
2m
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 3 mL of the medium that does not contains terbinafine (flask number 4) to all the first wells of the plate.
Equipment
Motorized Pipette Filler
NAME
Levo Plus
TYPE
DR. LAB
BRAND
7033100100
SKU
LINK
.
Equipment
Microwell plate
NAME
Thermo Scientific
BRAND
130185
SKU
LINK
15m
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.
Equipment
Motorized Pipette Filler
NAME
Levo Plus
TYPE
DR. LAB
BRAND
7033100100
SKU
LINK
Equipment
Microwell plate
NAME
Thermo Scientific
BRAND
130185
SKU
LINK
15m
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.
Equipment
Microwell plate
NAME
Thermo Scientific
BRAND
130185
SKU
LINK
Equipment
Motorized Pipette Filler
NAME
Levo Plus
TYPE
DR. LAB
BRAND
7033100100
SKU
LINK
15m
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.
Equipment
Motorized Pipette Filler
NAME
Levo Plus
TYPE
DR. LAB
BRAND
7033100100
SKU
LINK
Equipment
Microwell plate
NAME
Thermo Scientific
BRAND
130185
SKU
LINK
15m
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.
5m
Use of the plates
Use of the plates
Using a sterile swab, prepare a suspension (sterile water) 0.5 McF of the dermatophyte to be studied.
Transfer 25 µL 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.