Antibiogram testing of pure strains
OBJ: Testing the sensibility or resistance of a pure strain to different antibiotics
Duration: -
Adapted from: McFarland et al., https://pdf4pro.com/amp/view/mcfarland-standard-dalynn-f76d0.html, and from Matuschek et al., 2014 (DOI: https://doi.org/10.1111/1469-0691.12373)
Materials
- plate 90mm
- falcon tube 15mL
- couvette UV/VIS
- Mueller Hinton media
Equipment
- dry incubator at 37°C
- shaking incubator at 37°C
- spectrophotometer UV/VIS
- paper disks
Solutions
- Antibiotic solutions to be tested with a working concentration
Procedure
- Glycerol stock (-80 °C) of your stain
- Streak on Mueller-Hinton agar plate (12h at 37 °C dry incubator)
- Inoculate a single colony in 5ml of mueller hinton liquid media (~12 h at 37 °C shaking incubator). you need to know the hours passed between the starting of the inoculum and the lecture at the spectrophotometer
- Read the OD 625 nm value at spectrophotometer. dilute the sample 1:10 (100 µL of sample in 900 µL of liquid media; 0.5 mcfarland = 1 - 2 x 108 CFU/mL → OD = 0.08 - 0.1)
- Dilute your sample until you reach an OD 625 nm value comprises between 0.08 and 0.1
- Prepare plate with a base of Mueller-Hinton agar 1.8 % (10/15 mL)
- Add on the surface 10ml of mueller hinton agar 0.7 % with 1 mL of your sample (9 mL of MH agar 0.7 % + 1 mL of sample + shaking)
- After media solidification, add paper disks and wet with antibiotic (liquid volume required for wet the disk is 5 µL)
- Incubate for 12 h at 37 °C dry incubator
- Check for halo presence/absence or measure the diameter
Expected results (quantitative information, graphics, images)
Describe the expected results and if possible include a visual example (i.e. a picture, table or graph)
Common problems, troubleshooting and solutions
- List all common problems and solutions