79- Inoculate the bacterial strain of interest in 3–5 mL of appropri-
ate growth medium according to its metabolic and environ-
mental requirements. - Inoculate 0.1 mL of an overnight culture in 8 mL of medium
for 2 h at 37 °C to reach the exponential phase. - Standardize bacterial suspensions by adjusting the concentra-
tion of the inocula to ca. 2 × 107 bacteria/mL using OD 600. - Dilute to a final concentration of 7.5 × 105 bacteria/mL.
- Load the bacterial suspension in a transparent flat bottom
96-well plate. - Prepare the antibiotic dilution range in growth medium using
series of doubling dilution (e.g., 16, 8, 4, 2, 1, 0.5 μg/mL)
and add a proper volume to each well. - Centrifuge urine samples at 2400 × g for 5 min at room tem-
perature to remove leukocytes and epithelial cells. - Collect the supernatant, mix 50% (vol/vol) with CAMHBT
and incubate for 2 h at 37 °C. - Adjust bacterial concentration to 0.5 of the McFarland
standard. - Inoculate 0.1 mL in 11 mL of MH broth and mix
thoroughly. - Load the bacterial suspension in a transparent flat bottom 96
well plate. Prepare the antibiotic dilution range in growth
medium using series of doubling dilution (e.g., 16, 8, 4, 2, 1,
0.5 μg/mL) and add a proper volume to each well. - Centrifuge blood samples at 200 × g for 5 min to remove
blood cells. - Resuspend the bacterial pellet in CAMHBT and incubate for
2 h at 37 °C. - Inoculate 0.1 mL in 11 mL of MH broth and mix through.
- Load the bacterial suspension in a transparent flat bottom
96-well plate.
Prepare the antibiotic dilution range in growth medium using
series of doubling dilution (e.g., 16, 8, 4, 2, 1, 0.5 μg/mL) and
add a proper volume to each well. - Place the oCelloScope in a standard laboratory incubator for
biological applications for precise temperature regulation.
Prior performing the AST experiment, it is recommended to
let the instrument and the sample container equilibrate inside
the incubator for at least 2 h. This step allows the prevention
of condensation forming on the on the microtiter plate lid,
3.1 Guideline
for Preparation of Pure
Cultures of Bacterial
Strains
3.2 Guideline
for Preparation
of Urine Samples
3.3 Guideline
for Preparation
of Blood Samples
3.4 Automated
Time-Lapse Analysis
Using the oCelloScope
System
Real-Time Digital Bright Field Technology for Rapid Antibiotic Susceptibility Testing