Table 2.
Troubleshooting guide for the method developed to assess antibiotic resistance using the in vitro human digestion model.
| Procedure | Problem | Cause | Solution |
|---|---|---|---|
| Preparation of gut microbiota for digestion | Incorrect gut microbial population | Insufficient gut microbial population (108–1010 CFU/mL) for use in the in vitro human digestion model | Adjust the experiment timing. If the gut microbial population does not reach the required count, cultivate it for few more hours |
| Enzyme activity (amylase, pepsin, lipase, trypsin) | Assay not working | · Data read at wrong wavelength | · Check the wavelength in the data and filter settings of the microplate reader |
| · Using for different type plate | · In case of colorimetric method, use clear 96-well plates. (Fluorescence, black plates with clear bottoms; luminescence, white plates) | ||
| Irregular values in samples | · Samples made in an incorrect buffer | · Use assay buffer in the specific kit or refer to instructions | |
| · Poorly dissolved sample and reagents | · Dissolve all samples and reagents completely and mix gently before testing | ||
| Assay failure during measurement of linear pattern for standard curve | · Incompletely dissolved standards | · Completely mix all components before making standard stock | |
| · Incorrect pipetting of standard or reagents | · Avoid pipetting small amounts to prevent bubble generation | ||
| · Possible measurement of well plate in the presence of bubbles | · Inject all mixtures gently against the wall of the well plates | ||
| · Calculation errors | · Check calculations and values by referring to the data sheet | ||
| In vitro human digestion steps | Preparation of samples for analyzing changes at each digestion step | To analyze changes (quantitative or structural changes) in the samples at each digestion step (mouth, stomach, small intestine, and large intestine), the number of samples prepared must be more than the number of treatments | For analysis at each digestion step, the sample must be collected and analyzed at the end of each step (mouth, stomach, small intestine, and large intestine); thus, the number of samples prepared should be four times greater than the number of treatment groups |
| E.g., a total of 36 samples should be prepared for 3 treatments × 3 replication experiments (3 treatments × 3 replications × 4 digestion steps) | |||
| Short digestion time in mouth | Short digestion time (<5 min) in the mouth may be a result of considerable time spent adding the digestive enzyme | Do not experiment with too many samples at the same time; add the samples first, followed by enzymes randomly and quickly | |
| Difficulty in handling dialysis tube | It is not easy to tie the dialysis tube, and there may be many digested samples (including digestive juices) on the outer surface of the tube after the step involving digestion in the small intestine | The dialysis tube should be tied with a clip (not straps). After digestion in the small intestine, it should be cleaned with a phosphate buffer solution (pH 7) and then used for the experiments | |
| Effects of dilution | Increasing the sample volume by adding digestive juices | Before analysis, use a buffer that does not influence the data to adjust sample volume | |
| Agar disk diffusion | Difficulty in measuring inhibition zone | · Overlap with inhibition zone disks | · Use no more than 5 disks on a 100-mm agar plate or no more than 12 disks on a 150-mm agar plate |
| · No inhibition zone possessing a fully round shape | · Do not place disks on the edge of the agar plate | ||
| · Irregular inhibition zone shape | · Each disk must be pushed down using forceps to confirm complete contact with the agar surface | ||
| Disappearance of microorganism | · No growth of the test microorganism | · Check the optimal incubation conditions (e.g., temperature, time) and dilute appropriately to the optimal microorganism concentration | |
| MIC assay | Failure in determining MIC | Simply ensuring that the appropriate antibiotic has been selected | Ensure that the appropriate dose according to sensitivity testing is used |
| Inappropriate MIC | MICs are lower or higher than expected | Repeat testing using McFarland 0.5 turbidity or values at 600 nm. Check inoculation procedure and inoculum preparation steps |
MIC, minimum inhibitory concentration.