1. Transform samples into the liquid phase.
(For liquid samples like water and drinks, can skip this step and perform the filtration directly.) For solid samples like food and tissues, a stomacher is needed. The samples can be placed in a stomacher bag with filters, and fill the bags with 3 times the volume of saline. Make sure the saline can cover the surface of the samples. Run the stomacher at top speed for 5-10 min, depend on the sample type. Take the saline solution for further steps.
(This step is only suitable for samples with a low concentration of bacteria. If the samples already contain a high concentration of bacteria, the sample can be subjected to drop-plating directly.) Set the sterile filtration system. The system can be clean one-time use filter unit, or glass filter holders and containers autoclaved. For samples contain less solid particles, filtered with a 0.22 um filter membrane directly. For samples difficult to filter, filter with 0.45 µm membrane first, and then perform with the 0.22 um membrane again. The membranes were then transferred to a sterile tube containing 5 ml saline (0.85% NaCl) solution, and bacteria were scraped from the membrane with a culture loop and suspended in the saline (0.85% NaCl) solution.
(The bacteria solution can be frozen at -80 for DNA extraction and metagenomic sequencing. The solution can also be mixed with glycerol or DMSO for enrichment and isolation in the future).
3. 3×4 drop-plating with 96-well plate and 8-channel pipette.
(The low concentration sample can load to the 96-well plates directly.)The sample position designed for the 96-well plate is shown as in Fig S1. As detailed, 100 µl of concentrated bacteria saline solution from each sample was loaded in Raw 1. An 8-channels micropipette was used to add 90 µl of saline to the 3rd, 5th, and 7th row, while left the 2nd, 4th,6th row empty. The concentrated bacteria solution was 10-fold serial diluted by taking 10 µl of each sample in Row 1 with multi-channel pipettes and mixed with 90 µl saline solution in Raw 3 homogeneously, and repeatedly diluted with saline solution in Row 5 and Row 7. Then the 8-channel pipette was turned to the horizontal direction to take 10 µl from the original samples and each dilution, drop on a selective agar according to the requirement. The dropping was repeated 3 times on one piece of agar. The plates were dried under air-flow in the biosafety cabinet (BSC) and turned over for culture at a suitable temperature.
4.Isolation, pure culture, and confirmation.
Two to 20 colonies from the highest two dilution drop on selective plates were then picked and grew on nutrition-rich agar to purify. The single colonies can be further purified by re-streaking and subculture. The purified isolates can be stocked or subjected to the confirmation test.
For resistant bacteria, a disc diffusion test or microdilution test (MIC) can be applied to confirm the susceptibility.
The bacteria can also be collected for DNA extraction and whole-genome sequencing.
The bacteria can also be used for PCR tests directly. Briefly, One or two colonies can be picked and resuspend in 20 ul water. Vortex to the top speed and take 1-2 ul as PCR template.
After confirmation, the number of "True" positive bacteria can be recorded and applied for calculation.
Ci is the number colony confirmed as "True" positive. Wi is the volume of the original sample (ml). The highest dilution for the effective count was recorded with dilution factor: Di.
Mean density= (Ci*100*Di)/(Wi/5)
The bacteria concentration is calculated with the unit of log10 CFU/ml or CFU/L.