Sterilize instrument

1. Fill sheath tank with 70% ethanol, empty the in-line sheath filter of PBS, and pressurize the cytometer.

2. Allow ethanol to fill the sheath lines while bleeding the air from the in-line sheath filter, and establish a sheath stream. Run the ethanol through the cytometer for 1 hr. Proceed with normal shutdown procedures and leave 70% ethanol in the cytometer sheath lines for 24 hr.

3. Remove the ethanol from the sheath tank and in-line sheath filter, and replace with filtered, autoclaved PBS. Pressurize the system and allow the PBS to fill the sheath lines while bleeding the air from the in-line sheath filter. Establish a sheath stream and allow the system to run fluid for 15 min.

4. Place the system in vacuum mode, cut the sheath fluid line near the flow cell, and insert the male and female luer lock fittings. Add 0.5% sodium hypochlorite to the line closest to the flow cell and allow the solution to pass through the sheath line into the flow cell to be removed through the vacuum line. Next, attach the Sterivex GP filter where the sheath line was cut so that the male side of the filter is nearest the flow cell body and represents the outlet side of the filter. Establish a sheath fluid stream.

5. Run sheath fluid through the lines for 1 hr.

6. Sterilize the sample line by running 70% ethanol through the line for 5 min. Back flush sheath fluid through the line to remove the ethanol solution. Sterilize the sample uptake probe with 70% ethanol and allow the probe to air dry prior to putting a sample on the instrument.

For safety reasons, be sure to turn off the voltage to the sorting plates during this step.

Assess instrument sterility

7. Test sterility of the sheath fluid by using the sort test option on the sorter: in the sheath mode, sort 1 to 100,000 test drops increasing in 10-fold increments into 2 ml LB medium (for each sort) in 15-ml conical test tubes.

8. Test sterility of the sample line by back flushing a few drops of sheath fluid through the sample line into 2 ml LB medium in 15-ml conical test tubes.

9. Test the air around the sorter by leaving 2 ml LB medium open in the sort compartment for a period of time similar to that required for the sort process.

10. Incubate all three samples 18 hr at 37°C with shaking. The medium should remain clear.

In situations where contamination appears in the LB medium, the contamination probably resides within the Sterivex filter or after the filter. Replace the filter, sample tubing, and sheath fluid line between the filter and the flow cell body. Soak the flow cell body in a 0.5% sodium hypochlorite for 15 min, then rinse thoroughly in sterile water prior to reassembly onto the sorter. Repeat steps 7 to 10.

See Critical Parameters and Troubleshooting section (below) for discussion of precautions to be taken to prevent contamination.

Sorting of Bacteria

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