How to clean glassware

LTEE flasks and lids

LTEE glassware—including both flasks and bottles—is kept separate from other lab glassware to help maintain consistency in the experiment. LTEE flasks should always be kept separate from other 50 mL flasks when autoclaving, and they can be distinguished from other 50 mL flasks by their glass lids. Keep the flasks and lids either combined or adjacent to each other to avoid and mixups.

1. Use 100% ethanol and kimwipes to remove population labels from the lids. Note: red sharpie labels (Ara- populations) tend to require more kimwipes to clean than black sharpie.
2. Autoclave trays of flasks containing LTEE cultures on a 121ºC liquid cycle for a 40 minute sterilization time.
3. Dump autoclaved flask contents down the sink.
4. Rinse both the flask and lid each at least 3 times with DI water (NO TAP WATER, NO DISHWASHER)
5. Invert flasks and place in plastic drying racks.
6. Cover the bottom of an autoclave tray with 2 layers of paper towels and place the lids on the paper towels.
7. Put the flask racks and the bin of glass lids into the 37ºC standing incubator to expedite the drying process.
8. When dry, combine lids and flasks in a clean autoclave pan, place a piece of autoclave tape on the corner, and autoclave on a gravity cycle at 121ºC for a 30 min sterilization time and 60 min dry time.

LTEE media bottles

1. Remove any tape labels and ensure the cap is unscrewed.
2. Autoclave the bottle at 121ºC for a 40 minute sterilization time.
3. Dump remaining contents of the bottle into the sink.
4. Rinse the inside of the bottle 3 times with DI water (NO TAP WATER, NO DISHWASHER) and put the bottle cap in a bin with other small items to dishwash.
5. Invert the bottle and place it in a cup cage in the 37ºC standing incubator to dry.
6. When the bottle is dry (usually takes at least overnight), cap it quickly to avoid dust or other particulates from entering the bottle, and set it on the LTEE empty bottles shelf for future use.

Small and medium flasks (50 and 250 ml respectively)

The 50 and 250 ml flasks are used in quantity in many experiments and need to be cleaned and sterilized on a regular basis.

Summary: autoclave (wet) → rinse/wash → autoclave (dry) → autoclave cabinet

Detailed procedure:

1. Remove any labels using 100% ethanol and a kimwipe.
2. Autoclave trays of flasks that were used for growing microbial cultures (including lids) on liquid cycle at 121ºC for a 40 min sterilization time.
3. After autoclaving, remove lids and dump the sterilized contents of the flasks down the drain.
4. Rinse each flask a few times with tap water, and place rinsed flasks in a dishpan. If there is any noticeable debris in the flasks, let them soak full of water and/or scrub them with a brush. Do not use soap, which can inhibit the growth of microbes!
5. Put flasks into the dishwasher and wash using cycle NDB (non-detergent Barrick). This takes about 45-60 minutes. Flask lids, spatulas, culture tube lids, and other small non-glass items can be dishwashed together beside the flasks.
6. After washing and letting them dry, place flasks into a stainless steel tray. Put clean dry lids onto the flasks. Place a new strip of autoclave tape on the corner of the tray.
7. The clean flasks are now ready for sterilization. Autoclave on gravity cycle with a 30 min sterilization time and 60 min dry time.
8. If necessary, place the autoclaved trays of lidded flasks in a standing incubator to dry, or let flasks air dry. Then, place in the black autoclave cabinet.

Large flasks (>250 ml)

Flasks larger than 250 ml are used mainly for making solid media. Since making the media involves autoclaving, these are typically not autoclaved before storage. If you need to grow a culture in a large flask, you are responsible for autoclaving it ahead of time.

Summary: autoclave (wet) → rinse/wash → air dry → glassware shelf

Detailed procedure:

1. Autoclave flasks that were used for growing microbial cultures on liquid cycle at 121ºC for a 40 min sterilization time.
2. Dump the sterilized contents of the flasks down the drain, and rinse each flask a few times with tap water.
3. Put flasks into the dishwasher and wash using cycle NDB. This takes about 45-60 minutes.
4. If necessary, place trays of flasks in a standing incubator to dry, or let them air dry. Then, place on the non-sterile glassware shelf.
5. Flasks can be used directly for making media, since you will be sterilizing the media by autoclaving it. If you are using large flasks to culture microbes, you will need to pre-sterilize your flasks (with a foil cover) on a gravity cycle for a 30 min sterilization time.

Test tubes

Summary (test tube): autoclave → dump → glass waste

Summary (plastic lid): autoclave → dishwash and dry → add to new test tube → autoclave (dry) → sterile cabinet

Detailed procedure:

1. Gather test tubes in a metal rack, place in an autoclave bin, and autoclave at 121ºC on a liquid cycle for a 40 min sterilization time.
2. Ensure the drain of the sink has a strainer in the bottom to catch pipette tips from tubes.
3. Dump contents of test tube (including any pipette tips) and rinse the tube out 2-3 times with tap water. If any residue remains at the bottom of the tube, you will need to clean this using a scrub brush. You can use soap for more stubborn residue since these tubes won't be reused for microbial culture.
4. Once the tubes are clean of residue, dispose of the tube (not including lids) in a glass waste bin.
5. Put the lids together in an autoclave bin to take to the dishwasher. Culture tube lids can be dishwashed with flask lids, media bottle caps, spatulas, and other non-glass small items. The one exception is that the 2-dram glass vials used for SOC can also be put with everything else, as they're small and less brittle than flasks or culture tubes.

Rattler Plating Bead Cleaning Protocol

Note: Contrad 70 is a severe skin irritant and should be handled carefully with proper PPE including gloves, goggles, and a lab coat.

1. Gather used beads in an autoclave bin until the bottom of the bin is covered in a layer no more than ~3 beads deep.
2. Submerge all beads in a mix of DI water and contrad 70 (5% v/v).
3. Stir the beads gently in the solution for 5 minutes, making sure not to cause splashes.
4. Every hour for the next 4 hours (or more if you prefer), stir the beads for 5 more minutes.
5. Let the beads sit in the bin overnight.
6. Stir the beads for 5 minutes every 2 hours for the entire day.
7. Let beads sit in the bin overnight again.
8. Transfer the beads to a strainer or sieve. We have two large flat-bottom sieves that are generally used for this purpose.
9. Ensure the sink you use to rinse the beads has a strainer/stopper at the bottom that won't allow beads to pass through.
10. Rinse beads in the sieve with DI water for at least 10 minutes until all trace of detergent is gone. Continuously stir the beads in the sieve with a gloved hand during this process.
11. Shake beads very well to remove as much residual water as possible.
12. After rinsing, the beads should be allowed to dry in the 37ºC standing incubator in autoclave bins with 2 layers of lab diapers at the bottom.
13. After beads are dry, transfer them into 250 mL bottles and autoclave.