This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)

Operation method

Scheme 11.5 Preparation and sterilization of ultrapure water (UPW)

Materials and Instruments

Graduated glass borosilicate or sterilized plastic screw-top bottles Screw-top lids Test strips Tape Labels Pure water equipment Sterilizing pots Journals

Move

1. Label the bottle with the date, volume and lot number.

2. the next morning after the overnight cycle, drain approximately 50 ml of water from the water purifier and determine the conductivity (or electrical resistance) and total organic carbon (TOC) levels using the appropriate instruments and record them in your journal.

3 . If the quality of the water meets the limited criteria (resistance ≥ 10 MΩ/cm and TOC ≤ 10 μg/L at 25°C), it is collected directly into the labeled bottle.

4. Fill to a certain volume, e.g. 430-450 ml if used to dilute a 10-fold concentrated medium, leaving 10 % space if it is to be used for sterilization.

5. Place a sterilization indicator in one of the bottles (check it after sterilization and discard the bottle).

6 . Seal the bottle with a screw cap, or loosen the cap if alkaline glass is used, i.e., if there is a possibility that the bottle may break.

7. Arrange the bottles in the sterilizer with the bottle containing the sterilization indicator in the middle.

8. Close the sterilizer and check the settings: 121°C, 100 kPa (15 ^lb/in2 ), postvac deselected for 20 min.

9 . Start the sterilization process.

10. After the cycle is complete, check the printout to verify that the correct sterilization process was performed and record it in this book.

11. Place and cool to below 50°C.

12. Open the sterilizer to remove the bottles. If the caps were loosened during autoclaving, tighten the caps when the bottles are brought to room temperature. Check the sterilization indicator to confirm that the sterilization conditions have been met and record in the journal.

13. Store bottles at room temperature for a short period of time.

14. replenish to the incubation room for back-up as needed, confirming that the bottles have been sterilized by checking the sterilization indicator tape.

Caveat

If the cap is opened during the sterilization process, screwing the cap back on while the bottle is still hot may allow some of the cap lining to fall into the bottle as it cools or the contents shrink. Similarly, when a bottle is opened for the first time, air can enter the bottle very quickly, which can cause contamination. Therefore, bottles that are opened during the sterilization process should be allowed to cool to room temperature in a sterile environment, such as in a sterilizer or in a laminar flow hood before being uncapped.

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