Check the design of the formulation: if the formulation is complete, it can be used directly after the addition of serum; if the formulation is incomplete (e.g., missing glutamine), add the appropriate concentrate.

Operation method

Program 11.1 Preparation and Sterilization of Glassware

Principle

Check the design of the formulation: if the formulation is complete, it can be used directly after the addition of serum; if the formulation is incomplete (e.g., missing glutamine), add the appropriate concentrate.

Materials and Instruments

Culture medium storage solution Glutamine Serum Antibiotics Penicillin Streptomycin
Pipette

Move

1. Check the formulation of the medium and decide what additives, e.g. glutamine, are required.

2. Place the medium in an ultra-clean bench and add other supplements or additives if required.

3 . If the caps are sealed with polyethylene, remove the polyethylene and scrub with 70 % ethanol.

4. Open the bottle.

5. Add the appropriate amount of each additive to the bottle and make the correct dilution; if preparing 100 ml, follow the recipe below:

Glutamine, 200 mmol/L, 1 ml
Antibiotics (if required), 0.5 ml (one of each)
Serum, 10 ml (10%)

(a) Use a different pipette for each additive.

(b) After adding an additive move it to the side of the ultra-clean bench so that you know which additive has been added.

(c) After the medium has been dispensed, remove all additives and supplements from the ultraclean table.

6. This protocol is specifically for MEM prepared in Hank's Salt Solution, which contains a low concentration of sodium bicarbonate (4 mmol/L ). If using Earle's solution MEM or other media with a high carbonate concentration (23 mmol/L), fill with 5% _CO2 gas after preparation. Do not aerate serum-containing media directly, as the media will foam and spill out of the neck of the bottle, increasing the risk of contamination.

7. Close the bottle.

8 . Change label, record additives, date and sign.

9. Store media at 4°C or use directly.

10. If using a new medium for the first time, aspirate some into a culture bottle or dish and incubate under standard conditions for at least 1h (preferably overnight) to confirm that the pH is at the correct level. If not, adjust the pH of the medium and repeat the process, or change the _CO2 concentration.

Caveat

1. Supplements added to the medium (e.g., serum or antibiotics) are not included in the original formulation. This must be outlined in the publication, and other additives added (e.g., glutamine or NaHCO3Other added additives (e.g., glutamine or NaHCO 3) are part of the formula, not supplements. They do not need to be specified in the publication unless there is a change in concentration.

2. Changing the temperature of the incubator CO2concentration also affects the rest of the media in the incubator. Changing the CO2concentration should be considered a one-time adjustment and should not be used to adjust for batch-to-batch variations, which are best adjusted by the addition of a sterile acid or base.

Common Problems

Preparing media with 10X concentrate is a good compromise between the economy and convenience of preparing media with dry powder and using 1X working solution. The design of Option 11.8 can be combined with Exercise 10.

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