Monolayer culture cells changing culture medium experiment
Monolayer culture cells changing culture medium experiment
Observe the culture with the naked eye and inverted microscope, and if indicated, e.g., decreased pH, remove the old medium and add as fresh medium, then place back into the incubator. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition
Operation method
Scheme 13.1 Experiment on changing culture medium of cells in monolayer culture
Principle
Observe the cultures with naked eye and inverted microscope, if there are indications, such as PH decreasing, then remove the old medium and add as fresh medium, and then put it back into the incubator for incubation. Move Materials For more product details, please visit Aladdin Scientific website.
Aseptic
Cultured cells: A549 cells at 2 x 10^4 ml inoculated into 25 cm2 culture flasks 4 days after ......4 flasks
Growth medium ........................... .............................. ...100ml
Example: Eagle 1 x MEM with Hanks salts and 4 mmol/L H2CO3, no antibiotics
If the training program can be picked up, two media prepared with contact 3, 1 bottle kept at 4°C for a week and 1 bottle kept at 37°C for a week
Pipettes, various scales, well cleaned and stuffed with cotton. If they are glass, sort them into square pipette boxes according to size 1ml, 5ml, 10ml, 25ml. If plastic, individually packaged and sorted on a shelf
If a vacuum pump or vacuum line is available, use pipettes that are not cotton plugged to remove culture solution
Non-sterile
Pipette or ear bulb (Figure 5.5, Figure 6.6) 
Tubing connecting the waste bottle to the vacuum line, or the waste bottle to the peristaltic pump (see Figures 5.1 to 5.3) 
Spray cans filled with 70% ethanol
Lint-free swabs or rags
Absorbent paper towels
Straw tubes with water and sanitizer
Ethanol-resistant markers
Notebook, pens, procedures, etc.
Procedures
1. Make sure it is clean and wiped with 70% ethanol and prepare the ultra-clean bench.
2. Reagents and materials needed for the experiment, bottles are wiped with 70% ethanol and immediately placed in the ultra-clean bench (see Scheme 6.1).
3. Carefully observe the cultures for signs of contamination or decline (Figures 13.1 and 19.1).
4. Check the aforementioned criteria for changing the medium - pH and cell density or concentration - and use your knowledge of the behavior of the culture to decide whether to change the medium. Weakly must change the medium, then proceed as follows:
5. place the culture in a sterile work area.
6. Open the lid.
7. take a sterile pipette and insert an ear bulb or pipette, or choose a pipette that is connected to a vacuum or pump that is not plugged with cotton.
8. Suction the old culture medium to the waste tank (Fig. 6.8) or, more preferably, remove the culture medium by pipetting through a pipette connected to the outside pump inside the ultra-clean table (Fig. 5.2, Fig. 5.3). 
9. Discard the pipette.
10. Uncap the culture solution bottle.
11. Remove the new pipette and add the same volume of fresh medium. It is important to preheat the medium to 37°C if there is no need to check cell growth. Cap the bottle.
12. Discard the pipette.
13. Cap the culture bottle and the culture medium.
14. put the culture back into the incubator.
15. complete the observation and fluid change records on the recording sheet or lab notebook.
16. clear away all pipettes, glass supplies, etc. and wipe down work surface.
Tips
When cultures are of low density or slow growth, it is best to perform a half-volume change - i.e., aspirate only half of the old medium in step 8 and make up the same volume of fresh medium in step 11.