Heredity Biosciences, Bhubaneswar
1. How do I select the right culture medium?
Use the medium recommended by the cell line supplier or standard references such as ATCC. Do not change media suddenly, as it may affect morphology, growth, and cell function.
2. What should I do if contamination appears?
Discard contaminated cultures immediately. Clean the biosafety cabinet, incubator, and work surfaces with disinfectant. Check media, serum, PBS, and trypsin by sterility testing.
3. Why does medium turn yellow?
Yellow medium usually indicates acidic pH due to overgrown cells, high metabolism, or contamination. Observe cells and subculture or discard if contamination is suspected.
4. Why does medium turn purple?
Purple medium indicates alkaline pH, usually due to CO₂ loss or loose bottle handling. Equilibrate medium in a CO₂ incubator before use.
5. When should I change the medium?
Usually every 2–3 days, depending on cell growth, colour change, and cell density. Fast-growing cells need more frequent medium changes.
6. How do I know when to subculture cells?
Subculture adherent cells when they reach around 70–90% confluency, depending on the cell type. Avoid over-confluency.
7. How long should trypsin digestion continue?
Stop trypsinization when cells become rounded and start detaching. Over-digestion damages cells, while under-digestion reduces recovery.
8. Why are cells not attaching after thawing?
Possible causes include poor freezing conditions, slow thawing, old cells, DMSO toxicity, or unsuitable medium. Follow “slow freezing and rapid thawing.”
9. Why do cells die after initial attachment?
This may occur due to harsh trypsinization, poor serum quality, contamination, incorrect medium, or stress during thawing.
10. Should DMSO be removed after thawing?
Yes, for most sensitive cells. After thawing, dilute cells in complete medium and replace medium after 12–24 hours.
11. Why is slow freezing important?
Slow freezing prevents large ice crystal formation inside cells and improves post-thaw viability.
12. Why is rapid thawing important?
Rapid thawing reduces ice crystal damage and limits DMSO exposure time.
13. What is the standard freezing medium?
Commonly used freezing medium is 70% complete medium, 20% FBS, and 10% DMSO. Some sensitive cells may require optimized freezing medium.
14. Can cells be stored long-term?
Yes, cells should be stored in liquid nitrogen for long-term preservation. Avoid long storage at −80°C.
15. How can I detect contamination?
Bacterial contamination causes turbidity. Fungal contamination shows filaments or floating particles. Mycoplasma usually requires PCR or staining-based detection.
16. Can mycoplasma be seen under a microscope?
Usually no. Mycoplasma contamination is difficult to identify visually and requires specific testing.
17. Should antibiotics be added routinely?
Routine antibiotic use is not recommended. It may hide poor aseptic technique and does not prevent fungal or mycoplasma contamination.
18. What is the role of serum?
Serum provides growth factors, hormones, proteins, attachment factors, and nutrients required for cell growth.
19. Can serum batches be changed?
Avoid frequent serum batch changes. Different batches may affect cell growth, morphology, and experimental results.
20. Why does serum show precipitates?
Serum precipitates may be fibrin, calcium phosphate, lipids, or proteins. Usually they do not affect cell growth. Gentle thawing helps reduce precipitation.
21. Is heat inactivation of serum always required?
No. It is required only for specific immunological or sensitive experiments. Unnecessary heat inactivation may reduce serum quality.
22. What is the role of L-glutamine?
L-glutamine supports energy metabolism, protein synthesis, and nucleic acid synthesis in growing cells.
23. What is the role of phenol red?
Phenol red acts as a pH indicator. Yellow means acidic, red/orange means normal, and purple means alkaline.
24. Why is CO₂ required in the incubator?
CO₂ maintains medium pH through the sodium bicarbonate buffering system. Most mammalian cultures use 5% CO₂.
25. When should 5% or 10% CO₂ be used?
CO₂ level depends on bicarbonate concentration in the medium. Most standard media use 5% CO₂, while some high-bicarbonate media may require 10%.
26. What is the role of EDTA in trypsin?
EDTA helps detach cells by chelating calcium and magnesium ions, improving trypsin activity.
27. How should cells be centrifuged?
Use gentle centrifugation, commonly around 1000 rpm for 5 minutes, depending on cell type and protocol.
28. How do I distinguish live and dead cells?
Live cells appear clear and bright. Dead cells appear dark, floating, or granular. Trypan blue staining confirms viability.
29. How is cell viability checked?
Mix cell suspension with trypan blue and count using a hemocytometer. Live cells remain unstained, while dead cells appear blue.
30. Why do cells grow unevenly?
Uneven growth may occur due to poor mixing after seeding, movement before attachment, uneven incubator shelves, or low medium volume.
31. What is the difference between adherent and suspension cells?
Adherent cells grow attached to a surface, while suspension cells grow floating in the medium.
32. What is primary culture?
Primary culture means cells freshly isolated from tissue and grown in vitro before long-term adaptation.
33. What is a cell line?
A cell line is obtained after subculturing primary cells. It may be finite or continuous depending on lifespan.
34. What is the difference between finite and continuous cell lines?
Finite cell lines divide for limited passages, while continuous cell lines can proliferate indefinitely.
35. What is the difference between 2D and 3D culture?
2D culture grows cells on flat surfaces. 3D culture provides a tissue-like environment and better mimics in vivo conditions.
36. How should new cells be handled after arrival?
Check packaging, leakage, medium colour, cell morphology, and density. Disinfect the vessel and allow cells to recover in the incubator.
37. How should cryopreserved cells be handled after arrival?
Check dry ice and vial integrity. Store immediately in liquid nitrogen or thaw as per protocol.
38. Can culture medium be changed if stock is unavailable?
Avoid sudden medium change. Use the recommended medium or gradually adapt cells if change is necessary.
39. How long can complete medium be stored?
Complete medium with serum and supplements is usually stored at 2–8°C and used within 2–4 weeks.
40. What records should be maintained?
Maintain records for media preparation, cell passage, cell thawing, freezing, viability, contamination, equipment monitoring, and waste disposal.
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