Preventing and Handling Black Dots in Cell Culture
News 14 10 月, 2025
When culturing cells, many researchers notice small black dots under the microscope and wonder — are they cell debris or contamination?
How to Identify the Cause
1. Observe movement:
Particles showing slow Brownian motion are usually harmless cell debris, while rapidly moving dots may indicate bacterial contamination.
2. Check the medium:
Contaminated media quickly turn yellow and cloudy due to bacterial growth (within 12–48 hours). Clear, reddish media usually indicate healthy cultures.
3. Test on a plate:
Inoculate suspected material on a microbial plate. Visible colonies = contamination. No colonies = debris.
Causes, Prevention, and Treatment
1. Cell Debris:
Caused by over-digestion (e.g., trypsinization too long) or rough pipetting.
- Prevention: Be gentle; control enzyme digestion time.
- Treatment: Wash with PBS, centrifuge gently, and re-seed in fresh media.
2. Bacterial Contamination:
Leads to cloudy medium and cell death.
- Detection: Inoculate 1 mL of supernatant into LB broth; if it turns cloudy overnight, contamination confirmed.
- Action: Discard contaminated cultures immediately.
3. Fungal Contamination:
Visible as filamentous hyphae or white floating particles.
- Action: Discard affected cultures and disinfect incubator and workspace.
4. Mycoplasma Contamination:
Common but hard to detect; requires PCR or fluorescence methods.
- Treatment: Repeated passaging and medium changes can help clear it in 2–3 weeks.
5. Apoptotic Bodies:
Formed when cells undergo apoptosis due to stress factors like pH shifts, toxins, or overcrowding.
- Prevention: Avoid overgrowth; maintain optimal medium pH and low-endotoxin serum.
6. Serum Precipitates:
Calcium phosphate precipitates can appear as black dots but are harmless.
- Tip: Avoid unnecessary heat inactivation of serum.
Tips to Prevent Contamination
Common contamination points:
- Incomplete disinfection after thawing cells in a water bath.
- Contaminated medium bottles or pipettes.
- Touching sterile areas with gloves or sleeves.
Best sterile practices:
- Disinfect all surfaces with 75% ethanol before and after use.
- Keep workspace clean and uncluttered.
- Prepare all materials in advance to minimize movement.
- Regularly disinfect incubators and water baths.
Medium handling tips:
- Always disinfect bottle surfaces.
- Avoid leaving caps open too long.
- Use fresh pipettes each time and never share reagents between cell lines.
- Observe cultures daily for changes in clarity.
Summary
Cell culture contamination can come from many sources — bacteria, fungi, mycoplasma, or even handling errors. Maintaining strict aseptic technique, regular observation, and careful reagent management are key to keeping your cultures clean and healthy.