How to Do a Proper Cell Culture Quick Check?

Performing a proper cell culture quick check is essential for maintaining the health and consistency of your cell lines.

 

Performing a proper cell culture quick check is essential for maintaining the health and consistency of your cell lines. This process helps to identify contamination, assess cell morphology, and determine cell density. Here is a step-by-step guide to conducting a thorough cell culture quick check:

 

1. Preparation

 

Materials Needed:

 

Cell culture flask or dish

Inverted microscope

Hemocytometer (optional for cell counting)

Trypan blue or other viability stain (optional)

Personal protective equipment (PPE) such as gloves and lab coat

Sterile pipettes and tips

70% ethanol or another suitable disinfectant

 

Sterilization:

Ensure all equipment and workspace are sterilized to prevent contamination.

Wipe down work surfaces with 70% ethanol.

Use sterile pipettes and tips to handle cultures.

Cell Culture Flask

2. Visual Inspection

 

Check Flask or Dish:

Inspect the culture flask or dish for any visible contamination such as turbidity, unusual colors, or fungal mycelium.

Check the color of the medium; a significant color change can indicate pH changes, often due to contamination.

 

Use an Inverted Microscope:

Observe the cells under an inverted microscope. Ensure the microscope is properly calibrated and the lenses are clean.

Look for signs of bacterial or fungal contamination, such as small, fast-moving particles or filamentous structures.

 

3. Assessing Cell Morphology

 

Cell Health:

Evaluate the morphology of the cells. Healthy cells should have a consistent shape typical of their type (e.g., fibroblasts should be spindle-shaped, epithelial cells should be cobblestone-like).

 

Look for any abnormal changes in morphology, such as rounding up of cells, granularity in the cytoplasm, or vacuolization, which can indicate stress or contamination.

 

Confluence:

Estimate the confluence of the culture (the percentage of the surface area covered by cells). This can inform you about the growth stage and whether the culture needs passaging.

 

For adherent cells, a confluence of 80-90% typically indicates it's time to passage the cells to avoid overgrowth and maintain optimal conditions.

 

Cell Culture Dish

 

4. Cell Counting (Optional)

 

Preparing Cell Suspension:

If cell counting is needed, prepare a single-cell suspension. For adherent cells, detach them using trypsin or another dissociation reagent, then neutralize the trypsin with complete medium.

 

Gently pipette the cell suspension to ensure a homogenous mixture.

 

Using a Hemocytometer:

Mix an aliquot of the cell suspension with trypan blue or another viability dye to distinguish live cells from dead ones.

Load the mixture onto a hemocytometer and count the cells under a microscope. Calculate the cell concentration and viability.

 

5. Checking for Contamination

 

Bacterial or Fungal Contamination:

Besides visual inspection under the microscope, look for sudden pH shifts in the medium, which might indicate contamination.

Perform regular mycoplasma tests using specific kits or PCR methods, as mycoplasma contamination is common and not visible under a microscope.

 

Serological Pipette

6. Record Keeping

 

Document Observations:

Keep detailed records of your observations, including cell morphology, confluence, and any signs of contamination.

Note the passage number and date of observation to track the cell line's history and performance over time.

 

7. Maintenance and Troubleshooting

 

Addressing Issues:

If contamination is detected, discard the contaminated cultures and disinfect the incubator and workspace thoroughly.

If cells appear unhealthy, review your protocols, including medium components, incubation conditions, and handling procedures, to identify and correct potential issues.

 

8. Routine Monitoring

 

Regular Checks:

Perform these checks routinely (e.g., every 2-3 days) to ensure early detection of problems.

Consistent monitoring helps maintain cell health and integrity, leading to reliable experimental results.

 

Summary

 

Conducting a proper cell culture quick check involves careful preparation, visual inspection, assessment of cell morphology, optional cell counting, checking for contamination, diligent record-keeping, and troubleshooting. Routine and meticulous checks are critical to ensuring the health and reliability of cell cultures. By following these steps, researchers can maintain robust and contamination-free cell cultures, supporting successful experiments and reproducible results.

 

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