Contamination can come from:

• Thawing process
• Pipettes/tips
• Media or reagents
• Cryovial surface
• Your hands/gloves
• Specific well position on the 6-well plate

Best Practices and Tips

A. Cryovial Thawing

• Spray liberally with 70–75% ethanol and wipe thoroughly, especially around the cap threads.
• Avoid placing the cap face-down on the hood surface.
• Consider using pre-warmed media with antibiotics (at least initially).

B. Sterile Technique Review

• Use filtered tips and ensure your pipette is not touching the inner rim of wells.
• Change gloves if touching non-sterile surfaces (e.g. fridge, incubator).
• Limit talking or movement while working under the hood.

C. Alcohol Lamp?

• Yes, alcohol flaming can be useful for sterilizing metal tools (forceps, glass pipettes), but not necessary for routine pipetting with plastic consumables.
• Make sure hood airflow is not disrupted by flames—some BSCs (biosafety cabinets) advise against open flame use.

D. Plate Layout Consideration

• Does contamination always happen in the same well (e.g., corner or edge)?
  • Sometimes, airflow in the incubator or uneven plate handling leads to local contamination.
  • You can try rotating well usage to see if the contamination follows the location.

E. Culture Media & Reagents

• Ensure your media and reagents are sterile, not expired, and not cross-contaminated.
• Filter sterilize any home-made media with a 0.22 µm filter.

F. Surface Sanitation

• Clean your biosafety cabinet before and after use with 70% ethanol and optionally a sporicide weekly (e.g., Virkon or bleach).
• Allow 5–10 minutes for hood airflow to stabilize before use.

Other Ideas

• Consider adding a low concentration of antibiotic-antimycotic (e.g., Pen/Strep) during thaw and early seeding.
• Use Mycoplasma detection kits monthly, even if no visible signs appear.
• If using shared incubators or hoods, mark your items and monitor other users’ habits.

Summary Recommendations:

Before Starting (Preparation)

Turn on the biosafety cabinet and wait 10 minutes for airflow to stabilize.

Clean the working surface with 70% ethanol.

Only bring necessary materialsinto the hood.

Wear fresh gloves, a mask, and sleeve covers if available.

Thawing the Cryovial

Remove the cryovial from liquid nitrogen or the freezer.

Disinfect thoroughly with 70–75% ethanol, including the cap.

Quickly thaw in a 37°C water bath(2–3 minutes max).

Wipe the cryovial again with ethanol before opening it inside the hood.

Never place the cap facing down on the hood surface.

Handling Inside the Hood

Open consumables (tips, media) without touching sterile areas.

Use sterile filtered pipette tips.

Change tips for each new step.

Avoid reaching over sterile items.

Do not talk while working under the hood.

Seeding Into 6-Well Plate

Ensure the plate is new and sterile.

Gently distribute cells into wells evenly(avoid air bubbles).

If possible, rotate well positionsto rule out location-specific contamination.

Use medium containing antibiotics at least for the first day.

Check visually for signs of contamination (e.g., cloudiness, filaments, floating particles).

After the Procedure

Close all tubes and containers properly.

Dispose of tips and waste in proper bins.

Wipe down the hood with ethanol.

Record procedures and observations in the lab notebook.

Ongoing Monitoring

Check cultures daily under the microscope (morphology and contamination).

Perform Mycoplasma tests at least once per month.

Label all flasks/plates with date and name.

Definitely No; Becoz the alcohol lamp may have the chances to melt/ destroy the cryocans,as these are made by PP and also some times inactivates/kills live cells ,so instead It is better to Do all the culture procedure under controlled aseptic conditions using 70% or 75% ethanol for making aseptic sterilization in Biosafety cabinet.

Alcohol does NOT sterilize.