Basics of Aseptic Cell Culture Techniques

Overview

This lecture explains basic sterile (aseptic) technique for cell culture to prevent contamination and ensure reliable experimental results.

Importance of Sterile Technique

• Sterile technique prevents contamination of cell lines and primary cells by bacteria, fungi, and mycoplasma.
• Contamination can ruin experiments and make data non-reproducible.
• Microorganisms are present in air and on lab surfaces, so a controlled environment is essential.
• Good technique is more crucial than sterile packaging alone for maintaining sterility.

Biosafety Cabinet (Cell Culture Hood)

• Biosafety cabinet provides a sterile environment using laminar air flow as an air barrier.
• Also called cell culture hood or tissue culture hood in lab practice.
• Turn on cabinet lights and air filter before starting any cell culture work.
• Adjust the glass sash height to the correct marked position for proper operation.
• Run the cabinet filter for at least 15 minutes before beginning work.

Biosafety Cabinet Setup and Use

• Spray 70% ethanol on gloved hands before working inside the cabinet.
• Spray the interior surfaces of the cabinet with 70% ethanol and wipe with lint free wipes or paper towels.
• Avoid lateral arm movements that disrupt the laminar air curtain inside the cabinet.
• Enter and exit the front of the cabinet straight on to maintain airflow pattern.

Personal Protective Equipment (PPE) and Safety

• Before starting work, wash hands thoroughly to reduce microbial load.
• Wear PPE: lab coat, long sleeves, gloves, and safety glasses at all times.
• PPE protects both samples from contamination and you from exposure to materials.
• Follow biosafety guidance from your local lab safety officer for all procedures.
• Comply with local rules on handling, storage, and disposal of biological waste.

Disinfection of Materials and Workspace

• All items entering the cabinet should be disinfected with 70% ethanol and wiped.
• Disinfect packaged consumables, media bottles, reagent bottles, and small tubes thoroughly.
• Pay special attention to disinfecting the caps and necks of bottles and flasks.
• Clean any accidental drops or spills immediately using ethanol-sprayed wipes.

Water Bath Practices

• When pre-warming bottles in a water bath, use bactericidal and fungicidal additives.
• Replace water in the water bath regularly to prevent microbial growth.

Organization Inside the Biosafety Cabinet

• Avoid cluttering the cabinet with too many items at the same time.
• Overcrowding can disturb the air curtain and increase contamination risk.
• Plan each step ahead and only bring in items needed for that step.
• Keep pipettor and pipette tips on one side, adjusted for right or left hand.
• Place other items toward the back, leaving the center as the main work area.

Placement and Handling of Caps

• When removing caps from bottles or tubes, do not place them face up.
• Place caps face down on the cabinet surface to minimize contamination of interiors.
• Optionally hold the cap in the non-dominant hand while pipetting, with practice.

Pipetting Technique and Tip Use

• For serological pipettes, open the end of the wrapper only and attach to pipettor.
• Insert the pipette end firmly into the pipettor before use.
• Rotate the pipette so the volume gradations are clearly visible while pipetting.
• Avoid touching the pipette tip to the exterior surfaces of any container.
• Avoid contact between pipette and interior surfaces of bottles or flasks, especially the neck or cap.

Pipette Tips and Cross-Contamination

• Use each pipette tip only once to prevent cross contamination.
• Discard pipette tips immediately after a single use into appropriate waste.

Handling and Labeling of Vessels

• Replace caps on flasks, bottles, and tubes as quickly as possible after use.
• Make sure caps are properly threaded and tightly closed to maintain sterility.
• Label cell culture vessels, bottles, and tubes as needed before removing from the hood.
• Clear and accurate labeling helps track samples and prevent mix-ups.

Workflow, Efficiency, and Cleanup

• Move through each step deliberately and efficiently to reduce exposure time.
• Allow sufficient time for each step; rushing increases human error risks.
• As with any lab skill, proficiency in aseptic technique comes with practice.
• After removing all items, wipe the interior of the cabinet again with ethanol.

Shared Cell Culture Room Etiquette

• Cell culture rooms are usually shared spaces among multiple researchers.
• Be considerate when scheduling time in the biosafety cabinet and plan ahead.
• Keep the area tidy and organized for the next user.
• Communicate any issues or problems to coworkers to maintain smooth experiments.

Summary Table of Key Sterile Technique Practices

Key Terms & Definitions

• Sterile technique (aseptic technique): Procedures used to keep cell cultures free from microbial contamination.
• Biosafety cabinet (cell culture hood/tissue culture hood): Enclosed workspace providing laminar air flow to protect samples.
• Laminar air flow: Smooth, directed airflow that acts as a barrier against airborne contaminants.
• Mycoplasma: Small bacteria lacking a cell wall that can contaminate and damage cell cultures.

Action Items / Next Steps

• Practice proper arm movement, pipetting, and cap handling techniques regularly.
• Review and follow your institution’s specific biosafety and waste disposal guidelines.
• Plan and organize materials before using the biosafety cabinet to minimize clutter.
• Maintain regular cleaning routines for the biosafety cabinet and water baths.