Aseptic Techniques: Cell Culture Basics

Summary

This video script emphasizes the criticality of maintaining sterile environments in cell culture to prevent contamination. It outlines the necessity of personal protective equipment, proper hand hygiene, and cleaning protocols. The script details the correct use of a cell culture hood, including setup, handling of pipettes and media, and the importance of aseptic technique. It also highlights the proper disposal of contaminated items and the importance of cleanliness post-experimentation.

Takeaways

• 🔬 Maintaining a sterile environment is crucial for successful cell culture to prevent microorganism contamination.
• 🧼 Always wear personal protective equipment (PPE) when working in the lab, including closed-toe shoes and clothes covering your legs.
• 👀 Review the Material Safety Data Sheets (MSDS) before handling any media or reagents to ensure safety.
• 🧼 Wash hands thoroughly before handling cells or performing cell culture work to remove bacteria and dead skin particles.
• 🚫 Avoid using alcohol spray in areas with open flames due to fire hazards.
• 🧹 Clean the cell culture hood before and after use with 70% ethanol to maintain a sterile environment.
• 🛡️ Keep the cell culture hood work surface uncluttered to prevent accidental contamination and ensure proper airflow.
• 🔧 Use a pipette aid for proper handling of pipettes to avoid contamination during cell culture procedures.
• 🏺 Only open media, reagents, and supplies within the cell culture hood to maintain sterility.
• 🔄 Practice proper pipetting technique and avoid touching non-sterile surfaces to prevent cross-contamination.
• 🗑️ Dispose of any contaminated pipettes immediately and do not reuse them to maintain a sterile field.

Q & A

Why is it crucial to keep cells free from microorganism contamination during cell culture?

Keeping cells free from microorganism contamination is crucial because contamination can disrupt the cell culture's integrity, leading to inaccurate experimental results or complete loss of the culture.

What is the role of personal protective equipment (PPE) in cell culture?

PPE is essential for protecting both the researcher and the cell culture from contamination. It includes items like lab coats, gloves, and safety goggles, which prevent the introduction of contaminants from the researcher to the cell culture.

Why is it important to review the Material Safety Data Sheet (MSDS) before working with media or reagents?

Reviewing the MSDS is important to understand the potential hazards of the chemicals being used, how to handle them safely, and what precautions to take to minimize risk to both the researcher and the cell culture.

What does it mean to work in a sterile technique?

Working in a sterile technique means performing procedures in a way that minimizes the risk of introducing contaminants into the cell culture. This includes working slowly and deliberately, using sterile equipment, and maintaining a clean work area.

Why should you clean the cell culture hood before and after use?

Cleaning the cell culture hood before and after use helps to prevent the buildup of contaminants that could otherwise be introduced into the culture during the next session.

What is the significance of using 70% ethanol for hand washing in a cell culture lab?

A 70% ethanol wash is effective at killing microorganisms on the skin. It is used to remove bacteria and other contaminants before handling cells or culture materials to prevent their introduction into the culture.

How should you handle items placed in the cell culture hood to maintain sterility?

Items placed in the cell culture hood should be cleaned with ethanol before placement. They should be handled with sterile techniques, avoiding contact with non-sterile surfaces, and placed in the hood with a forward motion to not disrupt the airflow.

Why is it advised not to overcrowd the work surface in a cell culture hood?

Overcrowding the work surface can increase the risk of contamination through accidental touching and disrupt the airflow, which is necessary to maintain a sterile field within the hood.

What is the correct way to open a pipette package without contaminating the pipette?

The correct way is to grasp the pipette high on the neck, insert it into the pipette aid, turn the desired measurement marks toward you, and then discard the wrapper without letting the pipette touch anything non-sterile.

Why is it important to avoid touching the inside edge of the cap when handling media or reagents?

Touching the inside edge of the cap can contaminate it, as this part comes into direct contact with the media or reagents. It's crucial to replace the cap as soon as possible to maintain sterility.

How should you transfer large volumes of liquid between containers in a sterile manner?

The best way to transfer large volumes is to use a sterile pipette. If that's not possible, pour quickly and deliberately using the front corner of the bottle to channel the liquid, ensuring accuracy and minimizing the risk of contamination.

Outlines

00:00

🔬 Sterile Technique for Cell Culture

This paragraph emphasizes the criticality of maintaining a sterile environment in cell culture to prevent contamination by microorganisms. It outlines the necessity of using personal protective equipment (PPE), consulting safety teams, and reviewing Material Safety Data Sheets (MSDS). It advises wearing closed-toe shoes and covering legs, washing hands, and using a 70% ethanol wash. The paragraph also details the importance of cleanliness, including cleaning the cell culture hood and items placed within it. It instructs on proper setup within the hood, avoiding overcrowding, and maintaining a sterile field. The handling of pipettes and bottles of medium is demonstrated, highlighting aseptic techniques such as not touching non-sterile surfaces and discarding contaminated items. The paragraph concludes with advice on transferring liquids and cleaning up after work, ensuring all items are closed and the workspace is sanitized.

05:00

🎵 Music Interlude

This paragraph is a placeholder for a musical interlude, likely used to transition between sections of the video or to provide a brief moment of auditory relief. It does not contain any spoken content but serves as an integral part of the video's pacing and mood.

Keywords

💡Cell Culture

Cell culture refers to the process of growing cells outside their natural environment, typically in a laboratory setting. It is a fundamental technique in biological research, medicine, and biotechnology. In the video, successful cell culture is emphasized as being heavily dependent on maintaining a sterile environment to prevent contamination, which is a critical aspect of the video's theme.

💡Sterile Environment

A sterile environment is one that is free from living organisms such as bacteria, viruses, and fungi. This is crucial in cell culture to ensure that the cells grow without being compromised by contaminants. The video script highlights the importance of maintaining a sterile environment by following strict guidelines and using personal protective equipment (PPE).

💡Personal Protective Equipment (PPE)

PPE includes protective clothing, helmets, goggles, gloves, and other garments or equipment designed to protect the wearer's body from injury or infection. In the context of the video, PPE is essential for anyone working in a laboratory to prevent contamination of the cell cultures and to protect the individual from potential hazards.

💡MSDS Information

MSDS stands for Material Safety Data Sheet, which is a document that provides information on the potential hazards of substances and how to handle, store, and dispose of them safely. The video script mentions reviewing the MSDS before working with any media or reagents, which is a critical step in ensuring safety in the laboratory.

💡Ethanol Wash

An ethanol wash is a common method used to disinfect surfaces by killing microorganisms. In the video, a 70% ethanol wash is recommended for cleaning hands and surfaces to reduce the risk of contaminating cell cultures. This is a practical example of how to maintain sterility in the lab.

💡Sterile Technique

Sterile technique refers to the methods and procedures used to prevent contamination in a laboratory setting. The video script provides detailed instructions on how to perform sterile techniques, such as cleaning the hood, handling pipettes and media bottles, and transferring liquids, all of which are essential for successful cell culture.

💡Cell Culture Hood

A cell culture hood, also known as a laminar flow hood, is a controlled environment where the air is filtered to remove particles and provide a sterile workspace. The video script describes the proper setup and use of a cell culture hood, including not overcrowding the work surface and maintaining the correct airflow.

💡Pipette Aid

A pipette aid is a device that helps in handling pipettes without contaminating them or the surrounding environment. The video script mentions using a pipette aid to control pipetting, which is an example of a tool that facilitates sterile technique and helps in the accurate handling of liquids in cell culture.

💡Media and Reagents

Media and reagents are substances used in scientific experiments, particularly in cell culture, to provide nutrients and other necessary components for cell growth. The video emphasizes the importance of keeping media and reagents sterile by only touching them with sterile objects and opening them within the cell culture hood.

💡Aseptic Technique

Aseptic technique is a set of practices designed to prevent contamination of sterile materials and environments. The video script demonstrates aseptic techniques such as supplementing media with non-essential amino acids, transferring liquids, and handling containers in a way that maintains sterility.

💡Contamination

Contamination in the context of cell culture refers to the unwanted presence of microorganisms or other foreign substances that can compromise the integrity of the experiment. The video script provides various examples of how to avoid contamination, such as proper hand washing, using ethanol to clean surfaces, and handling materials in a sterile field.

Highlights

Importance of maintaining cell cultures free from microorganism contamination.

Achieving sterility through proper knowledge of sterile environments and techniques.

Wearing personal protective equipment (PPE) is mandatory in the laboratory.

Reviewing Material Safety Data Sheets (MSDS) before working with media or reagents.

Washing hands and using 70% ethanol to reduce contamination risks.

Cleaning the cell culture hood before and after use to maintain sterility.

Avoiding the use of spray alcohol in areas with open flames due to fire hazards.

Cleaning each item placed in the hood with ethanol to remove dust and contaminants.

Proper setup of the cell culture hood to prevent overcrowding and maintain airflow.

Keeping the front sash of the cell culture hood in the lower position during work.

Using sterile media, reagents, and supplies to prevent contamination.

Demonstrating proper pipette handling and bottle opening techniques for aseptic supplementation.

Avoiding touching the pipette to non-sterile surfaces to prevent contamination.

Using aseptic technique when transferring liquids between containers.

Wiping spills immediately with ethanol to maintain a clean work area.

Closing all containers tightly and cleaning the work surface after finishing to prevent cross-contamination.

The necessity of following aseptic techniques to ensure the integrity of cell cultures.

Transcripts

successful cell culture depends heavily

on keeping the cells free from

microorganism contamination this can be

accomplished with proper knowledge of

sterile environments working slowly and

deliberately and following all of the

guidelines for sterile technique always

wear basic personal protective equipment

or PPE when working in the laboratory

talk to the safety team at your

Institute for your required PPE also

remember to review the MSDS information

before working with any media or

reagents wear closed toed shoes and

clothes that cover your legs

washing your hands before handling cells

or performing culture work removes

bacteria and microscopic dead skin

particles dead skin cells can be a

potential source of contamination

a 70% ethanol wash kills microorganisms

that could contaminate the cultures

cleanliness is one of the most important

parts of sterile technique always clean

the hood before and after use spray

alcohol should not be used in any area

where a flame is being used due to the

fire hazard the outside of containers

carry dust and contaminants remember to

clean each item placed in the hood with

ethanol you may choose to spray the item

before placing it in the hood or

immediately after as demonstrated as you

clean the item and place it into the

cell culture hood put it in the correct

position enter the hood with your hands

in a forward motion try not to sweep

across the front and disrupt the airflow

proper setup of the cell culture hood

includes not overcrowding the work

surface not only does this increase the

risk of contamination through accidental

touching but it also interrupts the

airflow through the chamber which will

not maintain the sterile field for the

same reason it is important to keep the

front sash in the lower position when

working the basic idea is to keep your

media and reagents sterile by only

touching them with sterile objects using

sterile media reagents and supplies is a

big step in keeping your cultures free

from contamination

notice how the cell culture hood is set

up to make the following actions easier

the pipette aid is on the right side so

you can easily control the pipetting in

your right hand reagents are in the

center back so you can easily open the

bottles and pipette from them it is very

important to have the setup in this

manner so you're not crossing your hands

or supplies over top of sterile items

will now demonstrate the basic technique

of handling a pipette and a bottle of

medium while aseptic Lee supplementing

DMM with non essential amino acids when

opening the pipette package be sure the

pipette does not touch anything non

sterile grasp the pipette high on the

neck insert the pipette into the pipette

aid turn the desired measurement marks

toward you and then discard the wrapper

discard any pipette you accidentally

contaminate only open your media

reagents and supplies in the cell

culture hood opening these items outside

the sterile field will result

contamination when holding the cap it is

important not to touch the inside edge

or you could contaminate it replace the

cap as soon as possible

if you must set the cap down in order to

free your hand set it down with the

interior surface facing down when

pipetting try not to touch the pipette

to anything non sterile particularly the

outside of containers or contamination

could result each pipette should be used

one time and discarded or washed if

using glass pipettes reusing pipettes by

dipping directly into another media

bottle or by leaving it standing in a

media bottle increases your chances of

spreading contamination gently mix the

contents after supplementation labeling

the bottle after supplementation is a

good laboratory practice sometimes

you'll need to transfer large volumes of

liquid from one container to another the

best way to transfer is always to use a

sterile pipette but with practice it is

possible to aseptic leap or instead you

should pour quickly and deliberately

using the front corner of the bottle to

channel the liquid and improve the speed

and accuracy of your pour any spills

should be wiped with ethanol immediately

when passages of aseptic technique are

the same only open the containers in the

sterile field

do not cross your arms or other items

over and open a flask

don't rush but work at a good pace and

with deliberate motions do not reuse

pipettes and do not use items that were

inadvertently contaminated when finished

make sure everything is closed tightly

before removing the cell culture hood

anything opened outside the sterile

field will now be non sterile and should

not be used for cell culture work wipe

down the work surface with ethanol again

and straighten up the hood before you

leave

you

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