Cell Culture Techniques and Practices Quiz
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What is a key advantage of using Serum Free Media (SFM) in cell culture?

  • Ability to formulate with specific growth factors (correct)
  • Lower cost compared to serum-containing media
  • Higher cell growth rates for all cell types
  • Easier to optimize for any cell type
  • Which of the following is NOT a disadvantage of Serum Free Media?

  • Limited availability for all cell types
  • Higher cost compared to serum-containing media
  • Improved growth rates for all cell types (correct)
  • More difficult to optimize for specific conditions
  • What pH range is optimal for cell culture media to support cell growth?

  • 6.0 - 6.5
  • 7.5 - 8.0
  • 7.0 - 7.5
  • 7.2 - 7.4 (correct)
  • How can changes in pH affect cell culture?

    <p>They can alter enzyme activity and protein conformation</p> Signup and view all the answers

    What is a common challenge associated with optimizing Serum Free Media for specific cell types?

    <p>Extensive testing may be required for optimization</p> Signup and view all the answers

    What is a critical design feature for the flooring in a cell culture room?

    <p>Vinyl/acrylic coating for dustproofing</p> Signup and view all the answers

    Why is it recommended to separate tissue culture labs from preparation and sterilization areas?

    <p>To protect against contamination</p> Signup and view all the answers

    What type of air pressure should a cell culture lab maintain relative to its surrounding areas?

    <p>Positive pressure</p> Signup and view all the answers

    What is one essential service requirement for a cell culture lab?

    <p>Gas piped directly into the facility</p> Signup and view all the answers

    Which material is recommended for the service bench in a cell culture lab?

    <p>Stainless steel for sterility</p> Signup and view all the answers

    What is the purpose of having a buffer zone with a HEPA filter in a cell culture lab?

    <p>To prevent outside air from contaminating the lab</p> Signup and view all the answers

    How should the layout of a small cell culture lab be designed for accessibility?

    <p>With storage accessible from the sterile working area</p> Signup and view all the answers

    What indicates the need for proper drainage in the preparation/washup area of a cell culture lab?

    <p>To prevent water accumulation</p> Signup and view all the answers

    What is the primary feature that distinguishes Class II biosafety cabinets from Class I cabinets?

    <p>They provide protection to the sample, user, and environment.</p> Signup and view all the answers

    Which of the following types of biosafety cabinets are specifically designed for handling highly infectious agents?

    <p>Class III Biosafety Cabinet</p> Signup and view all the answers

    What type of laboratory equipment is commonly used alongside Class II BSCs in cell culture labs for the manipulation of materials?

    <p>Serological pipettes</p> Signup and view all the answers

    Which feature is NOT characteristic of Class III biosafety cabinets?

    <p>They are suitable for low-risk work.</p> Signup and view all the answers

    What is the primary purpose of serum in complete media?

    <p>To promote cell proliferation and attachment</p> Signup and view all the answers

    What is the main purpose of the HEPA filters in Class II biosafety cabinets?

    <p>To filter air and remove contaminants.</p> Signup and view all the answers

    Which of the following is an example of complete media?

    <p>MEM with 10% calf serum</p> Signup and view all the answers

    Why are serum-free media (SFM) preferred in certain applications?

    <p>They provide a more defined and reproducible composition</p> Signup and view all the answers

    For which of the following processes is a pipette controller most suitable?

    <p>Dispensing and withdrawing mediums and reagents.</p> Signup and view all the answers

    What is a potential disadvantage of using serum in cell culture?

    <p>It can introduce variability into experiments</p> Signup and view all the answers

    Which characteristic is true regarding serological pipettes?

    <p>They are made of plastic and are sterilized for one-time use.</p> Signup and view all the answers

    What is the primary function of a CO2 incubator in cell culture?

    <p>To maintain a specific concentration of CO2 for cell growth</p> Signup and view all the answers

    Which of the following components is NOT typically found in complete media?

    <p>Haloalkanes</p> Signup and view all the answers

    What is the role of an inverted microscope in cell culture laboratories?

    <p>To provide routine views and detect cell changes.</p> Signup and view all the answers

    Which of the following is a characteristic of a water bath used in cell culture preparation?

    <p>It warms solutions to a desired temperature before use</p> Signup and view all the answers

    What is one key advantage of serum-free media over serum-containing media?

    <p>Lower risk of contamination</p> Signup and view all the answers

    Which of the following nutrients is primarily sourced from glucose in complete media?

    <p>Energy</p> Signup and view all the answers

    What role does a centrifuge play in the cell culture process?

    <p>It helps in washing and harvesting cells</p> Signup and view all the answers

    What type of media is often used for applications like drug discovery and tissue engineering?

    <p>Serum-free media</p> Signup and view all the answers

    What is the significance of a liquid nitrogen storage Dewar in a culture lab?

    <p>It preserves samples by maintaining ultra-low temperatures</p> Signup and view all the answers

    How does a CO2 incubator contribute to preventing cell death?

    <p>By controlling humidity to keep cells hydrated</p> Signup and view all the answers

    What temperature should CO2 incubators typically maintain for optimal mammalian cell growth?

    <p>37°C</p> Signup and view all the answers

    Why is it important to remove residual media and contaminants after cell culture?

    <p>To minimize the risk of contamination in further experiments</p> Signup and view all the answers

    What temperature does liquid nitrogen maintain for preserving biological samples?

    <p>-196°C</p> Signup and view all the answers

    What is the primary purpose of using liquid nitrogen to store biological samples?

    <p>To keep samples viable and functional for future use</p> Signup and view all the answers

    How does the flash freezing process during cryopreservation benefit biological samples?

    <p>It minimizes ice crystal formation to preserve sample integrity</p> Signup and view all the answers

    Which statement correctly describes T25 and T75 flasks?

    <p>They provide a specific surface area for cell growth</p> Signup and view all the answers

    What function do Falcon tubes serve in a biological lab setting?

    <p>Prepared for cell culture media, centrifugation, and sample storage</p> Signup and view all the answers

    What is the primary feature of multiwell plates in cell culture?

    <p>They allow for simultaneous experiments with multiple cell cultures</p> Signup and view all the answers

    Which of the following describes basal media in cell culture?

    <p>They contain essential nutrients for cell growth and maintenance</p> Signup and view all the answers

    What is a key characteristic of serum-free media (SFM)?

    <p>They do not contain serum, making them suitable for specific cell types</p> Signup and view all the answers

    Which of the following types of media includes essential nutrients for cell growth?

    <p>Basal Media</p> Signup and view all the answers

    Study Notes

    Cell Culture Room Design

    • The layout of the cell culture room depends on the scale of operations and number of users.
    • The room should be designed for easy cleaning.
    • Lab furniture should fit tightly to the floor, with space underneath for cleaning.
    • The floor should be made of vinyl or acrylic for dustproofing.
    • The floor should have a slight slope towards the drain.
    • Tissue culture should be separated from preparation and sterilization areas.
    • There should be no stairs for cart/material movement, with rooms on the same floor.

    Build Requirements

    • The design should consider the number of users.
    • Space availability is important.
    • The location of preparation areas needs to be carefully planned.
    • Adequate storage space is crucial.
    • Accessibility for all users is necessary.
    • Adequate containment and sterility are paramount.

    Small Cell Culture Lab Layout

    • The layout should be self-contained, suitable for 2-3 people.
    • Essential equipment includes a regular incubator, humid CO2 incubator, CO2 supply, refrigerator, freezer, benches, a cell counter, centrifuge, water purifier, prep bench, water bath, a sterilizing oven, and an autoclave.
    • There should be designated soaking and washing sinks.
    • The room should contain an air extract, a laminar flow hood (biological safety cabinet), and a service cart.

    Cell Culture Lab with Adjacent Prep Room

    • Medium-sized cell culture lab should be close to preparation, microscope, and 37°C rooms.
    • The preparation area should be next to the wash up and sterilization area.
    • Storage and incubators should be accessible from the sterile working area.
    • The design should include a bench for a cell counter, microscope, and other instruments
    • Sterile storage for glassware, plastic, pipettes, caps, and syringes should also be present.
    • Additional equipment like small centrifuges can be accommodated.

    Advantages of Separate Adjacent Room

    • Separate rooms protect against contamination, keeping cell culture stocks separate from other lab reagents and equipment. This is a safety precaution.

    Services

    • The cell culture room needs water, electricity, gas (methane/propane/carbon dioxide), and compressed air.
    • The electricity supply needs to be sufficient to power all equipment.
    • Proper drainage is vital, especially in the preparation/washup area.
    • Gas lines, such as for carbon dioxide, are needed.

    Air Pressure Balance

    • Proper air pressure is crucial for the safety of the cell culture lab and the environment.
    • Tissue culture labs need positive pressure relative to surrounding work areas (negative pressure would draw in contaminants).
    • The preparation area should have negative pressure.
    • The microscope room should ideally have positive pressure.
    • Hot room needs negative pressure

    Ventilation

    • The cell culture lab should maintain positive pressure.
    • The design should adapt to negative pressure if containment is necessary.
    • The lab should contain a buffer zone to accommodate filtering air.
    • Laminar flow hoods should continually duct air out.
    • Alternative air ducts can be installed for emergencies.

    Layout

    • The room design should have a sterility gradient.
    • The preparation area is adjacent to wash-up and sterilization areas.
    • Storage and incubators are located in accessible sterile areas

    Service Bench

    • The service bench needs to be positioned and easily accessible in the sterile handling area.
    • The bench should have space for cell counters, microscopes, and other necessary instruments.
    • Sterile supplies (glassware, plastics, pipettes, caps, syringes) need designated storage.
    • Additional equipment like small centrifuges can be incorporated.

    Horizontal Laminar Flow

    • There are two horizontal laminar flow chamber types.
    • Airflow is directed from the side facing the users, and the airflow is not circulated.
    • Aseptic environments are maintained because the airflow is stable.
    • The airflow is directed towards the user, which can be a disadvantage.
    • They are generally cheaper to obtain

    Vertical Laminar Flow

    • Sterile air flows from the top to the bottom (working area).
    • The airflow can be recirculated or vented.
    • The air drawn into the front of the chamber creates a sterile working area without harming the user.
    • It is suitable for hazardous materials like radioactivity, viruses, and animal cells, especially in Class II biosafety cabinets.
    • Some have UV lights for sterilization which can be switched on before commencing work.
    • The exhaust air filters through HEPA filters.

    Biosafety Cabinets

    • Containment devices filter air using HEPA filters and protect users, environments, and products from biological hazards.
    • Biosafety cabinets create an enclosed space for sterile manipulation.
    • A stable airflow prevents contamination.
    • HEPA filtration ensures sterile air.

    Type & Class of Biosafety Cabinets

    • Class I, II, and III biosafety cabinets are crucial for cell culture.

    Class I Biosafety Cabinet

    • Class I cabinets provide basic user and environmental protection.
    • They have a HEPA filter and blower to create negative pressure to prevent contaminants from escaping.
    • Suitable for low-to-moderate risk tasks (aerosols, powders, and liquids).

    Class II Biosafety Cabinet

    • Class II provide increased protection to users, environment, and samples.
    • They have HEPA filters for air filtration and create negative pressure.
    • Suitable for low to high-risk materials, including infectious agents, toxic chemicals, and carcinogens.
    • Three subtypes exist: Type A2, Type B1, and Type B2.

    Class III Biosafety Cabinet

    • Class III cabinets are highly specialized and provide the most protection to users, the environment, and the sample.
    • They are entirely enclosed and have airtight seals.
    • Suitable for high-risk materials, including highly infectious agents and radioactive materials.

    Essential Equipment in Cell Culture Lab

    • Laminar flow hood (BSc Type II) is vital for sterile environments.
    • Pipette controllers are for precise liquid transfers.
    • Inverted microscopes allow for viewing cells in their original vessels.
    • Water baths are used for heat or cool certain solutions.
    • CO2 incubators provide controlled environment.
    • Refrigerators, freezers, and centrifuges are critical.
    • Liquid nitrogen storage dewars are for cryopreservation

    Pipette Controller

    • Pipette controllers are for precise dispensing and withdrawal of medium, reagents, and cells.
    • They can transfer volumes from 1 to 100 mL and can be built-in or separate.

    Serological Pipettes

    • Disposable serological pipettes are sterilized and packed individually.
    • They are made of plastic and range from 1 to 100 mL.

    Inverted Microscope

    • For routine cell cultures, observing and detecting morphological changes and contamination are crucial.
    • Inverted microscopes are excellent for viewing cell cultures in their original containers which are often larger than conventional slides.

    Water Bath

    • Water baths are used for heating or cooling liquids, critical in keeping cell culture solutions at stable low temperatures to prevent bacterial growth.
    • Media and buffers are kept in water baths for stable temperatures before being added to cells.

    CO2 Incubator

    • CO2 incubators provide a controlled environment for cell culture, replicating living conditions.
    • Maintaining stable temperature, humidity, and pH is essential to mimic in vivo conditions.

    Centrifuge

    • Cell washing is done to remove cell culture waste and excess fluids such as media.
    • Centrifugation helps in concentrating cells to make analysis easier.
    • After treatment, cell harvesting uses centrifugation to isolate cells.

    Liquid N2 Storage Dewar

    • Dewars store biological samples like cells and tissues at ultra-low temperatures (-196°C).
    • Essential for preserving sample integrity over time.
    • It is used in cryopreservation to freeze samples rapidly to minimize ice crystal formation

    Consumables

    • T25 and T75 flasks are commonly used for cell growth and maintenance.
    • Falcon tubes are used in cell culture media preparation and storage.
    • Multiwell plates facilitate multiple cell culture experiments and provide easy observation and manipulation.
    • Serological pipettes are for precise liquid transfers throughout cell culture work.

    T25 and T75 Flask

    • T25 and T75 flasks offer convenient, reliable ways to culture cells in a controlled environment.
    • They provide a defined surface area for cell growth, important for proper cell handling.
    • They are made of polystyrene, compatible with various cell types and are reliable in cell culture use.

    Falcon Tubes

    • Falcon tubes serve to prepare and store cell culture media; these can be used to separate cells based on cell density or size during centrifugation.
    • Falcon tubes, and other specialized containers are for temporary storage before further experiments.

    Multiwell Plate

    • Multiwell plates facilitate cell culture and observations under a microscope for a wide range of biological experiments.
    • They are excellent for observing and manipulating cells in different experimental conditions and for drug testing.

    Media Formulations for Cell Culture

    • Cell culture media formulations vary greatly, dependent on the type of cell and experimental process. Types include basal media, complete media, and serum-free media.

    Types of Cell Culture Media

    • The different types of media are basal, complete and serum-free media.

    Basal Media

    • Basal media contains the essential nutrient components for cellular growth and maintenance.
    • Includes amino acids, vitamins, glucose, minerals, and salts.

    Complete Media

    • Complete media contains all the fundamental nutrients and supplemented with serum or other compounds.
    • Includes growth factors, hormones, and additional biomolecules derived from animal blood (serum).

    Complete Media Constituents

    • Essential amino acids (like cysteine, arginine, glutamine, tyrosine).
    • Vitamins (including B vitamins, choline, folic acid, and inositol).
    • Salts, glucose, organic supplements (like proteins, peptides, nucleosides) are important for growth and function. Serum with its growth factors and adhesion factors is also a vital part of complete media.

    Serum Free Media

    • Serum-free media, lacking serum (animal blood derivatives), uses defined components (growth factors, hormones, lipids) instead.

    Advantages of Serum Free Media

    • Serum-free media have well-defined compositions, simplifying standardization.
    • It reduces variability in cell culture experiments associated with serum use.
    • Serum-free media lowers risk of contamination from viruses, bacteria.
    • Serum-free media allows for more specific control of cell growth factors for particular experiments.

    Disadvantages of Serum Free Media

    • Serum-free media is generally more expensive than media containing serum.
    • Some cell types may grow less effectively in serum-free media.
    • Optimizing serum-free media for specific cell types requires extensive testing

    Physicochemical Properties

    • pH in cell culture media must be kept between 7.2 and 7.4 to ensure optimal cell growth, since changes in pH can affect cellular function and overall protein integrity.
    • Phenol indicator dyes, like phenol red, are used to monitor pH.
    • Osmolality of cell culture media is similar to cells, to prevent osmotic stress.
    • Media have buffering capacity (via agents like bicarbonate) to maintain optimal pH, even with changes in CO2 levels.

    Sterility

    • Cell culture media must be sterile to avoid contamination by bacteria, fungi, or other microorganisms to avoid affecting or harming cell culture.
    • Media sterilization techniques are either by filtration or autoclaving. Sterility is crucial.

    Gas Exchange

    • Animal cells need oxygen for respiration and produce carbon dioxide as a byproduct.
    • Cell culture is typically maintained in a CO2 incubator to provide a controlled atmosphere for gas exchange.
    • Maintaining a 5%-10% CO2 concentration in the incubator is crucial for optimal conditions.

    Nutrient Composition

    • Cell culture media need an optimized nutrient composition specific to the cell type and experimental conditions.
    • The concentration and source of each nutrient (amino acids, vitamins, minerals) in the media can significantly impact cell growth, viability, and function.

    Conclusion

    • The composition and physicochemical properties of cell culture media are key to growing and maintaining animal cells in vitro.
    • Adjusting these properties is crucial for optimizing cell viability, growth rate, and function, in addition to the reproducibility and reliability of cell culture experiments.

    Aseptic Technique and Safety

    • Aseptic techniques aim to prevent contamination of cell cultures through meticulous sterilization and handling.
    • Steps for contamination risk mitigation include using sterile equipment, maintaining sterile work areas, and performing procedures under appropriate conditions.
    • Personal protection/safety equipment (such as sterilized gloves, lab coats, face masks, and covered shoes) are essential to maintain a sterile and healthy work environment for cell culture procedures.

    Objectives

    • Minimizing contamination risk (bacteria, mycoplasma, yeast, and fungal spores) is paramount.
    • Employing sterile technique (good techniques, microscope observations) helps reduce contamination risks.
    • Preventing contamination involves using sterile reagents and specialized materials.

    Element of Aseptic Environment

    • Maintaining a clean work environment through laminar airflow is critical.
    • Surfaces are cleaned with 70% alcohol to maintain sterility.
    • Sterile reagents and media are used, and all materials are sterilized before use in biosafety cabinets/laminar flow hoods.
    • Strict personal hygiene practices, hand washing, and sterilized/gloved handling are essential.
    • Outside reagents should have undergone sterile controls to maintain media purity.
    • Cell lines must be quarantined and monitored for contamination before being used in the cell culture media.
    • Sterile handling procedures (swabbing surfaces with 70% alcohol, capping bottles carefully, flaming glassware) are critical to prevent contamination.
    • Pipettes should utilize cotton plugs.

    General Safety & Risk Assessment

    • Cell culture work requires proper training and safety procedures to mitigate exposure to potential hazards.
    • All personnel working with cell cultures should be trained on appropriate safety procedures, especially if new to cell culture.
    • Safe handling and disposal of glassware and sharp objects are vital to avoid accidents.
    • Hazardous materials or specific risks associated should be clearly understood when handling specimens.
    • Proper identification, control, and containment of chemicals/gases are important in cell cultures.

    Chemical Toxicity

    • Appropriate use and handling (e.g., nitrile gloves for DMSO) is crucial in cell cultures, particularly with substances that can transport through skin.
    • Handling gaseous substances (CO2, O2, N2) needs proper containment, including pressurization.
    • Proper ventilation and immediate evacuation procedures are necessary if leaks occur.
    • Appropriate safety equipment (fire blankets, eye protection) is needed.

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    Description

    Test your knowledge on cell culture techniques with this comprehensive quiz. Explore the advantages and challenges of using Serum Free Media, optimal conditions for cell growth, and essential design features of a cell culture lab. Perfect for students and professionals in the field of cell biology!

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