Animal Cell and Tissue Culture - Lecture 4
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Animal Cell and Tissue Culture - Lecture 4

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Questions and Answers

What is the purpose of using a 0.2 µm filter in the process of cell culture?

  • To remove bacteria and fungi larger than 0.2 µm (correct)
  • To eliminate viral particles from the culture
  • To sterilize the cells before culturing
  • To filter out cell culture media additives

    • Which factor is NOT a requirement for successful cell culture?

  • pH and buffering
  • Oxygen levels (correct)
  • Temperature and humidity
  • Culture surface

    • What does granularity around the nucleus indicate in cultured cells?

  • Excess nutrient in culture medium
  • Toxicity or contamination (correct)
  • Normal cell morphology
  • Healthy cell replication

    • Why is it crucial to perform regular media changes in cell culture?

    <p>To avoid metabolic by-products and replenish nutrients</p> Signup and view all the answers

    What size filter must be used to effectively remove viruses from cell culture?

    <p>0.1 µm</p> Signup and view all the answers

    What is the main reason for using aseptic techniques in cell culture?

    <p>To prevent contamination by faster-growing organisms</p> Signup and view all the answers

    Which pH indicator is commonly used in cell culture media to monitor cell health?

    <p>Phenol red</p> Signup and view all the answers

    Which factor is NOT typically included in the requirements for successful cell culture?

    <p>Increased nutrient density</p> Signup and view all the answers

    What color does phenol red turn when the pH falls below 6.8?

    <p>Bright yellow</p> Signup and view all the answers

    What type of contaminants are most commonly encountered in cell culture?

    <p>Bacteria and fungi</p> Signup and view all the answers

    What can cause a change in phenol red's color during cell culture?

    <p>Overgrowth of contaminants</p> Signup and view all the answers

    What is one of the sources of contamination in cell culture?

    <p>Free organisms in the air</p> Signup and view all the answers

    What might be a consequence of contamination in cell culture?

    <p>Overgrowth of contaminants leading to cell death</p> Signup and view all the answers

    What is a common method for detecting mycoplasma contamination in cell cultures?

    <p>Fluorescent DNA staining with Hoechst 33258 dye</p> Signup and view all the answers

    Why are mycoplasma bacteria difficult to detect in cell cultures?

    <p>They lack a cell wall and are very small</p> Signup and view all the answers

    What visual characteristic may indicate fungal contamination in cultures?

    <p>Thin, wisp-like filaments or white fluffy balls</p> Signup and view all the answers

    What is a common media additive used to prevent bacterial contamination?

    <p>Penicillin and streptomycin</p> Signup and view all the answers

    What challenge arises when the contaminant is cryptic?

    <p>They grow too slowly for detection</p> Signup and view all the answers

    Which antibiotics are increasingly used to control mycoplasma contamination?

    <p>Gentamicin and kanamycin</p> Signup and view all the answers

    What is one consequence of using antibiotics in cell cultures?

    <p>They can induce cryptic contaminations</p> Signup and view all the answers

    How may fungal colonies appear under microscopic examination?

    <p>As dense clusters or distinct colors</p> Signup and view all the answers

    What is a characteristic microscopic feature of contaminated cultures viewed under a 100X objective?

    <p>Individual bacteria can be resolved and distinguished</p> Signup and view all the answers

    What indicates a heavy yeast contamination in the medium?

    <p>White powder-like particles floating in the medium</p> Signup and view all the answers

    How do yeast cells typically appear under a 20X microscope?

    <p>As individual ovoid or round particles</p> Signup and view all the answers

    What is a sign of bacterial contamination when observing cultures by eye?

    <p>Cloudy/turbid media with slight film or scum</p> Signup and view all the answers

    Which of the following statements about molds is correct?

    <p>Molds form multicellular filaments called hyphae</p> Signup and view all the answers

    What is a common misconception about the pH change during yeast contamination?

    <p>Yeast contamination results in no change in pH unless it's heavy</p> Signup and view all the answers

    With respect to contamination by bacteria, what does the shimmering effect indicate?

    <p>Moving bacteria in the culture</p> Signup and view all the answers

    Which of the following can be observed in a flask contaminated with yeast?

    <p>Individual ovoid particles budding off</p> Signup and view all the answers

    What is the main risk of using cell lines with very short doubling times?

    <p>They can cross-contaminate other cell lines.</p> Signup and view all the answers

    Which of the following is NOT recommended to prevent cross-contamination?

    <p>Use the same pipettes for different cell lines.</p> Signup and view all the answers

    What is the purpose of a laminar flow hood in cell culture?

    <p>To create a sterile environment free from contaminants.</p> Signup and view all the answers

    Which method is most effective for sterilizing heat-sensitive media?

    <p>Filter sterilization.</p> Signup and view all the answers

    What is a Class II biological safety cabinet primarily used for?

    <p>To minimize exposure to airborne contaminants for the user.</p> Signup and view all the answers

    What is the recommended action before and after using a cell culture hood?

    <p>Perform UV irradiation.</p> Signup and view all the answers

    Why is autoclaving an important step in preparing for cell culture?

    <p>It sterilizes instruments and glassware used in cell cultures.</p> Signup and view all the answers

    What is the purpose of using a high-efficiency particulate air (HEPA) filter in a cell culture hood?

    <p>To trap airborne pollutants including microorganisms.</p> Signup and view all the answers

    Study Notes

    Animal Cell and Tissue Culture - SIO2004 Lecture 4

    • Course: SIO2004 Animal Cell and Tissue Culture
    • Lecture: 4
    • Program: Biotechnology
    • University: University of Malaya
    • Instructor: Dr. Nuradilla Mohamad Fauzi

    Requirements for Successful Cell Culture

    • Culture surface: Essential for cell growth
    • Gas phase: Proper gas composition, crucial for cell function
    • Temperature and humidity: Precise control of temperature and humidity regulate cell growth
    • Media: Contains amino acids, vitamins, salts, energy sources,
    • pH and buffering: Maintenance of a stable pH is vital
    • Osmotic balance: Maintaining correct osmotic pressure for cell health
    • Serum factors: Growth factors, hormones, lipids needed for cellular processes
    • Sterility: Crucial to avoid contamination of cultures

    Sterility

    • Contaminating organisms: Bacteria, yeast, etc., multiply faster than animal cells
    • Contamination consequences: Overwhelms the culture, damages or kills cells
    • Aseptic technique: "Non-dirty techniques" required to avoid contamination
    • Antimicrobials: Additives to media to control contamination
    • Antibiotics: Protect against bacteria
    • Antimycotics/anti-fungals: Protect against fungi
    • Interference: Some antimicrobials may affect certain cell types or experiments

    Contamination

    • Contaminating organisms: Bacteria, fungi (filamentous and yeast), mycoplasma, viruses, other cell types
    • Contamination sources: Air (organisms, dust, aerosols), surfaces/equipment, human operator
    • Contamination implications: Cells' growth & overall health are impacted by uncontrolled contaminants

    Phenol Red: pH Indicator

    • Indicator use: In cell culture media, to indicate the pH
    • Color transition: Gradual shift from yellow to red, then to bright pink/fuchsia (above pH 8.2), and bright yellow (below pH 6.8)
    • Physiological pH: Cell function optimal pH, appears bright red (~7.4)
    • Health indicator: Change in indicator color suggests issues (problems, waste, contaminants)

    Bacterial contamination

    • Characteristics: Unicellular, various shapes (spheres, rods, spirals), micrometers in size
    • Detection: Cloudy/turbid media, film/scum on surface, color change (orange/yellow pH decrease), granular spaces, shimmering under microscopy
    • Resolution: Resolving individual bacteria types (rods vs. cocci) under higher magnification (100x)
    • Association: Some bacteria clump or associate with cultured cells

    Yeast contamination

    • Characteristics: Unicellular microfungi, few to 40 micrometers in size
    • Detection: White powder-like particles floating in the medium. Under microscopy (20X objective), yeast appears as individual ovoid or round particles that may bud off smaller particles.
    • pH impact: Little change in pH unless heavy contamination; pH often decreases with severe contamination
    • Visual cues: Easy to see with naked eye if colony is big enough, white fluffy balls

    Mold (Filamentous Fungi) Contamination

    • Characteristics: Eukaryotic microorganisms, grow as multicellular filaments (hyphae) forming a network (mycelium)
    • Detection: Similar to yeast, the pH of the culture remains stable until initial stages of contamination, then it may rapidly decrease. Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as denser clumps of spores (appearing blue or green). Presence of white fluffy balls - easily detectable with naked eyes.

    Mycoplasma Contamination

    • Characteristics: Genus of bacteria lacking cell walls; unaffected by many common antibiotics
    • Detection difficulties: Extremely tiny size (under a micrometer) makes detection difficult until significant density is reached.
    • Infection symptoms: No visible symptoms until high density leading to cell culture deterioration
    • Detection methods: Fluorescent DNA stains (Hoechst 33258), PCR, and microbiological culture.

    Media Additives to Prevent Contamination

    • Antibiotics: Penicillin and streptomycin combination (PenStrep), effective and generally not toxic
    • Antifungals: Fungizone, nystatin
    • Anti-mycoplasma: Gentamycin, kanamycin (increasingly common use due to increase in human tissues/cells being used in culture)

    Detecting Contamination

    • Rapidly growing organisms: Usually obvious and easily detected
    • Cryptic contamination: Challenging to spot especially if the contaminant is a slow-growing organism, or too small to be seen under the microscope (e.g. mycoplasma)
    • Antibiotic impact: Antibiotic use can mask presence of contamination

    Cross-contamination

    • Cell strain characteristics: Valuable for rapid experiments, but heightened risk of cross-contamination
    • Extensive contamination: Potential cross-contamination of different strains, especially with cell lines like HeLa
    • Prevention: Authenticate cell lines, do not share or reuse related material, do not mislabel.

    Aseptic Technique

    • Controlled environment: Traffic, dedicated room, air flow critical
    • Sterile materials: Cell media, reagents, equipment essential for contamination prevention
    • Solution storage: Proper storage to avoid airborne contamination
    • Equipment handling: Avoid repeated use of equipment (e.g., pipette tips)
    • Sterilization: crucial to prevent contamination

    Hoods for Cell Culture

    • Principle: Creates a controlled environment free of microorganisms and spores
    • Laminar Flow Hood / Biological Safety Cabinet (BSC): Most used, Class II & III for maximum protection to the environment and the worker
    • HEPA filter: Critical to trap pollutants, allergens, and microorganisms

    Sterilization

    • UV Radiation: Useful for cell culture hoods and sometimes rooms
    • Gamma Radiation: Using toxic gas at high pressure
    • Autoclaving: Heating to 121°C for a given time to sterilize instruments and glassware
    • Filtration: Fine sterile filters for heat-sensitive solution components (0.2 µm for bacteria/fungi, 0.1 µm for viruses)

    Autoclave Machines

    • Equipment types: Different designs/models available; varying capacity and features

    Filters

    • Various filter types: Used in sterilization process
    • Applications: Filter sterilization, specifically media and solutions, also for removing particles from liquids

    Media Changes

    • Purpose: Replenishes nutrients, avoid harmful metabolic by-products
    • Adherent cultures: Straightforward aspiration to remove the old media, place fresh medium.
    • Suspension cultures: Centrifugation to separate the cells from the media and resuspend in fresh media

    Check the Morphology of Cells in Culture

    • Regular examination: Crucial for recognizing issues early and for evaluating cell health
    • Degradation signs: (granularity, detachment, cytoplasmic vacuolation) can point to deterioration
    • Reasons for deterioration: Various possibilities (contamination, senescence, toxins in the media)

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    Animal Cell and Tissue Culture Lecture 4 PDF

    Description

    This quiz focuses on the requirements for successful animal cell and tissue culture as discussed in Lecture 4 of the SIO2004 course. Topics include culture surface, gas phase, temperature control, media composition, pH maintenance, osmotic balance, serum factors, and sterility. Test your knowledge on these critical aspects of cell culture.

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