Mammalian Cell Culture Overview
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Questions and Answers

What is the primary characteristic of viruses affecting mammalian cell cultures?

  • They take over the host cell's machinery to reproduce. (correct)
  • They reproduce independently in culture.
  • They always cause visible changes to the host cells.
  • They are larger than bacteria.

Which method is NOT commonly used to detect viral contamination in cell cultures?

  • PCR with appropriate viral primers
  • Electron microscopy
  • Culture on nutrient agar (correct)
  • ELISA assays

What type of effect can some viruses have on infected cells that can be observed under a microscope?

  • Cytopathic effect (correct)
  • Increased motility of cells
  • Color change in the medium
  • Cell growth enhancement

What challenge do viruses pose in cell culture laboratories?

<p>Their small size makes them hard to detect. (A)</p> Signup and view all the answers

What health risk can arise from using virally infected cell cultures?

<p>They present a health hazard to laboratory personnel. (C)</p> Signup and view all the answers

What indicates a positive result for endotoxin in the LAL test?

<p>Formation of a clot (D)</p> Signup and view all the answers

Which of the following can be a source of endotoxin in cell culture?

<p>Plasticware and glassware (D)</p> Signup and view all the answers

What effect can endotoxin have on cell cultures?

<p>It acts as a powerful mitogen (B)</p> Signup and view all the answers

Which type of contamination is particularly problematic due to its small size?

<p>Mycoplasma (A)</p> Signup and view all the answers

What is the key approach to prevent contamination in mammalian cell cultures?

<p>Routine testing for contamination (B)</p> Signup and view all the answers

What indicates that a mammalian cell culture needs replacing or sub-culturing?

<p>Indicators turning yellow (C)</p> Signup and view all the answers

What is the doubling time for mammalian cells?

<p>12-48 hours (B)</p> Signup and view all the answers

Which characteristic is indicative of bacterial contamination in mammalian cell cultures?

<p>Culture looks cloudy (D)</p> Signup and view all the answers

How do yeast appear under a microscope when they contaminate a culture?

<p>As individual ovoid or spherical particles (D)</p> Signup and view all the answers

What happens to the pH of a culture contaminated by mold in initial stages?

<p>Remains stable (A)</p> Signup and view all the answers

What is a common result of heavy yeast contamination in a culture?

<p>Increased turbidity (B)</p> Signup and view all the answers

Under microscopic evaluation, how do bacteria generally appear?

<p>As individual microorganisms (A)</p> Signup and view all the answers

What leads to the yellow color in the culture medium?

<p>Microorganism overgrowth (C)</p> Signup and view all the answers

What is the primary component of endotoxin in Gram-negative bacteria?

<p>Lipid A (C)</p> Signup and view all the answers

Which method is NOT used to detect Mycoplasma contamination in cell cultures?

<p>Limulus amoebocyte lysate (LAL) assay (A)</p> Signup and view all the answers

What effect can endotoxin have on the host organism?

<p>Induce fever (B)</p> Signup and view all the answers

What is a reliable method for avoiding cross-contamination in cell cultures?

<p>Obtaining cell lines from reputable cell banks (B)</p> Signup and view all the answers

What is a key feature of the Limulus amoebocyte lysate (LAL) assay?

<p>It produces a gel clot in the presence of endotoxin. (B)</p> Signup and view all the answers

Which method is NOT used for detecting cell line contamination?

<p>Temperature analysis (D)</p> Signup and view all the answers

How long does the microbiological broth-agar colony assay take before results are available?

<p>25 days (B)</p> Signup and view all the answers

What is the recommended action when faced with microbial contamination?

<p>Discard the culture and its medium (D)</p> Signup and view all the answers

Why is mycoplasma considered a troublesome contaminant in cell cultures?

<p>It is unaffected by many common antibiotics (C)</p> Signup and view all the answers

What major issue does mycoplasma contamination cause in cell cultures?

<p>Induced cellular changes affecting metabolism and growth (D)</p> Signup and view all the answers

What is the primary challenge in detecting mycoplasma contamination?

<p>Its small size and varied shapes (A)</p> Signup and view all the answers

Which of the following statements about viral contamination is true?

<p>Disposal of contaminated cultures is the only option for viral contamination (B)</p> Signup and view all the answers

What is the major reason for avoiding the treatment of cultures with antibiotics during contamination?

<p>It leads to the development of antibiotic-resistant microorganisms (A)</p> Signup and view all the answers

What is the primary purpose of creating an aseptic environment in mammalian cell culture?

<p>To reduce or eliminate contamination (A)</p> Signup and view all the answers

Which of the following is NOT a source of biological contamination in mammalian cell cultures?

<p>Chemical agents (D)</p> Signup and view all the answers

What is a strong indicator of bacterial contamination in mammalian cell cultures?

<p>Sudden change in pH (A)</p> Signup and view all the answers

Which of the following statements about phenol red is true?

<p>It changes color based on the pH of the medium. (A)</p> Signup and view all the answers

Which contaminant is particularly noted as problematic in mammalian cell cultures?

<p>Mycoplasma (D)</p> Signup and view all the answers

What is the core aim of discussing contamination in mammalian cell cultures?

<p>To identify common contamination sources (A)</p> Signup and view all the answers

In what form does contamination in cell cultures occur?

<p>Either biological or chemical sources (A)</p> Signup and view all the answers

Which of the following reflects the appearance of phenol red in media at a pH of 7.4?

<p>Red (D)</p> Signup and view all the answers

What type of contamination could arise from cross-contamination in cell cultures?

<p>Introduction of different cell lines (A)</p> Signup and view all the answers

Which of the following practices is essential to prevent contamination in mammalian cell cultures?

<p>Maintaining an aseptic environment (D)</p> Signup and view all the answers

Flashcards

Viruses

Infectious agents that invade host cells and hijack their machinery to reproduce.

Virus Detection Challenge

The difficulty in detecting viruses in cell cultures due to their extremely small size.

Cytopathic Effect

The visual changes that infected cells exhibit due to viral replication.

Viral Contamination Detection Methods

Techniques used to detect viral contamination in cell cultures, including electron microscopy, ELISA assays, and PCR.

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Viral Contamination Health Hazard

The risk posed by using contaminated cell cultures to laboratory personnel, especially when human or primate cells are involved.

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What are Mycoplasma?

Mycoplasma are tiny bacteria that can contaminate cell cultures and cause significant changes in gene expression profiles.

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How can Mycoplasma be removed?

Mycoplasma can be removed from cell cultures by filtration using a 0.1 µm filter.

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How can Mycoplasma contamination be detected?

Methods like immunoassays, PCR, DNA fluorescence staining, and microbiological broth-agar colony assays can be used to detect Mycoplasma contamination in cell cultures.

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What is endotoxin?

Endotoxin is a component of the outer membrane of Gram-negative bacteria, specifically the lipopolysaccharide (LPS) component.

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How is endotoxin detected?

The Limulus Amebocyte Lysate (LAL) assay uses an extract from the blood cells of the horseshoe crab to detect the presence of endotoxin. If endotoxin is present, a gel clot forms.

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LAL Endotoxin Test

A test that detects the presence of endotoxins in cell culture media or supernatants. It involves incubating the sample with a reagent containing a specific enzyme. If endotoxins are present, the enzyme cascade is triggered, resulting in clot formation.

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Endotoxin

A component of the outer membrane of gram-negative bacteria, which can trigger immune responses in mammals and affect cell growth.

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Mycoplasma Contamination

A type of biological contamination that can be particularly hard to detect due to its small size. It can affect cell growth and metabolism.

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Effects of Endotoxin on Cell Culture

A range of effects that endotoxins can have on cell culture. This includes changes in cell membrane and morphology, stimulation of cell division, and alterations in cell adhesion.

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Routine Testing for Contamination

Routine testing is vital to monitor for any contaminants in cell cultures. This helps ensure the integrity and reliability of cell culture experiments and prevents potential issues.

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What is cross-contamination in cell cultures?

A situation where a cell culture is contaminated with other cell lines.

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Mammalian Cell Culture

The removal of animal cells from their natural environment and their subsequent growth and cultivation in a controlled, artificial environment.

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How can we prevent cross-contamination in cell culture?

Obtaining cells from reputable sources, regularly checking cell characteristics, and maintaining a sterile environment.

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Contamination in Mammalian Cell Culture

The presence of any biological entity other than the desired mammalian cells in a cell culture.

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What is DNA fingerprinting in cell culture?

A technique used to identify different cell lines using DNA patterns, like fingerprints.

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Aseptic Environment

A sterile environment that minimizes the risk of contamination in cell culture.

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What is karyotype analysis in cell culture?

Examining the arrangement of chromosomes in cells to identify specific cell lines.

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What is isotype analysis in cell culture?

Analyzing the types of antibodies produced by a cell line to determine its identity.

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Cross Contamination

A type of contamination in cell culture where a different cell line is introduced, leading to a mix of unwanted cells.

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Mycoplasma

A common contaminant in mammalian cell cultures that can be difficult to detect and eliminate.

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Why are mycoplasmas difficult to detect and treat?

Mycoplasmas lack a cell wall, which is how many antibiotics work. They're too small to easily detect with microscopes.

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Phenol Red

A pH-sensitive indicator dye used in cell culture media, turning yellow in acidic conditions and purple in alkaline conditions.

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How do mycoplasmas harm cell cultures?

Mycoplasmas can affect cell growth and metabolism, potentially leading to cell death.

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Contamination Control

Maintaining a controlled environment to prevent the entry and growth of unwanted organisms in cell culture.

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Cryopreservation

A process that involves freezing cells at very low temperatures for long-term storage.

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Innate Immune Response

The natural defense mechanisms of the body that provide immediate protection against invading pathogens.

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Adaptive Immune Response

The specific immune response that targets specific pathogens and remembers past infections.

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Bioassay

A biological assay that uses living cells to measure the effects of a substance, often used in drug development.

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Turbidity in mammalian cell cultures

Contamination in mammalian cell cultures can be identified by the presence of turbidity, which is a cloudy appearance of the media. This turbidity is caused by the growth of contaminating microorganisms, such as bacteria, yeast, or mold.

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Microscopic evaluation of contaminated cultures

When a culture is contaminated with bacterial species, the presence of individual microorganisms can be observed under a microscope. This helps determine the type of contamination.

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Acidic pH in bacterial contamination

Bacterial contamination in mammalian cell cultures leads to a rapid decrease in pH, resulting in the indicator solution turning yellow. This acidic environment indicates contamination.

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Yeast contamination characteristics

Yeast contamination of mammalian cell cultures is characterized by slow pH changes until the contamination becomes significant. The pH usually increases slightly, and under microscopy, the yeast cells appear as individual ovoid or spherical particles that may bud.

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Mold contamination characteristics

Mold contamination in mammalian cell cultures leads to a rapid increase in pH once the contamination has progressed. Microscopically, the mycelia of mold appear as thin, wisp-like filaments or denser clumps of spores.

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Mammalian cell vs. bacterial cell growth rate

Mammalian cells divide much slower than bacterial cells, making them susceptible to contamination. The rapid growth of bacteria can quickly overtake the culture, leading to changes in pH and media clarity.

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Types of contaminants in mammalian cell cultures

Contamination of mammalian cell cultures can be caused by various microorganisms, including bacteria, yeast, and mold. Each type of contaminant exhibits distinct characteristics that can aid in identification.

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pH change due to mammalian cell waste

Mammalian cell waste products gradually lower the pH of the culture medium, turning the indicator solution orange. This signals the need for media replacement or sub-culturing to maintain a healthy environment for the cells.

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Study Notes

Weekly Schedule

  • Schedule for lectures and labs throughout a semester is provided
  • Dates for lectures related to topics such as mammalian cell use, cell culture lab materials, contamination control, nutrient uptake, cell culture media, data analysis, and various recap sessions are detailed.
  • Dates for reading week and supplementary lectures on growing mammalian cells and cryopreservation are also included
  • Revision sessions are planned for the final week of the semester.

Contamination in Mammalian Cell Cultures

  • Lecture Overview: Introduction discussing the importance of the topic, identifying common sources of contamination in a cell culture laboratory, and conclusion to reinforce key messages.

  • Introduction: Mammalian cell culture involves removing cells from animals and cultivating them in a suitable artificial environment (vitro). An aseptic environment minimizes contamination risks. Contamination can originate from biological or chemical sources.

  • Mammalian Cell Culture Biological Threats: Contamination arises from any biological entity other than the desired mammalian cell. This includes:

    • Other cell lines (cross-contamination)
    • Yeast
    • Mold
    • Viruses
    • Bacteria (Mycoplasma is a particularly problematic bacteria)

  • Characteristics of Bacterial Contamination: pH changes, typically a sudden change, can indicate bacterial contamination. Phenol red in the media shows pH changes as the media changes from yellow to red then purple. Rapidly overgrowing slower-dividing cells can lead to acidification. Mammalian waste products slowly lower pH which indicates the need for replacing or re-culturing the cells. Bacterial contamination is shown with turbidity (cloudy media) and microscopic analysis can identify contaminating microorganisms.

  • Yeast Contamination: Yeast contamination leads to cloudy cultures, especially in advanced stages. pH increases with heavy infestations. Microscopic evaluation identifies yeast as individual ovoid or spherical particles.

  • Mold Contamination: pH remains stable initially but increases as contamination worsens, leading to turbid cultures. Mycelia appear as thin filaments or dense spore clumps.

  • Virus Contamination: Viruses are minute, difficult-to-detect, infectious agents that commandeer host cell mechanisms for reproduction. Viral infections are often difficult to discover and potentially harmful to lab personnel, especially when working with primate/human cells. Viral infections can result in cytopathic effects (morphological alterations of infected cells) viewable via microscopy. Electronic microscopy, along with ELISA and PCR assays with specific primers are crucial for virus detection.

  • Cross-Contamination: Methods to prevent cross-contamination include using reputable cell banks, routinely checking cell characteristics, and maintaining an aseptic environment. Analytical approaches such as DNA fingerprinting, karyotype analysis, and Isotype analysis help identify cross-contamination.

  • Eradication of Contamination: Microbial contamination is typically eradicated by discarding the culture, medium, and related materials, not with antibiotic treatment which can create antibiotic resistant microorganisms. Decontamination is uncommon unless absolutely essential to save the cell strain. Viruses are currently not reliably eradicated and disposal is the method of choice.

  • Mycoplasma: Mycoplasma are small bacteria lacking a cell wall. They pose challenges in research and biopharmaceutical production, show varying shapes, and are resistant to many common antibiotics.

  • Endotoxin: Lipopolysaccharide (Lipid A) found in Gram-negative bacterial walls, endotoxin can cause systemic effects (fever, shock, blood coagulation, etc.) in specific types of host. LAL tests (using horseshoe crab blood) detect it and quantify it in samples. Endotoxin can affect cells with many factors (direct/indirect) and many variable effects.

  • Conclusion: Contamination arises from diverse sources (chemical and biological) with bacterial, fungal, viral, and cellular contamination being common. Mycoplasma are notable because of size and unusual properties. Endotoxins show significant properties, and routine diagnostic testing is essential in cell culture work.

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Lecture 5: Contamination in Mammalian Cell Cultures (PDF)

Description

This quiz covers essential topics related to mammalian cell culture, including contamination sources, aseptic techniques, and nutrient requirements. Participants will learn about the significance of maintaining a contamination-free environment and review critical concepts through a structured weekly schedule. Ideal for students looking to solidify their understanding of cell culture practices.

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