Cell Culture Techniques Quiz

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the typical seeding concentration for most continuous cell lines during subculture?

1 × 10^4 to 5 × 10^4 cells/mL (correct)

1 × 10^6 cells/mL

1 × 10^3 cells/mL

1 × 10^5 cells/mL

What happens if morphological deterioration is observed in cells?

Cells will enter apoptosis if not addressed. (correct)

Cells will improve their growth rate.

Cells will multiply exponentially.

Cells will survive indefinitely.

During which phase do cells grow exponentially after subculture?

Death phase

Lag phase

Stationary phase

Log phase (correct)

Which type of cells typically requires a seeding concentration of around 1 × 10^5 cells/mL?

Fragile cultures like endothelium Signup and view all the answers

What is the recommended medium volume-to-surface area ratio for cell subculturing?

0.2-0.5 ml/cm² Signup and view all the answers

Which condition may become a limiting factor in deep medium exceeding 5mm in depth?

Gaseous diffusion Signup and view all the answers

What should be done after centrifuging the cell suspension?

Remove supernatant and resuspend in complete growth medium Signup and view all the answers

Which step is performed first in the general protocol for subculturing cells?

Prewarm materials at 37° on waterbath Signup and view all the answers

Which of the following is a common source of contamination in cell cultures?

Improper aseptic techniques Signup and view all the answers

What is the purpose of using a hemocytometer?

To count cell density Signup and view all the answers

How long should cells be incubated with trypsin for detachment?

2-3 minutes Signup and view all the answers

What preventive measure can help mitigate risks from airborne contaminants?

Regular air quality monitoring Signup and view all the answers

What should be implemented to prevent cross-contamination in the lab?

Designating specific areas for different tasks Signup and view all the answers

What issue can arise from inadequate maintenance of laminar flow hoods?

Compromised airflow and filtration Signup and view all the answers

What is a crucial practice for maintaining aseptic techniques during cell handling?

Wearing appropriate personal protective equipment Signup and view all the answers

What should be done to mitigate contamination risk when moving personnel between different work areas?

Implement proper protocols for equipment movement Signup and view all the answers

What is the primary purpose of cryopreservation in cell line maintenance?

To allow for long-term storage of cell lines Signup and view all the answers

What is the main purpose of subculturing animal cells?

To facilitate cell growth and prevent overcrowding Signup and view all the answers

Which of the following techniques is crucial for controlling sterility in cell line maintenance?

Working in a laminar flow hood Signup and view all the answers

What is the primary consequence of not changing the culture media regularly?

Accumulation of waste products and cell death Signup and view all the answers

What is a critical factor in determining the appropriate seeding density for subculturing?

Cell growth rates Signup and view all the answers

How does cell counting contribute to routine maintenance of a cell line?

It helps assess cell density and viability Signup and view all the answers

What factor should influence the frequency of subculturing animal cells?

The growth characteristics of specific cell types Signup and view all the answers

Why is regular record-keeping important in cell line maintenance?

To maintain consistency in experimental protocols Signup and view all the answers

What happens during the subculturing process of suspension cells?

Cells are diluted to maintain optimal density Signup and view all the answers

What could indicate the need for medium replacement in cell culture?

A pH drop below 6.5 Signup and view all the answers

Which of the following correctly describes how subculture prevents overgrowth?

It transfers cells to new vessels, preventing nutrient depletion Signup and view all the answers

Which method can be used to confirm the identity of a cell line and prevent cross-contamination?

Short tandem repeat (STR) profiling Signup and view all the answers

What does regular mycoplasma testing help to ensure in cell line maintenance?

Reliability and reproducibility of results Signup and view all the answers

During which condition is subculturing usually performed?

When cells have reached a desired confluency or density Signup and view all the answers

What is one major benefit of performing subculturing regularly?

It allows for continuous production of cells for experiments Signup and view all the answers

Which of the following statements is true regarding thawing cryopreserved cells?

Careful thawing is necessary for high post-thaw viability Signup and view all the answers

Which of the following observable signs may indicate contamination in cultured cells?

Changes in cell morphology Signup and view all the answers

What could the presence of floating debris in a culture suggest?

Microbial contamination Signup and view all the answers

Which phenomenon is characterized by cells clustering together in culture?

Cell clumping due to contaminants Signup and view all the answers

What does cloudiness in culture medium typically indicate?

Microbial contamination Signup and view all the answers

How can cross contamination affect experimental reliability?

Compromises the integrity of results Signup and view all the answers

What is a consequence of misidentification of cell lines due to cross contamination?

Unreliable experimental results Signup and view all the answers

Which of the following best describes a common cause of cross contamination?

Contactor surfaces and equipment Signup and view all the answers

What is a significant effect of slow or altered growth in cell cultures?

Indicates possible contamination Signup and view all the answers

What is a key method for preventing temperature fluctuations in cold storage units?

Regular monitoring and maintenance of cold storage units Signup and view all the answers

What is one way to prevent cross-contamination in cold storage?

Labeling and segregating stored materials Signup and view all the answers

Which of the following is crucial for ensuring the quality of sterile materials?

Quality control of purchased sterile materials Signup and view all the answers

How can mishandling of sterile materials be prevented?

Proper training of personnel in handling and storage Signup and view all the answers

What is a recommended practice to prevent contamination in cell lines?

Regular authentication of cell lines Signup and view all the answers

What type of contamination can mycoplasma cause in cell culture?

A significant concern as these small bacteria lack a cell wall Signup and view all the answers

Which method is effective for detecting fungal contamination?

Visual inspection and microscopic examination Signup and view all the answers

What is a risk associated with viral contamination in cell cultures?

It can lead to misidentification of cell lines Signup and view all the answers

Study Notes

Culturing and Subculturing of Animal Cells

Subculture is the process of transferring a portion of an existing cell culture to a new vessel to promote growth.
It involves removing cells from the original vessel and transferring them to fresh growth medium or a new substrate.
Subculturing is typically performed when cells have occupied most of the available space.

Importance of Subculture

Preventing overcrowding and overgrowth of cells
Removing accumulated waste products from dead cells
Providing fresh nutrients and growth factors to support continued cell growth
Enabling cell propagation for experimental purposes, creating larger populations with sufficient numbers for downstream assays or applications.

Factors Influencing Subculturing Frequency

Growth characteristics of specific cell types
Required cell density/confluency
For suspension cultures, dilution of cell culture to maintain optimal density is often required.

Routine Maintenance of Cell Lines

Media Changes

Essential to provide fresh nutrients and growth factors
Maintain optimal pH and osmolality
The frequency depends on the cell type and growth rate
Prolonged exposure to low-nutrient or acidic conditions should be avoided
Some cells require media changes every 3-7 days depending on proliferating rate.

Subculturing

The process of transferring a portion of cell culture into a new vessel.
The process promotes cell growth and prevents overcrowding.
Involves detaching adherent cells or diluting suspension cells in fresh growth medium.
Performed when cells reach desired confluency or density.

Cell Counting

For assessing cell density and viability
Methods include manual counting (e.g., hemocytometer) or automated cell counters.
Helps determine appropriate seeding density for subculturing
Monitors cell growth rates and population doubling times.

Sterility and Contamination Control

Maintaining aseptic conditions is crucial to prevent contamination.
Involves working in a laminar flow hood, employing sterile techniques, and routinely testing for contamination
Examples of contamination include bacteria, fungi, and mycoplasma.
Sterility checks may use culture media or specific tests like polymerase chain reaction (PCR).

Cryopreservation and Thawing

Cryopreservation allows long-term storage of cell lines.
Freezing cells in liquid nitrogen or ultra-low-temperature freezers.
Regular cryopreservation helps maintain cell line stability and genetic integrity.
Thawing cryopreserved cells should be done carefully for optimal post-thaw viability and recovery.

Record-Keeping

Maintaining accurate records of cell line activities (passage numbers, media, subcultures, dates, cell counts)
Good record keeping helps with traceability, troubleshooting.
Ensures consistency in experimental protocols.

Authentication and Quality Control

Ensuring cell identity and preventing cross-contamination.
Methods include short tandem repeat (STR) profiling, isoenzyme analysis and DNA sequencing.
Implementing quality control measures, such as, mycoplasma testing, and regularly monitoring cell line behavior.
This aids in the reliability and reproducibility of experimental results.

Replacement of Medium

Required when pH drops below a certain level (7.0 to 6.5), cell viability will be lost.
High cell concentration necessitates changes for nutrient exhaustion
Some cell types consume medium faster than others.
Morphological deterioration or apoptosis may necessitate changing the medium.

Normal Growth Rate of Cells After Subculture

After seeding, there is a lag phase followed by an exponential growth phase
Feeding (medium change) is usually performed during exponential growth.

Cell Concentration at Subculture

Most continuous cell lines are subcultured at seeding concentrations between 1 x 10⁴ to 5 x 10⁴ cells/mL
Finite fibroblast cells are subcultured at similar concentrations
Fragile cultures (endothelium, early passage epithelial) are subcultured at around 1 x 10⁵ cells/ml.

Volume, Depth, and Surface Area

Usual ratio of medium volume to surface area is 0.2-0.5 mL/cm².
Oxygen requirement of cells affects the optimum ratio
Cells with high oxygen requirements perform better in shallow media (2mm), while celles with low oxygen requirements perform better in deep media (5mm).
Depth exceeding 5mm can affect gaseous diffusion and might limit growth.

Medium volume for subculturing

Appropriate medium volumes for various flask sizes are listed, correlating area size to volume e.g. 25cm² flask = 5-10 mL; 75 cm² flask = 10-30mL

General Protocol for Subculturing Monolayer of Cells

Detailed steps, from pre-warmed materials to the final dilution of the remaining solution to seed new flasks.

How to Subculture (Passage) Primary Cells?

Detailed Video Protocol (link provided)

Contamination in Cell Culture

Source of Contamination (General)

Operator techniques that include improper aseptic techniques.
Environmental factors such as airborne contaminants (bacteria, fungi, or viruses).
Cross-contamination, through movement of personnel between different work areas without proper decontamination.
Issues with maintaining laminar flow hoods.
Cold storage fluctuations.
Quality of sterile materials, contaminated or improperly sterilized materials.
Contaminated cell lines, that can include bacteria, fungi, mycoplasma, or viruses.

Contamination Source Details (Specific)

Operator Techniques: Issues include failure to maintain proper sterile techniques during handling and manipulation. Prevention involves training in aseptic techniques, appropriate personal protective equipment (PPE), and reinforcement of good laboratory practices.
Environment: Issues include airborne particles carrying bacteria, fungi, or viruses settling onto open cultures Prevention includes laminar flow hoods, regular air quality monitoring, and maintaining a clean laboratory environment.
Cross-Contamination: Issues from personnel movement between work areas without proper decontamination. Prevention involves implementing protocols for equipment and personnel movement, and designating specific areas for different tasks.
Use of Laminar Flow: Issues include inadequate maintenance leading to compromised airflow and filtration. Prevention involves regular maintenance and certification of laminar flow hoods, any needed repairs, adhering to recommended workspace capacity, and organizing work areas efficiently.
Cold Stores: Issues include temperature fluctuations affecting stored material integrity and requiring careful material organization to maintain proper airflow. Prevention includes regular monitoring, maintaining cold storage units, and appropriate material storage to prevent cross-contamination, strict labeling, segregation, storage and regular cleaning and maintenance.
Sterile Materials: Issues include use of contaminated or improperly sterilized materials, necessitating quality control of purchased sterile materials, proper autoclaving procedures and adherence to expiration dates, and handling procedures, preventing mishandling of sterile materials during use by training personnel in the correct handling, storage and use of aseptic techniques.
Cell Lines: Issues include introducing contaminated cell lines containing bacteria, fungi, mycoplasma, or viruses. Prevention includes regular authentication of cell lines, quarantine of newly acquired cell lines, adherence to recommended culture conditions, cross-contamination between cell lines leads to misidentification, preventing the issues by regular monitoring, proper labeling, and adherence to cell line authentication protocols.

Types of Contamination

Details provided for bacteria, fungi, mycoplasma, and viruses, including detection methods.

Detection of Microbial Contamination in Animal Cell Culture

Detailed methods for microscopic examination, cultural methods, nucleic acid-based (PCR), and immunological methods (ELISA) for detecting contamination.
Types of contamination in bacterial contamination, mycoplasma, yeast and overall contamination observation.

Cross-Contamination in Cell Culture

Inadvertent transfer of biological material, such as cells or microorganisms, from one culture to another.
Reasons for cross-contamination are through contact with contaminated surfaces, equipment, or through air.
Cross contamination can cause misidentification of cell lines, which is crucial for accurate experimental design.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

Test your knowledge on cell culture practices, including subculturing, seeding concentrations, and contamination control. This quiz covers essential techniques and protocols for maintaining continuous cell lines. Challenge your understanding of the best practices in laboratory settings.