Podcast
Questions and Answers
What is the primary reason for sanitizing the microbiological safety cabinet before starting work?
What is the primary reason for sanitizing the microbiological safety cabinet before starting work?
What is the role of 70% alcohol in aseptic techniques?
What is the role of 70% alcohol in aseptic techniques?
Which step is NOT recommended when sanitizing gloves before handling cell culture materials?
Which step is NOT recommended when sanitizing gloves before handling cell culture materials?
Why should movements inside and around the microbiological safety cabinet be slow?
Why should movements inside and around the microbiological safety cabinet be slow?
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How should liquid cell culture waste be handled before disposal?
How should liquid cell culture waste be handled before disposal?
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What is the necessity of periodic cleaning or fumigation of the cabinet?
What is the necessity of periodic cleaning or fumigation of the cabinet?
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What should be done if gloves become contaminated during cell culture work?
What should be done if gloves become contaminated during cell culture work?
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Why is trypsin/EDTA used in the subculturing process?
Why is trypsin/EDTA used in the subculturing process?
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What is the primary purpose of using trypsin/EDTA in the subculturing process?
What is the primary purpose of using trypsin/EDTA in the subculturing process?
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Which equipment is essential for assessing cell confluency during the subculture process?
Which equipment is essential for assessing cell confluency during the subculture process?
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What is the consequence of failing to confirm the absence of contaminants before subculture?
What is the consequence of failing to confirm the absence of contaminants before subculture?
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How is trypsin inactivation achieved post cell detachment?
How is trypsin inactivation achieved post cell detachment?
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What role does Trypan Blue play in cell quantification?
What role does Trypan Blue play in cell quantification?
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What should be done after adding trypsin/EDTA to ensure effective cell detachment?
What should be done after adding trypsin/EDTA to ensure effective cell detachment?
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What is the best method to determine the appropriate seeding density for a new flask?
What is the best method to determine the appropriate seeding density for a new flask?
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If proteases are unsuitable, what alternative methods can be used to detach cells?
If proteases are unsuitable, what alternative methods can be used to detach cells?
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What is the purpose of Newton's refraction rings during haemocytometer preparation?
What is the purpose of Newton's refraction rings during haemocytometer preparation?
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Why is it recommended to count over 100 cells when performing cell quantification?
Why is it recommended to count over 100 cells when performing cell quantification?
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What typical components are found in freeze medium for cryopreservation?
What typical components are found in freeze medium for cryopreservation?
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At what confluency should adherent cells be harvested for optimal cryopreservation?
At what confluency should adherent cells be harvested for optimal cryopreservation?
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What is the consequence of not achieving more than 90% cell viability before freezing?
What is the consequence of not achieving more than 90% cell viability before freezing?
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What information must be labeled on cryopreservation ampoules?
What information must be labeled on cryopreservation ampoules?
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What is the role of a passive freezer like the Nalgene Mr Frosty box?
What is the role of a passive freezer like the Nalgene Mr Frosty box?
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How should cells be resuspended for cryopreservation after centrifugation?
How should cells be resuspended for cryopreservation after centrifugation?
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What is the purpose of using sterile gloves during cell culture procedures?
What is the purpose of using sterile gloves during cell culture procedures?
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Which of the following actions should be avoided when using a microbiological safety cabinet?
Which of the following actions should be avoided when using a microbiological safety cabinet?
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What should be done to cell culture waste before disposal?
What should be done to cell culture waste before disposal?
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Which component is used for disinfecting the cabinet surfaces after completing work?
Which component is used for disinfecting the cabinet surfaces after completing work?
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Which personal protective equipment is NOT specifically mentioned as necessary for cell culture practice?
Which personal protective equipment is NOT specifically mentioned as necessary for cell culture practice?
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Why is it important to air dry gloves after sanitizing them with 70% alcohol?
Why is it important to air dry gloves after sanitizing them with 70% alcohol?
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What is the recommended way to handle contamination of gloves during a procedure?
What is the recommended way to handle contamination of gloves during a procedure?
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During cell culture procedures, how should speech, sneezing, and coughing be managed?
During cell culture procedures, how should speech, sneezing, and coughing be managed?
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What is the purpose of washing the cell monolayer with PBS without Ca2+/Mg2+ before adding trypsin/EDTA?
What is the purpose of washing the cell monolayer with PBS without Ca2+/Mg2+ before adding trypsin/EDTA?
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Why might it be necessary to mechanically bring some cell lines into suspension instead of using proteases?
Why might it be necessary to mechanically bring some cell lines into suspension instead of using proteases?
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Which of the following is the correct volume of trypsin/EDTA recommended for a 25cm² flask?
Which of the following is the correct volume of trypsin/EDTA recommended for a 25cm² flask?
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What should be done after returning the flask to the incubator post-trypsin application?
What should be done after returning the flask to the incubator post-trypsin application?
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What is the reason behind using an inverted microscope during the subculture process?
What is the reason behind using an inverted microscope during the subculture process?
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What is the role of the Soyabean Trypsin Inhibitor in the procedure?
What is the role of the Soyabean Trypsin Inhibitor in the procedure?
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Why is it essential to assess the absence of contaminants before subculturing?
Why is it essential to assess the absence of contaminants before subculturing?
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What equipment is necessary for accurate cell quantification during the subculture process?
What equipment is necessary for accurate cell quantification during the subculture process?
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Why is it important to use a consistent number of cells in manipulations such as transfections or cryopreservation?
Why is it important to use a consistent number of cells in manipulations such as transfections or cryopreservation?
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What is the primary function of Trypan Blue solution in the cell quantification process?
What is the primary function of Trypan Blue solution in the cell quantification process?
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What is the recommended minimum number of cells to count for accuracy in cell quantification?
What is the recommended minimum number of cells to count for accuracy in cell quantification?
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During the procedure, what must be done to the haemocytometer before use?
During the procedure, what must be done to the haemocytometer before use?
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What is the purpose of using an inverted phase contrast microscope during cell counting?
What is the purpose of using an inverted phase contrast microscope during cell counting?
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What should be done to the coverslip of the haemocytometer before placing it on the chamber?
What should be done to the coverslip of the haemocytometer before placing it on the chamber?
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What is meant by 'Newton’s refraction rings' during haemocytometer preparation?
What is meant by 'Newton’s refraction rings' during haemocytometer preparation?
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What should be the volume of cell suspension added to each side of the haemocytometer chamber for counting?
What should be the volume of cell suspension added to each side of the haemocytometer chamber for counting?
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What is the recommended cell concentration for resuspension in freeze medium for cryopreservation?
What is the recommended cell concentration for resuspension in freeze medium for cryopreservation?
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Which component is commonly used in freeze medium to protect cells during cryopreservation?
Which component is commonly used in freeze medium to protect cells during cryopreservation?
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What percentage of viable cells is considered ideal for a good recovery after freezing?
What percentage of viable cells is considered ideal for a good recovery after freezing?
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What is the primary method for assessing cell confluency prior to cryopreservation?
What is the primary method for assessing cell confluency prior to cryopreservation?
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In the protocol, what role does the centrifuge play after harvesting cells?
In the protocol, what role does the centrifuge play after harvesting cells?
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What should be labeled on the cryopreservation ampoules?
What should be labeled on the cryopreservation ampoules?
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Why is it recommended to harvest adherent cells at 80-90% confluency?
Why is it recommended to harvest adherent cells at 80-90% confluency?
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What is a potential consequence of using a non-programmable freezer for cryopreservation?
What is a potential consequence of using a non-programmable freezer for cryopreservation?
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Study Notes
Protocol 1: Aseptic Technique and Good Cell Culture Practice
- Sanitizing the cabinet: Prevents contamination from bacteria, fungi, and other sources, ensuring a sterile cell culture environment.
- 70% alcohol use: Effectively disinfects surfaces and gloves by killing microorganisms without evaporating quickly, allowing sufficient contact time for disinfection.
- Sanitizing gloves: Spray with 70% alcohol, let air dry for 30 seconds before handling cell culture materials.
- Avoiding contamination: Avoid touching face/hair, move slowly, avoid rapid movements, and direct speech/sneezes/coughs away from the cabinet.
- Slow movements: Allow proper airflow circulation, preventing disruption of the sterile environment.
- Liquid waste handling: Discard liquid waste into sodium hypochlorite (10,000 ppm) and keep for at least two hours (preferably overnight) before discarding into the drain with copious amounts of water.
- Periodic cleaning/fumigating: Ensures long-term sterility by eliminating contaminants that accumulate over time.
Protocol 2: Subculture of Adherent Cell Lines
- Subculturing purpose: Prevents cell death by providing more space and nutrients when cells reach confluency or the medium is depleted.
- Trypsin/EDTA use: Detaches cells by breaking down proteins that mediate cell adhesion, but should not be used for cells sensitive to proteases or when enzymatic detachment removes membrane markers or receptors of interest.
- Subculturing materials/equipment: Pre-warmed medium, PBS (without Ca2+/Mg2+), trypsin/EDTA, trypsin inhibitor (if necessary), sterile tissue, PPE, microbiological safety cabinet, water bath, inverted microscope, centrifuge, pre-labelled flasks, haemocytometer/cell counter, pipettes, marker pen.
- Trypsin procedure: Remove spent medium, wash monolayer with PBS, add trypsin/EDTA, rotate, incubate for 2-10 minutes until cells detach. Neutralize trypsin activity with serum-containing medium (serum-free cultures use trypsin inhibitor).
- Contaminant confirmation: Confirm the absence of contaminants before starting subculture by visual inspection and/or other appropriate tests.
Protocol 3: Cell Quantification
- Cell quantification importance: Ensures consistent cell number to maintain optimal growth conditions, standardizing procedures for reproducible results.
- Trypan Blue purpose: Differentiates viable cells (bright, unstained) from non-viable cells (stained blue) in cell quantification.
- Haemocytometer preparation: Clean, moisten coverslip with water/exhaled breath, slide onto chamber until Newton's refraction rings appear.
- Newton's rings significance: Indicate proper coverslip placement for accurate cell counting.
- Cell counting procedure: Fill both sides of the chamber with 5-10 µL of cell suspension and view under an inverted microscope at x20 magnification.
- Cell count calculation: Count viable and non-viable cells in multiple squares, calculate concentration using the haemocytometer's formula, and determine the percentage of viable cells.
- Cell count recommendation: Recommend counting >100 cells for increased accuracy and reliability of cell concentration and viability data.
Protocol 4: Cryopreservation of Cell Lines
- Cryopreservation aim: Allows long-term storage of cell lines, maintaining viability and characteristics for future use.
- Optimal harvest time: Ideally at 80-90% confluency in log phase.
- Freeze medium purpose/composition: Protects cells from damage during freezing, containing 90% FBS and 10% DMSO or glycerol.
- Viability before freezing importance: High cell viability ensures better recovery and growth after thawing.
- Resuspension procedure: After centrifugation, resuspend cells at a concentration of 2-4 x 10^6 cells/mL in freeze medium.
Additional information
- Cryopreservation ampoule labeling: Include cell line name, passage number, lot number, cell concentration, and date.
- Passive freezer use: Ensure controlled cooling rate of approximately -1°C per minute, optimal for preserving cell viability during freezing, using a passive freezer like Nalgene Mr Frosty box.
- Frozen ampoule storage: Transfer to liquid nitrogen storage vessel after initial freezing at -80°C, ideally in the vapor phase.
- Cryopreserved ampoule record-keeping: Proper records ensure easy retrieval and prevent loss/misidentification, including storage locations.
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Description
This quiz covers essential practices for maintaining aseptic conditions in cell culture. Learn about sanitizing methods, proper glove usage, and techniques to minimize contamination. Ensure a sterile environment and understand waste disposal protocols for successful cell culture management.
