BACE Aseptic Technique and Contamination 3 — Questions and Answers
Question 1: Which classification of biosafety cabinet is appropriate for working with BSL-3 pathogens?
- Class I BSC
- Class II Type A1 BSC
- Class II Type B2 or Class III BSC (Correct answer)
- Any Class II BSC is sufficient
Correct answer: Class II Type B2 or Class III BSC
Class II Type B2 BSCs (100% exhaust) or Class III BSCs (completely enclosed glove boxes) provide the containment required for BSL-3 work.
Class I BSCs protect personnel only. Class II Type A1/A2 recirculate some HEPA-filtered air and are suitable for BSL-1/2 work. Class II Type B2 exhausts 100% of air to the outside via a hard-ducted connection, suitable for BSL-3. Class III BSCs are completely sealed glove boxes under negative pressure, required for the most dangerous pathogens.
Question 2: What is the significance of the 'dead zone' at the front edge of a biosafety cabinet work surface?
- It is the cleanest area farthest from the HEPA filter
- Air turbulence at the cabinet opening disrupts laminar flow making this area least protected (Correct answer)
- It is reserved for waste containers
- UV light exposure is strongest in this zone
Correct answer: Air turbulence at the cabinet opening disrupts laminar flow making this area least protected
The front edge of the BSC work surface is subject to air turbulence where room air meets cabinet airflow, disrupting sterile conditions — critical work should be performed further back in the cabinet.
The 'dead zone' (approximately the first 4 inches from the front sash) is where room air currents and the cabinet's downward laminar airflow interact, creating turbulence that can carry contaminants into the work area. Sterile materials and open vessels should always be placed behind this zone — ideally 4-6 inches from the front edge.
Question 3: When performing an autoclave validation, what biological indicator organism is typically used?
- Escherichia coli spores
- Geobacillus stearothermophilus spores (Correct answer)
- Staphylococcus aureus vegetative cells
- Candida albicans spores
Correct answer: Geobacillus stearothermophilus spores
Geobacillus stearothermophilus spores are the standard biological indicator for steam autoclave validation because they are among the most heat-resistant organisms known.
Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) produces extremely heat-resistant endospores. If a properly run autoclave cycle kills these spores, it will kill all less-resistant organisms. Biological indicators are used alongside chemical and physical indicators for a complete sterility assurance system.
Question 4: A technician must transfer a sterile solution from a large bottle to a smaller container. What is the best aseptic practice?
- Pour directly while holding both containers over the bench
- Work inside the BSC, flame bottle neck if glass, pour without touching inside surfaces, recap immediately (Correct answer)
- Use a syringe to withdraw through the bottle cap without opening
- Transfer on the open bench after wiping with 70% ethanol
Correct answer: Work inside the BSC, flame bottle neck if glass, pour without touching inside surfaces, recap immediately
Inside the BSC, careful handling prevents contamination; avoiding contact with internal surfaces and recapping immediately preserves sterility.
Aseptic transfers should always be performed in the BSC. For glass bottles, flaming the neck after opening kills surface organisms. Caps should be held in the hand and replaced immediately after pouring. Pour in a single smooth motion to minimize splashing and exposure time. Syringes through caps are appropriate only for appropriate liquid volumes and container types.
Question 5: Which of the following is NOT a physical indicator of contamination in a mammalian cell culture?
- Color change of phenol red pH indicator in the media
- Presence of floating particles or unusual debris
- Cells growing in a monolayer as expected (Correct answer)
- Visible mycelial filaments in the culture flask
Correct answer: Cells growing in a monolayer as expected
Cells growing in a normal monolayer is an expected, healthy finding — not an indicator of contamination. The other options all signal potential contamination.
Normal mammalian cell growth (monolayer formation, clear media, expected morphology) indicates an uncontaminated culture. Contamination signs include: unexpected pH change (media yellowing from bacterial acid production), floating debris, visible fungal hyphae or yeast colonies, media turbidity, and abnormal cell morphology. Regular visual inspection before any manipulation is a core aseptic technique practice.
Question 6: What is the purpose of using a 0.22 µm membrane filter when preparing cell culture media supplements?
- To remove endotoxins from the solution
- To sterilize the solution by removing bacteria and larger microorganisms (Correct answer)
- To concentrate the solution by removing water
- To remove pH indicator dyes from the supplement
Correct answer: To sterilize the solution by removing bacteria and larger microorganisms
A 0.22 µm filter physically removes bacteria and fungi, sterilizing heat-sensitive solutions that cannot be autoclaved.
Membrane filtration with 0.22 µm pore size is the standard method for sterilizing heat-sensitive solutions such as serum-free supplements, growth factors, antibiotics, and vitamins. The filter physically excludes bacteria (minimum ~0.3 µm) and fungi. Note that viruses and mycoplasma can pass through 0.22 µm filters. Endotoxin removal requires separate charged membrane filters.
BACE Aseptic Technique and Contamination 3
Which classification of biosafety cabinet is appropriate for working with BSL-3 pathogens?