Understanding Your BSL-2 Environment
BSL-2 (Biosafety Level 2) labs are designed for work with moderate-risk biological agents. Think common human pathogens, cell lines, parasites like Plasmodium falciparum, and anything involving human-derived samples. If you're culturing cells, you're automatically in BSL-2 territory, which means you have to follow specific safety protocols.
The cardinal rule? Everything that touches your cultures must be sterile, and everything that leaves must be decontaminated. Your success depends on maintaining this barrier between the sterile world inside your flasks and the microbial chaos of the outside environment.
🎥 Want to See It in Action?
Check out our video tutorial on Decontaminating cell culture rooms on the Adwoa Biotech YouTube Channel, where we talk through the process.
The Clean Bench vs. Biosafety Cabinet: Know the Difference
Before we talk decontamination, let's clear up a critical confusion that trips up many newcomers:
Laminar Flow Clean Bench
A laminar flow clean bench uses HEPA-filtered air to create a particle-free environment flowing across your work surface. Here's what you need to know:
Protects: Your cell cultures (product protection only)
Does NOT protect: You, the operator
Best for: Routine aseptic manipulations of non-infectious cultures
Critical rule: Once contamination is suspected, DO NOT open contaminated vessels inside the clean bench
Class II Biosafety Cabinet (BSC)
A biosafety cabinet provides both personnel and product protection through directional airflow and HEPA filtration.
Protects: Both you AND your cultures
Best for: Infectious materials, human-derived samples, pathogenic organisms
The rule: If you're working with anything that could make you sick (like Plasmodium or Schistosoma), use the BSC, not the clean bench
Pathogen work happens in the BSC. Routine cell maintenance can happen in the clean bench. If you have both available and you're working with infectious agents, always choose the BSC.
Common Contamination Types and What They Mean
Knowing your enemy helps you fight it:
Bacterial Contamination
Signs: Rapid media turbidity, pH changes (media turns yellow or pink), visible colonies
Common sources: Poor aseptic technique, contaminated reagents, airborne bacteria
Prevention: Sterile technique, routine antibiotic use (if appropriate), proper incubator maintenance
Fungal Contamination
Signs: Fuzzy or filamentous growth, slower than bacteria to appear
Common sources: Airborne spores, contaminated incubator water, poor air quality
Prevention: HEPA filtration, copper sulfate in incubator water, control room humidity
Mycoplasma Contamination
Signs: Subtle. Slight growth rate changes, morphology changes, often asymptomatic
Common sources: Contaminated cell lines, aerosols from infected cultures
Prevention: Regular testing, strict quarantine of new cell lines, never share media
Detection: Requires specific testing (PCR, fluorescent staining)—visual inspection isn't enough
Yeast Contamination
Signs: Budding cells visible under microscope, media turbidity, sometimes visible colonies
Common sources: Contaminated reagents, poor hand hygiene, environmental sources
Prevention: Proper hand washing, sterile technique, regular equipment cleaning
Your Disinfection Arsenal: Bleach and Alcohol
You've got two workhorses in your decontamination toolkit, and knowing when to use each one is crucial.
Bleach (Sodium Hypochlorite): The Heavy Hitter
Bleach is your go-to for spills, heavy contamination, and anything involving organic matter like blood or cell culture media. But it's not as simple as "spray and wipe."
Why Fresh Bleach Matters:
Bleach loses potency fast: yesterday's batch is probably useless today
Always prepare fresh bleach solutions daily
Label them clearly with the date and concentration
Concentration Guide:
10% bleach (1:10 dilution, ~0.5% sodium hypochlorite): For serious business—spills, blood cleanup, contaminated liquid waste, incubator decontamination
1% bleach (1:50 dilution, ~0.1% sodium hypochlorite): For routine bench wiping and daily maintenance
Contact Time is Everything: The surface must stay wet for the disinfectant to work:
Routine cleaning: minimum 10 minutes
Spills and heavy contamination: 20-30 minutes
If the surface dries before the contact time is up, you're not achieving proper disinfection
The Metal Problem: Bleach corrodes metal surfaces. After the required contact time, always rinse metal benches with sterile distilled water to prevent damage.
NEVER Mix Bleach: Seriously, never combine bleach with ammonia (found in glass cleaners) or acids. This creates toxic chlorine gas that can cause severe respiratory damage. When in doubt, stick to bleach and water only.
70% Alcohol (Ethanol or Isopropanol): The Daily Driver
Alcohol is perfect for:
Daily surface wiping before and after work
Equipment exteriors (pipettors, incubator handles)
Quick decontamination of non-porous surfaces
Final wipe-down after bleach treatment
Why 70% and not 100%? The water content is essential. It helps the alcohol penetrate microbial cell walls more effectively. Pure alcohol evaporates too quickly to be effective.
When Contamination Strikes: Your Immediate Response
You've just noticed fuzzy growth in your flask or cloudy media where it should be crystal clear. Don't panic, act systematically.
Step 1: Stop Everything
Immediately cease all culture work. Contamination can spread through aerosols, so the less you disturb things, the better.
Step 2: Quarantine and Label
Label all affected flasks or plates with "CONTAMINATED" and the date
Critical: Do NOT open contaminated vessels inside the clean bench
This is how contamination spreads to your entire workspace
Step 3: Safe Containment
Wipe the exterior of each contaminated vessel with 10% bleach
Seal everything in a biohazard bag
Autoclave at 121°C for 30 minutes before disposal
Step 4: Assess the Damage
Which equipment might be compromised? The incubator? The bench? Both?
Did you use any shared equipment (pipettors, racks) that need decontamination?
Are other cultures at risk?
Now you're ready for the systematic decontamination process.
Decontaminating Your 37°C Incubator
The incubator is often contamination ground zero: it's warm, humid, and perfect for microbial growth. Here's how to properly decontaminate it:
Monthly maintenance prevents this, but when contamination happens, follow this protocol:
Power Down: Switch off the incubator and let it cool slightly
Empty and Disassemble:
Remove all trays, shelves, and water pans
If autoclavable, autoclave them (121°C, 30 min)
If not autoclavable, soak in 10% bleach for 30 minutes
Drain the Humidity System:
Completely drain and discard water from the humidity pan
This water is often the primary contamination source
Primary Disinfection:
Wipe all internal surfaces with 10% bleach
Don't forget corners, door seals, and the back panel
Let it sit for a full 20 minutes (set a timer!)
Rinse and Dry:
Wipe down with sterile distilled water to remove bleach residue
Allow the interior to air-dry completely (this may take several hours)
Reassemble with Fresh Water:
Refill humidity pan with sterile distilled water containing 0.2% copper sulfate or a commercial antifungal agent
This helps prevent future contamination
Document:
Record the decontamination date and your initials in the incubator maintenance log
Note what type of contamination was observed
Pro tip: Replace incubator water weekly, not when it evaporates completely. Stagnant water is a contamination waiting to happen.
Decontaminating Your Laminar Flow Clean Bench
Your clean bench is your sterile sanctuary. Treat it with respect during decontamination.
Power Down Safely:
Turn off airflow and unplug the unit
Never spray liquids into a running clean bench
Initial Bleach Treatment:
Wipe all interior surfaces with 10% bleach
Focus on corners, back panels, and the work surface
Allow 10-15 minutes contact time
Remove Bleach Residue:
Wipe with sterile distilled water
Follow with 70% ethanol to ensure complete residue removal
Bleach residue can inhibit cell growth
Special Treatment for Fungal Contamination:
If you saw visible fungal growth (fuzzy colonies), add an extra step
After bleach treatment, wipe surfaces with 3% hydrogen peroxide
Then proceed with the ethanol wipe
Restart and Purge:
Turn airflow back on
Let it run for at least 15 minutes before resuming work
This purges any residual chemical vapors and re-establishes laminar flow
UV Treatment (if equipped):
Some benches have UV lights: run them for 30 minutes after cleaning
Remember: UV doesn't penetrate organic matter, so always clean first
Decontaminating Surrounding Surfaces and Equipment
Contamination doesn't respect boundaries. Your entire workspace needs attention:
Laboratory Surfaces
Benchtops: Wipe with 70% ethanol daily, 10% bleach if contaminated
Incubator exterior: 70% ethanol, paying attention to door handles
Pipettors and racks: 70% ethanol on exterior surfaces only (never submerge electronic pipettors)
Floors and Walls
Mop floors weekly with 0.5% sodium hypochlorite solution (10% bleach).
After contamination events, mop immediately
Don't forget the area under and around equipment
Hidden Culprits
CO₂ tubing filters: Replace if visibly moist or if they haven't been changed in 6 months
Air conditioning vents: Check monthly for visible fungal growth or debris
Drain systems: Ensure they're clear and not harboring stagnant water
The Art of Proper Waste Disposal
Your decontamination job isn't done until waste is properly segregated. Mixing waste streams is dangerous, expensive, and often illegal.
Critical safety note: Never try to sort or separate waste after it's been placed in a container. Segregate at the source, during your work.
Verify Before You Resume: The Sterility Check
After all that cleaning, how do you know it worked? Trust, but verify.
The Sterility Test
Prepare test media: Use the same media you culture with
Incubate in the cleaned incubator: Leave plates or flasks for 3-5 days at 37°C
Observe daily: Any cloudiness, color change, or visible growth means you need to repeat decontamination
Document results: Record verification in your lab decontamination log
Only after successful verification should you resume normal culture operations. Skipping this step risks recontaminating fresh cultures immediately.
Your Quick-Reference Maintenance Schedule
Here's your at-a-glance guide to keeping contamination at bay. Print this out and post it near your clean bench:
Pro tip: Set up recurring calendar reminders for these tasks. It's easy to let preventive maintenance slip when experiments are going well—but that's exactly when contamination strikes.
Record Keeping: Document Everything
If it isn't documented, it didn't happen. Maintain clear records of:
Every decontamination event (date, type of contamination, equipment affected, procedure followed)
Preventive maintenance activities (with dates and initials)
Sterility verification results
Spill incidents and responses
Equipment malfunctions or repairs
Why this matters:
Identifies patterns (e.g., recurring contamination in one incubator)
Demonstrates compliance during audits
Helps troubleshoot when new contamination appears
Protects you legally in case of exposure incidents
Keep logs for at least two years, and make them easily accessible to all lab users.
📥 Download Full Reference Guide PDF - Save this entire guide for offline access in the lab
References
World Health Organization (2020). Laboratory Biosafety Manual, 4th Edition. Geneva: WHO.
ATCC (2018). Cell Culture Basics: Handling and Decontamination Guidelines. Manassas, VA.
Centers for Disease Control and Prevention (CDC) (2020). Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. Washington, DC: U.S. Department of Health and Human Services.
Thermo Fisher Scientific (2021). CO₂ Incubator Cleaning and Maintenance Manual.
Panasonic Healthcare (2020). Incubator Operation and Maintenance Guidelines.
World Health Organization (2014). Practical Handbook of Laboratory Safety. Geneva: WHO.
CDC (2020). Guidelines for Safe Disposal of Laboratory Waste.
Invitrogen (2022). Cell Culture Contamination Control Guide.
