4.17 Aseptic Technique - Specific Tips

Maintaining purity of cultures by use of aseptic technique is essential to producing valid research results.

Work Area

• Culture space is in a separate room, or separated from other areas of activity in the lab.
• Access to culture space is limited or restricted during a work session.
• Culture area is kept as clean and uncluttered as possible.
• Air temperature in culture area is cool enough for lab workers wearing PPE to work without becoming overheated.
• Incubators, shakers, etc. are routinely cleaned and decontaminated.
• Certain incubators, BSCs, etc. are designated for use with specific culture types, i.e., a dedicated tissue culture BSC where no bacteria or fungal culture work is performed that would present a contamination risk.

Containment

• BSC blower velocity and directional air flow is qualitatively checked with every use.
• At least 5 minutes for air purge is provided in the BSC in between work sessions with different cell lines, and consider waiting up to an hour between sessions with different organism types (e.g., bacteria/tissue cells/ fungi, etc.).
• Open flames are not used in the BSC.
• Vortex mixers are positioned in the back third of the BSC work surface.
• BSC work surface is populated with needed supplies only for a work session.
• Items/supplies on BSC work surface are laid out in a “clean to dirty” arrangement, and the flow of work proceeds from the clean area to the dirty or contaminated area.
• The BSC work surface is not overcrowded with items, and is arranged so that an open space is provided for culture work to take place.
• Pipettes are discarded in a tray with disinfectant on the “dirty side” of the BSC work surface and the tray is removed after the work session; pipettes should not be discarded outside of the BSC during the work session.
• Each item is thoroughly spray-disinfected before placement in the BSC.
• Each contaminated item is thoroughly spray-disinfected before removal from BSC.

Reagents, Stock Solutions and Media

• Each lab worker uses his/her own stock solutions and media rather than sharing them among lab workers.
• Each cell line has stock solutions and media reserved for use only with that cell line.
• Lab workers aliquot stock solutions/reagents ahead of time rather than making stocks during culture sessions.
• Sterility of stock solutions are checked by culturing for no growth.

PPE, Hygiene and Behavior

• Hands, wrists and forearms are washed before donning PPE, and PPE is worn so that there is no exposed skin on forearms between glove and lab coat cuff.
• Disposable gloves that are resistant to ethanol are used.
• Gloved hands are sprayed with 70% ethanol before and frequently during culture work.
• Clean, cuffed lab coats are worn.
• Seat height is adjusted so that the lab worker’s face is fully above the opening to the work surface.
• Talking with coworkers is limited while culturing to better attend to the work.

Work Surfaces

• Surfaces are disinfected thoroughly and systematically, i.e, from one side to another, wiping over each section only once.
• A liberal amount of disinfectant is used and left on surfaces for needed contact time.
• Surfaces are disinfected before and after every separate culture session.
• Spills are cleaned up thoroughly and immediately.

Good Habits

• Culture containers are labelled well.
• Records for frozen cultures are well maintained.
• A log for media/reagent lot numbers is kept.
• An ample supply of frozen culture material is maintained.
• Culture workloads are manageable for lab workers.
• Lab workers strive to do one thing at a time, one culture at a time.
• Lab workers are proficient in aseptic pipetting, uncapping/capping, and pouring techniques with culture material.
• Lab workers avoid reaching over open or clean items during culture work.
• Lab workers evaluate their work flow periodically, and makes adjustments as needed.
• New lab workers are trained by a skilled worker before performing independent work at the bench.
• Training is documented.
• Proficiency checklists for training are utilized.
• A designated person in the lab manages training of new lab workers.

Detection and Management of Contamination

• When a contaminated culture is found, it is isolated from clean cultures. It is discarded and a new culture is started from frozen stock rather than trying to recover purity through aggressive antibiotic treatment.
• Cell cultures are kept antibiotic-free as much as possible so that low-level contamination can be more readily detected.
• Tissue cell cultures are examined microscopically every time they are handled;
• lab workers are familiar with the normal morphologies of cell lines used in the lab, and can recognize deviations caused by contamination or other abnormal conditions.
• Tissue cell lines are checked for bacterial/fungal contamination by culturing for these contaminants on a regular basis.
• Purity and identity of all cultured material is verified; bacterial/fungal stocks are checked for pure culture on a regular basis, and/or before experimentation.
• Cell cultures are checked for mycoplasma contamination at a regular interval.
• Vented, secure closures are used on culture flasks.
• Use safety caps/sealed rotors when centrifuging biohazardous material.