Lecture 4

Question 1

general requirements for aseptic technique

Answer 1

quiet area
clean work surface
good personal hygiene
sterile reagents and media

Question 2

aseptic technique of laminar flow hood

Answer 2

use filters, wipe surfaces with ethanol, UV lights (questionable effectiveness)
when working make sure to wipe with ethanol and bring only what you need. arrange workspace properly and don’t allow hands to pass over open flasks/dishes, clean spills immediately

Question 3

aseptic technique of reagents and media and cell cultures

Answer 3

commercial media are tested. swab outside with ethanol

cell cultures from other labs should be quarantined and kept away from own stock. test for mycoplasma

Question 4

aseptic technique of handling items

Answer 4

swabbing: use ethanol, keep all surface and bottles sterile, wear gloves
capping: all caps/bottles have to be free of chips. on open bench do not leave bottle open
flaming: removes dust and lint particles. don’t use in BSC or around alcohol
pipetting: hold lid or put upside down in BSC. tilt bottles to angle use appropriate size pipette, use glass or filtered plastic, micropipettors are a contamination hazard
pouring: a bit risky, only do if you pour everything at once

Question 5

aseptic personal practices

Answer 5

no eating, drinking, or food storage in lab
use PPE
decontaminate work surfaces
inspect equipment
decontaminate equipment after use
train workers!

Question 6

biosafety levels

Answer 6

1: separation of lab area from public, availability of autoclave decontamination, hand washing facility, bench tops impervious to water, heat and chemical resistant
2: same as 1 plus lockable doors, BSC, eye wash station
3/4: same as 2 plus entry through a series of two self closing doors, hand free sink, walls/floors/ceilings decontaminated, windows closed and sealed, special ventilation system, air treatment system, controlled access zones

Question 7

how is asepticness QC/validated

Answer 7

defined SOP and routine testing and documentation. three major elements to validate:
authentication, is the cell line what it’s claimed to be
provenance, how was the line derived and what has happened since its original isolation
contamination, is it free of all microbial contamination