“biosafety update 2009” consists of the
powerpoint presentations:
“Good Culture Practices” and “Misidentification
and Contamination” with their respective quizzes
Biosafety training starting in Jan 2009 will
consist of the in class session (offered monthly)
PLUS these two presentations. All three quizzes
are to be handed in at the same time – within a
week or so after the in class session.
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Good Culture Practices
self paced Powerpoint study
McMaster University Biosafety Office
please print off the quiz or obtain
copy from HSC 3N1C and return
or fax to 905 528 8539
2
guiding principles of good cell culture EU (2-7)
why worry? (NIH) (8-11)
information / SOP’s (12-14)
keep records (15-23)
good culture techniques (24 – 32)
risk of contamination (33 – 39)
test cells regularly (40 – 43)
contact us & training (44)
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Guiding principles for Good Cell
Culture Practice (EU)
1. understand the in vitro culture system you are
using and the relevant factors that could affect it
2. document assurance of the quality of all
materials and methods
3. document information to track materials and
permit repetition of the work.
4
4. use measures to protect individuals and the
environment from potential hazards
5. comply with laws, regulations and ethics
6. provide education and training to achieve good
cell culture practice
5
why practice quality assurance and
good culture practices?
“the maintenance of high standards is fundamental
to all good scientific research”
to maintain a reliable/reproducible program
to detect problems
to respond quickly when they occur
to prevent reporting erroneous results
6
goal of Good Culture Practices
training program
to improve the knowledge of basic biological
culture practices
to improve the likelihood that contamination will
be detected/reduced
to ensure that the intended cultures are used in
experiments
7
why are we worried about cell line
or culture contamination?
1) studies have shown that 20-35% of all cell lines
are contaminated (HeLa cells in many cases)
some cell lines have a short doubling time and are a
high risk for cross contamination into other cell lines.
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2) research results may not be accurate if you are
using a contaminated cell line
NIH has stated that “misidentification of cell cultures is a
serious problem” (Nov 2007)
please read these three articles:
“Cases of mistaken identity”, “Cell line cross contamination”
and “Open letter on cell line identification”
http://fhs.mcmaster.ca/safetyoffice/biosafety_cell_line.html
9
3) authentication of cultures may be a future
requirement for peer reviewed processes
(grants and publishing)
“NIH notice on authentication of cell lines”
http://fhs.mcmaster.ca/safetyoffice/biosafety_cell_line.html
10
4) PI’s are responsible for the accuracy and purity
of the cultures used in their research
- what detailed protocols/ practices are in place in your lab in
the form of a manual and SOP’s?
- take this quiz to see how many of the recommended
practices are currently in use in your lab (web link not yet
ready)
- visit a excellent McMaster research lab manual at
www.dwalab.com/labman for an example
11
typical SOP’s for a culture lab
• standards for the quality of culture vessels and surface
coatings
• sterility check for re-used items
• maintenance and sterility check for automatic pipettes
and pipettors
• maintenance and sterility check for incubators
• standards for culture reagents
• Visit an excellent McMaster research lab manual at
www.dwalab.com/labman as an example
12
Checking cell viability and count
in practice: learn how to do it at www.jove.com “basic
protocols”
good microscopy for good results
in practice: learn how to do it at www.jove.com “basic
protocols”
good freezing and recovery techniques are important for
healthy cells
in practice: learn how to do it at www.jove.com “basic
protocols”
13
when starting with a new line/strain
know the following:
•
•
•
•
•
•
•
•
authenticity
morphological appearance
viability
growth rate
passage number
differentiation state
contaminates present (or not)
appropriate +/- controls for the application
14
protocols to reduce the risk of
contamination / misidentification
A) keep excellent records
B) use good culture techniques
C) test cultured lines regularly
15
A) keep excellent records
1) obtain validated cell lines only from a reputable
source
in practice: do not rely solely on verbal assurances for the
identity of a cell line
in practice: have a documented provenance of the cell line,
the genotypic and phenotypic characteristics
in practice: consider purchasing new cell lines rather than
getting them from the “lab next door”
16
2) keep real time paperwork/inventories on
cultures
in practice: several inventory options will record similar
information, depending on the nature of your work
in practice: this will allow for tracking of lines and who has
used them, and how all material has been used
www.fhs.mcmaster.ca/safetyoffice/biological_inventory.html
17
sample inventory forms
any of these forms or equivalent can be used
www.fhs.mcmaster.ca/safetyoffice/biological_inventory.html
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3) have a standard coding and recording system
in the lab for storage of frozen lines
in practice: keep a location log book and ensure everyone
uses it
in practice: have a documented monitoring system for liquid
nitrogen refilling procedures
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4) do not use any cells/strain with questionable
labeling
in practice: if it is no longer readable, discard it,
in practice: if it is not in the logbook, discard it.
in practice: if the entry in the logbook does not match the
label on the vial, discard it.
20
5) label vials in advance when freezing or
subculturing
in practice: pre label all vials for freezing and freeze only
one cell line at a time.
learn about freezing cells at www.jove.com “basic
protocols”
in practice: fill no vials or culture flasks without pre labeling
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6) compare the label on the thawed vial with the
label on the growth chamber before making the
transfer.
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B) good culture technique
1) check and record that all reagents have been
tested for sterility
in practice: a central facility should keep records of all
sterility checks
in practice: incubate an uninoculated growth medium with
all reagents as a blank if there is no central prep facility
23
2) do not use the same reagent bottle (eg media)
for different cell/strain lines
in practice: do not share reagents among operators
in practice: it would be ideal to label each reagent bottle for
the cell line it is to be used for
24
3) always change pipets between reagents/cell
lines and plug all pipets
in practice: the plugged pipet decreases the chance that
contamination can enter into the pipetting unit.
in practice: if liquid enters the pipetting unit, change the
HEPA filter nose piece unit
25
4) work with only one cell/strain line at a time
in practice: finish all work with one line before moving on to
another, this significantly reduces the chance of cross
contamination
26
5) put media and reagents into flask, add cells last
in practice: this prevents a cell contaminated pipet entering
into the stock media and contaminating it.
27
6) handle rapidly growing cells at the end of the
work session (eg HeLa)
in practice: the cells are not available to contaminate
slower growing lines used earlier in the work session
28
7) newly isolated lines to be characterized as soon
as possible
in practice: the information garnered at this step will serve
as the identification tests for the future as finite cell lines
may change characteristics over time
In practice: avoid subjecting cells to fluctuating
temperatures or culture conditions as it may cause cells
to alter
29
8) quarantine all new cell/strain lines until they are
shown to be not contaminated
in practice: isolate new cells so that there will be no
opportunity for incubator cross contamination.
eg. use filtered flasks
30
9) re-evaluate your sterile technique regularly
in practice: institute regular sterility checks for all aspects of
your culture
eg. all reagents except cell lines in culture
eg. plates in incubator
eg. swabs of pipeter and surface of biological cabinet
31
Conditions that increase the risk
of contamination
1) new
- staff or student
in practice: individuals are most likely to have an incident
in the first three months of working.
- supplier or reagent,
In practice: quality control any new reagents before placing
them into general use or buy from a reputable source
- equipment
in practice: decontaminate and evaluate all new equipment
32
2) deteriorating conditions
in practice: aseptic technique is not followed
in practice: attention to detail is no longer followed
in practice: quality of lab air has changed due to local
sanitary conditions or external environmental conditions
(eg increased fungal spore count)
33
3) poor cleaning
in practice: protocols not followed or not available in a
written format eg. biohood decon
in practice: disinfectant not effective against the agent
being cultured
in practice: outdated disinfectant will be ineffective,
therefore, date all disinfectants on the date of
preparation eg. 10% bleach has a life span of several
hours
in practice: gloves not sufficiently sterilized before use
34
4) autoclave - lack of control
in practice: all individuals using an autoclave must receive
autoclave training
in practice: quality control for autoclaves requires regular
use of a biological indicator, with records
in practice: there should be an understanding of
appropriate packaging/loading for autoclaving
in practice: the steam for autoclaves should be quality
controlled.
35
5) dirty equipment
in practice: pipet tissue culture nose piece filters are to be
inspected regularly
in practice: incubator and water source and centrifuge need
to be decontaminated regularly
in practice: cold/ warm rooms need to be cleaned regularly
in practice: lab coats need a regular schedule of cleaning,
not just when they “look dirty”
36
6) lab conditions
in practice: increased general activity in lab will increase
risk of contamination
in practice: construction activity (ie on ductwork) may
release contaminant spores
in practice: increase in lab air currents are likely to affect
the biological cabinet air curtain
37
7) biological cabinet
in practice: blocking either front or back grill will increase
contamination
in practice: all biological cabinets should be certified
annually
in practice: two people working in one hood causes too
much disruption for good culture practices
38
C) test cultures regularly
1) every time a culture is handled
- check by eye for contamination
- check by phase contrast microscopy
in practice: learn at www.jove.com “basic protocols”
39
2) on regular intervals
- run a culture without antibiotics to reveal contamination
that may exist in the stock line, know what you are
dealing with
- if possible, use no antibiotics in culture
40
3) test cell regularly in an appropriate assay
in practice: do the on–line biosafety training part 2
41
4) run positive and negative controls and do not
ignore unexpected results.
in practice: consider human error
in practice: consider all reagents and equipment which
have been in contact with the culture. assume nothing.
in practice: consider that the cell line is not what you expect
it to be
42
visit our biosafety website at fhs.mcmaster.ca/safetyoffice
for 2009
1) “biosafety update 2009” consists of “Good
Culture Practices” and “Misidentification and
Contamination” and the quiz
2) “initial biosafety training” from now on will
consist of the in class session offered monthly
PLUS these two presentations
43