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7/29/2019 Chapter1 Laboratory
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HANOI UNIVERSITY OF AGRICULTURE
FACULTY OF BIOTECHNOLOGY
CHAPTER 1
Lecturer: Dr. Nguyen Huu Duc
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Contents
1.1. Planning
1.2. Construction and services
1.3. Layout of aseptic room or suite
1.4. Incubation
1.5. Preparation area
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The major requirement thatdistinguishes tissue culture from most
other laboratory techniques is the need
to maintain asepsis. Although it is
usually not economically viable to
create large sterile areas
The laminar-flow hoods has
greatly simplified the problem
and allows the utilization of
unspecialized laboratory
accommodation, provided that
the location is suitable
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Quarantine
Access
Renovations
Accommodation
Ventilation
Several considerations need to be taken into account in planning
new accommodation
When a new building is contemplated, there is more scope for
integrated and innovative design, and facilities may be positioned
for ergonomic and energy-saving reasons. The following items
should be considered:
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1.1.1. Ventilation
Pressure balance Laminar flow hoods
Ideally, a tissue culture
laboratory should be at
positive pressure relative
to surrounding work
areas, to avoid any influxof contaminated air from
outside
It is preferable to duct laminar-flow hoods to
the exterior to improve air circulation and
remove excess heat (300500 W per hood)
from the room. This also facilitates
decontamination with formaldehydeVenting hoods to the outside will probably
provide most of the air extraction required for
the room, and it remains only to ensure that
the incoming air, from a central plant or an air
conditioner 5
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Laminar-flow hoods are better left to runcontinuously, but if they are to be switched off when
not in use, then an alternative air extract must be
provided and balanced with the extract via the hoods
Laminar-flow hoods
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These considerations determine how
many laminar-flow hoods will be
required (based on whether people
can share hoods or whether they will
require a hood for most of the day)
and whether a large area will be
needed to handle bioreactors, animal
tissue dissections, or large numbers
of cultures.
As a rough guide, 12 laminar-flow
hoods in a communal facility can
accommodate 50 people with
different
The largest area should be given to
the culture operation, which has to
accommodate laminar-flow hoods,
cell counters, centrifuges,
incubators, microscopes, and some
stocks of reagents, media,
glassware, and plastics.
The second largest is for washup,
preparation, and sterilization, third
is storage, and fourth is incubation.
A reasonable estimate is 4:2:1:1, in
the order just presented
SpaceStaff numbers
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Hoods
The space between hoods shouldbe approximately 500 mm (2 ft),
to allow access for maintenance
and to minimize interference in
airflow between hoods
This space is best filled with a
removable cart or trolley, which
allows space for bottles, flasks,
reagents, and a notebook
Ensure tissue culture is reasonablyaccessible to, but not contiguous
with, the animal facility
Windows can be a disadvantage in
a tissue culture laboratory, leadingto heat gain, ultraviolet (UV)
denaturation of the medium, and
the incursion of microorganisms if
they are not properly sealed
Asepticarea
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What type of incubation
will be required in terms
of size, temperature, gas
phase, and proximity tothe work space?
Will regular, nongassed
incubators or a hot roomsuffice, or are CO2 and a
humid atmosphere
required?
Generally, large numbers of flasks or large-
volume flasks that are sealed are best
incubated in a hot room, whereas open plates
and dishes will require a humid CO2
incubator 9
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Preparation area
Facilities for washing up and for sterilization should belocated close to the aseptic area that they service and on an
outside wall to allow for the possibility of heat extraction
from ovens and steam vents from autoclaves
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Give your washup,
sterilization, and
preparation staff a
reasonable visual
outlook; they usually
perform fairly
repetitive duties
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What is the scale of the
work contemplated and
how much storage space
will this require fordisposable plastics?
What proportion of the
work will be cell line
work, with its
requirement for storage
in liquid nitrogen?
Will an elevator be
required, or will a
ramp suffice?
If a ramp will do, what
will be the gradient
and the maximum load
that you can expect to
be carried up that
gradient without
mechanical help?
Servicing aseptic areas Storage
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If a conversion of existing facilities is contemplated,
then there will be significant structural limitations;
choose the location carefully, to avoid space
constraints and
awkward
projections into the
room that will limit
flexibility12
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Make sure that
doorways are both wide
enoughand high enough andthat ceilings have sufficient
clearance to allow the
installation of equipment
such as laminar-flow hoods
(which may need additional
space for ductwork),
incubators, and autoclaves
doorways and spacingbetween equipment
provide access for
maintenance
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Newly introduced cell lines and biopsiesneed to be screened for mycoplasma
before being handled in the same room
as general stocks, and some human and
primate biopsies and cell lines may carry
a biohazard risk that requires
containment
What quarantine facilities will be required
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Small Tissue Culture Laboratory Medium-Sized Tissue Culture Laboratory
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Tissue Culture Lab with Adjacent Prep Room
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These questions will enable you to decide what size
of facility you require and what type of
accommodation - one or two small rooms or a suite
of rooms incorporating washup, sterilization, one ormore aseptic areas, an incubation room
A separate facility gives better contamination
protection, allows tissue culture stocks to be kept
separate from regular laboratory reagents and
glassware
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The rooms should be supplied with filtered air; adjacentrooms, which lead to the aseptic area, should be regardedas buffer zones and should also receive filtered air but at
positive pressure
Furniture should fit tightly to the floor or be suspendedfrom the bench, with a space left underneath for cleaning.
Cover the floor with a vinyl or other dustproof finish
If possible it is preferable for the tissue culture lab to beseparated from the preparation, washup, and sterilization
areas, while still remaining adjacent
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Combustiblegas
Power
Carbondioxide
Compressedair
Vacuum
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Power is alwaysunderestimated, interms of both the
number of outlets andthe amperage peroutlet.
Assess carefully theequipment that will be
required
Gas is moredifficult to judge, as
it requires someknowledge of thelocal provision of
power
Electricity
is cleanerand
generally
easier to
manage
from a
safety
standpoint,
but gas may
be cheaperand more
reliable
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Carbon dioxide should be piped
into the facility
The installation will pay for itselfeventually in the cost of cylinders
of mixed gases for gassing
cultures, and it provides a better
supply, which can be protected, for
gassing incubators
Compressed air is generally no
longer required. It is used to expel
cotton plugs from glass pipettes
before washing and may be
required for some types of
glassware washing machine
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A vacuum line can be very useful for evacuating culture flasks,
but several precautions are needed to run the line successfully
A collection vessel must be present with an additional trap flask,
with a hydrophobic filter between the flasks,
Vacuum Line Drawing
in order to
prevent fluid,
vapor, or some
contaminant from
entering the
vacuum line and
pump
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Six main functions need to be accommodated in the laboratory:
sterile handling, incubation, preparation, washup, sterilization,
and storage
Ifa single room is used, create a sterility gradient; theclean area for sterile handling should be located at one end of
the room, farthest from the door, and washup and sterilization
facilities should be placed at the other end, with preparation,
storage, and incubation in between
The preparation area should be adjacent to the washup and
sterilization areas, and storage and incubators should be readily
accessible to the sterile working area
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Sterile work should be located in a
quiet part of the tissue culture
laboratory and should be restricted
to tissue culture
Use a separate room or
cubicle if laminar-flow
hoods are not available
Cubicle
separate room 24
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The bench should be either
freestanding (away from the
wall) or sealed to the wallwith a plastic sealing strip or
mastic
Nothing should be stored onthe bench and any shelving
above should be used only in
conjunction with sterile work
The work area should be a plastic laminate-topped bench,
preferably plain white or neutral gray, to facilitate the
observation of cultures, dissection
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Laminar-flow hoods affordsgreater control of sterility at alower cost than providing aseparate sterile room. Because
only the operators arms enter thesterile area, with sterile air blownonto the work surface
Individual freestanding hoods are
preferable (they separate operatorsand can be moved around), butlaminar-flow wall or ceiling unitsin batteries can be used
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Chairs should be a suitable
height, with adjustable seat
height and back angle, and
able to be drawn up closeenough to the front edge of
the hood to allow
comfortable working well
within it
Select hoods that suite
your accommodation -
freestanding or bench
top - and allow plentyof legroom underneath
with space for pumps,
aspirators, and so forth
Laminar-flow hoods
should have a lateral
separation of at least
500 mm
A small cart, trolley, or
folding flap (300500
mm minimum) should be
provided beside each
hood for materials which
may be required but are
not in immediate use
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Should use a separate room as a quarantine and/or containment room
This is a separate aseptic room with its own laminar-flow hood (Class II
microbiological safety cabinet), incubators, freezer, refrigerator, centrifuge,
supplies, disposal and must be separated by a door or air lock from the
rest of the suite
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Newly imported cell lines or
biopsies can be handled here until
they are shown to be free ofcontamination, particularly
mycoplasma and proscribed
pathogens such as HIV or hepatitis
B
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It may be convenient to position a
bench for a cell counter,
microscope, etc., close to the
sterile handling area
The service bench should also
provide for the storage of sterile
glassware, plastics, pipettes, screw
caps, syringes..., in drawer units
below and open shelves above
It may also be used for other
accessory equipment, such as a
small centrifuge
1.3.4. Service Bench
service bench
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Hot room
Incubator
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Incubation may becarried out in
separateincubators or in athermostaticallycontrolled hot
room
If only one or two are required, incubators are inexpensive andeconomical in terms of space; but as soon as you require more thantwo, their cost is more than that of a simple hot room, and their use
is less convenient
Incubators also
lose more heatwhen they areopened and are
slower to recoverthan a hot room
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If you have the space within
the laboratory area or have
an adjacent room, it may be
possible to convert the area
into a hot room
The area need not be
specifically constructed as a
hot room, but it should beinsulated to prevent cold
spots being generated on the
walls
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Wooden furnishings should be avoided as much as possible
Incandescent lighting is preferable to fluorescent, which can causedegradation of the medium
The temperature of the hot room should be controlled within 0.5C
at any point
The hot room should have a small
bench, preferably stainless steel or
solid plastic laminate, and the bench
should accommodate an inverted
microscope, the flasks that you wish to
examine, and a notebook.
Alternatively, a small laminar-flow
hood and a small laminar-flow unit(1218 in) could be located in the
room
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Heat is best supplied via a fan
heater, domestic or industrial,
depending on the size of the
room. Approximately 23 kW
per 20m3 will be required,
depending on the insulation
A second fan, positioned on the
opposite side of the room and with
the airflow opposing that of the fan
heater, will ensure maximum
circulation. If the room is more than
22m, some form of ducting may be
necessary
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Thermostatsshould be of the proportional
controller type. When the door opens
and the room temperature falls,
recovery will be rapid; on the other
hand, the temperature will not
overshoot its mark
Ideally, there should be two
separate heaters (H1 and H2), each
with its own thermostat (HT1 and
HT2). One thermostat (HT1)
should be located diagonally
opposite and behind the opposing
fan (F1) and should be set at 37oC
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The other thermostat (HT2) should be located diagonally opposite H2
and behind its opposing fan (F2) and should be set at 36oC. Two safety
override cutout thermostats should also be installed and set at 38C
The thermostat sensors should be located in an
area of rapid airflow, close to the effluent from the
second, circulating, fan for greatest sensitivity38
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Overheating
The problem of unwanted heat gain isoften forgotten because so much care
is taken to provide heat
It can arise because of a rise in
ambient temperature in thelaboratory in hot weather or heat
produced from within the hot room
by apparatus such as stirrer motors,
roller racks, laminar-flow units
Try to avoid heat-producing
equipment in the hot room
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AccessIf a proportional controller, good
circulation, and adequate heating areprovided, an air lock will not be
required. The door should still be well
insulated, light, and easily closed
preferably, selfclosing
ThermometerA temperature recorder should be
installed and should have a chart that
is visible to the people working in the
tissue culture room. If possible, onehigh-level and one lowlevel warning
light should be placed beside the
chart or at a different, but equally
obvious, location 40
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The need for extensive preparation ofmedia in small laboratories can be
avoided if there is a proven source of
reliable commercial culture media
Smaller laboratories may to purchase
readymade media, then need only to
prepare reagents, such as salt
solutions and
ethylenediaminetetraacetic acid
(EDTA), bottle these and water, and
package screw caps and other small
items for sterilization
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If reliable commercial media are difficult to obtain, the
preparation area should be large enough to accommodate a coarse
Heat-stable
solutions and
equipment can be
autoclaved or dry-
heat sterilized atthe nonsterile end
of the preparation
area
and a fine balance, apH meter, and, if
possible, an
osmometer
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Washup and sterilization facilitiesare best situated outside the tissue
culture lab
If the sterilization facilities must be located in the tissue culture
lab, place them nearest the air extract and farthest from the
sterile handling area
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The washup area should have plenty of space for soaking glassware
and space for an automatic washing machine, should you require one
There should also be plenty of bench space for handling baskets ofglassware, sorting pipettes, and packaging and sealing packs for
sterilization. You will need spacefor a pipette washer and dryer
Autoclaves, ovens, and distillation apparatus should be located in a
separate room if possible, with an efficient extraction fan
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Sinks should be deep enough (450 mm) to allow manual washing and
rinsing of your largest items without having to stoop too far to reach
into them. They should measure about 900 mm from floor to rim
Each washing sink will
require four taps: a single
coldwater tap, a combined
hot-and-cold mixer, a cold tap
for a hose connection for arinsing device, and a
nonmetallic or stainless steel
tap for deionized water from a
reservoir above the sinkWashing Up Sink and Pipette Washer
Sinks should Stainless steel or
polypropylene
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Storage must be provided for the following items ensuring
sterile and nonsterile are kept separate and clearly labeled:
3Sterile liquids, at room temperature (salt solutions, water...), at 4C
(media), and at 20C or 70C (serum, trypsin, glutamine...)
Sterile and nonsterile glassware, including media bottles and pipettes1
3
2 Screw caps, stoppers, etc., sterile and nonsterile
4 Apparatus such as filters, sterile and nonsterile
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Liquid nitrogen to replenish freezers; the liquid
nitrogen should be stored in two ways
A piped supply of CO2 can be taken to work stations, or
else the CO2 supply can be piped from a pressurized tank of
CO2 that is replenished regularly
Cylinder storage for carbon dioxide, in separate
cylinders for transferring to the laboratory as required
Gloves, disposal bags, etc
Sterile disposable plastics (culture flasks and Petri dishes,
and syringes)8
7
6
5
9
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Adequate ventilation must beprovided for the room in which the
nitrogen is stored and dispensed,
preferably with an alarm to signify
when the oxygen tension falls below
safe levels
The reason for this safety measure is
that filling, dispensing, and
manipulating freezer stocks areaccompanied by the evaporation of
nitrogen, which can replace the air in
the room
Safety Note
liquid nitrogen
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Refrigerators and freezers should be located toward the
nonsterile end of the lab. They require maintenance and
periodic defrosting
Remember to allocate
sufficient space for storage
In general, separate 20C
freezers are better than a walk-
in 20C room. They areeasier to clean out and
maintain, and they provide
better backup if one unit failsfreezer
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Several independent refrigerators will occupy more
space than the equivalent volume of cold room, but
may be easier to manage and maintain in the event
of failure
Cold room
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