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

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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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