Setting Up a Tissue Culture Lab

Any laboratory, in which tissue culture techniques are performed, regardless of the
specific purpose, must contain a number of basic facilities. These usually include the
following:

• A general washing area

• A media preparation, sterilization, and storage area
• An aseptic transfer area
• Environmentally controlled incubators or culture rooms
• An observation/data collection area.

Washing Area

The washing area should contain large sinks, some lead-lined to resist acids and alkalis,
draining boards, and racks, and have access to demineralized water, distilled water, and
double-distilled water. Space for drying ovens or racks, automated dishwashers, acid
baths, pipette washers and driers, and storage cabinets should also be available in the
washing area.

Media Preparation Area

The media preparation area should have ample storage space for the chemicals, culture
vessels and closures, and glassware required for media preparation and dispensing.
Bench space for hot plates/stirrers, pH meters, balances, water baths, and media-
dispensing equipment should be available. Other necessary equipment may include air
and vacuum sources, distilled and double-distilled water, Bunsen burners with a gas
source, refrigerators and freezers for storing stock solutions and chemicals, a microwave
or a convection oven, and an autoclave or domestic pressure cooker for sterilizing media,
glassware, and instruments.
In preparing culture media, analytical grade chemicals should be used and good
weighing habits practiced. To insure accuracy, and exact step-by-step routine should be
developed for media preparation and a complete checklist required of all media preparers
even for the simplest media.
The water used in preparing media must be of the utmost purity and highest quality.
Tap water is unsuitable because it may contain cations (ammonium, calcium, iron,
magnesium, sodium, etc.), anions (bicarbonates, chlorides, fluorides, phosphates, etc.),
microorganisms (algae, fungi, bacterial), gases (oxygen, carbon dioxide, nitrogen), and
particulate matter (silt, oils, organic matter, etc.) Water used for plant tissue culture
should meet, at a minimum, the standards for type II reagent grade water, i.e., be free of
pyrogens, gases, and organic matter and have an electrical conductivity less than 1.0
μmho/cm.
The most common and preferred method of purifying water to type II standards is a
deionization treatment followed by one or two glass distillations. The deionization
treatment removes most ionic impurities, and the distillation process removes large

1
organic molecules, microorganisms, and pyrogens. Three other methods that will
produce type II purity water are absorption filtration, which uses activated carbon to
remove organic contaminants and free chlorine; membrane filtration, which removes
particulate matter and most bacterial contamination; and reverse osmosis, which removes
approximately 9% of the bacterial, organic, and particulate matter as well as about 90%
of the ionized impurities.

Transfer Area

Under very clean and dry conditions, tissue culture techniques can be successfully
performed on an open laboratory bench. However, it is advisable that a laminar flow
hood or sterile transfer room be utilized for making transfers. Within the transfer area
there should be a source of electricity, gas, compressed air, and vacuum.
The most desirable arrangement is a small dust-free room equipped with an overhead
ultraviolet light and a positive-pressure ventilation unit. The ventilation should be
equipped with a high-efficiency particulate air (HEPA) filter. A 0.3-μm HEPA filter of
99.97-99.99% efficiency works well. All surfaces in the room should be designed and
constructed in such a manner that dust and microorganisms do not accumulate and the
surfaces can be thoroughly cleaned and disinfected. A room of such design is
particularly useful if large numbers of cultures are being manipulated or large pieces of
equipment are being utilized.
Another type of transfer area is a laminar flow hood. Air is forced into the unit through
a dust filter then passed through a HEPA filter. The air is then either directed downward
(vertical flow unit) or outward (horizontal flow unit) over the working surface. The
constant flow of bacteria-free filtered air prevents nonfiltered air and particulate matter
from settling on the working surface.
The simplest type of transfer area suitable for tissue culture work is an enclosed plastic
box commonly called a glove box. This type of culture hood is sterilized by an
ultraviolet light and wiped down periodically with 95% ethyl alcohol when in use. This
type of unit is used when relatively few transfers are required.

Culture Room

All types of tissue cultures should be incubated under conditions of well-controlled

temperature, humidity, air circulation, and light quality and duration. These
environmental factors may influence the growth and differentiation process directly
during culture or indirectly by affecting their response in subsequent generations.
Protoplast cultures, low-density cell suspension cultures, and anther cultures are
particularly sensitive to environmental cultural condition.
Typically, the culture room for growth of plant tissue cultures should have a temperature
between 15° and 30° C, with a temperature fluctuation of less than ±0.5°C; however, a
wider range in temperature may be required for specific experiments. It is also
recommended that the room have an alarm system to indicate when the temperature has
reached preset high or low temperature limits, as well as continuous temperature recorder
to monitor temperature fluctuations. The temperature should be constant throughout the
entire culture room (i.e., no hot or cold spots). The culture room should have enough

2
fluorescent lighting to reach the 10,000 lux; the lighting should be adjustable in terms of
quantity and photoperiod duration. Both light and temperature should be programmable
for a 24-hr period. The culture room should have fairly uniform forced-air ventilation,
and a humidity range of 20-98% controllable to ±3 percent. Many incubators, large
growth chambers, and walk-in environmental chambers meet these specifications.

3
BASIC LABORATORY EQUIPMENT

Many tissue culture techniques require similar basic laboratory equipment. The
following items are commonly found in a laboratory for in vitro propagation of plant
materials:

QTY ITEM DESCRIPTION APPROX. ITEM FUNCTION

COST
1 Water Purification System; water should have a Purification of water for
resistivity of at least 200,000 ohms-cm and a media preparation
conductivity 5.0 micromhos/cm

1 Electronic Balance (0.01 g readability; 200 g 295.00 Measuring out biochemicals

minimum capacity) and media

1 pH meter (range 0-14 +/- 0.01; automatic 74.95 Measurement and adjustment
temperature compensation 0-600 C; one or two of media pH
point calibration)

1 Hot Plate/Stirrer (7” x 7” ceramic top; variable 295.00 Mixing & heating media and
heating range from ambient to 4000 C; variable stock
stirring speed from 50-150 rpm; chemically
resistant)

1 Refrigerator/freezer; capable of maintaining a 750.00 Storage of stock solutions,

refrigerator temperature of 0-50 C with a freezer media, hormones
temperature of approximately –200 C

1 Laminar Flow Transfer Hood; incoming air 3500.00 Provide a sterile atmosphere
should be HEPA filtered to remove 99.99% of to transfer cultures
particles larger that 0.3μm; should meet or
exceed the Class 100 Clean Standard 209D;
maintain a flow of 90 fpm +/- 20% at static
pressures of 0.6-1.2”

4 liter Isopropyl alcohol 10.00 Used to sterilize instruments

and work areas

1 roll Aluminum foil, heavy duty; (18” x100 ft roll) 2.50 Used to wrap instruments
prior to sterilization, cover
vessels

12 Beakers, 250 mL 7.75 Mixing solutions

12 Beakers, 1000 mL 13.75 Mixing solutions

6 Beakers, 2000 mL 32.25 Mixing solutions and media

6 Beakers, 4000 mL 46.25 Mixing solutions and media

4 ea Bottle, Water; pre-labeled for use in dispensing 6.00 Rinsing instruments, beakers,
water; 500 mL capacity transplants from tissue
culture

4
QTY ITEM DESCRIPTION APPROX. ITEM FUNCTION
COST
4 ea Bottle, Isopropyl Alcohol; pre-labeled for use in 6.00 Rinsing sterile hood work
dispensing isopropyl alcohol; 500 mL capacity surfaces

1 case Bottle, 100 mL; Type 1 borosilicate glass with 24.00 Storage of stock solutions,
volume graduations; supplied with 33-340 black sterile distilled water, media
polypropylene cap with rubber liner; non-sterile

1 case Bottle, 500 mL; Type 1 borosilicate glass with 50.00 Storage of stock solutions,
volume graduations; supplied with 33-340 black sterile distilled water, media
polypropylene cap with rubber liner; non-sterile

2 Brush, flask or bottle Cleaning glassware

1 case Culture tubes, 25 x 150 mm, borosilicate glass; 146.50 Starting cultures in Stage I
500 tubes/case

1 case Culture tube racks; holds 40, 25 mm culture 90.50 Holding culture tubes
tubes; withstands temperatures up to 1210 C

500 Closure, for 25 mm culture tubes, 500 each 57.50 Sealing culture tubes

1 case Culture vessel, baby food jar; glass culture 42.50 Culture vessel for
vessel; autoclaveable; uses Magenta B Cap (C Maintaining plant cultures
903) as closure; 110 mm height; 100/case

1 case Culture vessel, baby food jar; glass culture 32.50 Culture vessel for
vessel; autoclavable; uses Magenta B Cap (C Maintaining plant cultures
903) as closure; 72 mm height; 100/case

1 case Magenta B Cap; autoclavable closure for baby 215.50 Closure for baby food culture
food jars; fits both C 904 and C 900; clear Vessel
polypropylene closure; 100/case

2 Culture vessel; autoclavable culture vessel and lid Culture vessel for
cases made from clear polypropylene; round vessel maintaining plant cultures
measures; 250/case

1 gal Detergent 5.75 Cleaning glassware

1 case Culture dishes, disposable, sterile, 100 x 25 mm 173.50 For Stage I cultures sterile
surface for cutting explants

1 gal Disinfectant, commercial brand 2.00 Disinfects explants

1 Erlenmeyer flask, wide mouth, 1000 mL 18.50 Mixing media

1 Erlenmeyer flask, wide mouth, 2000 mL 25.50 Mixing media

1 Erlenmeyer flask, 4000 mL Mixing media

1 Erlenmeyer flask, 6000 mL Mixing media

5
QTY ITEM DESCRIPTION APPROX. ITEM FUNCTION
COST
1 case Filtration system, vacuum; disposable, plastic, 63.00 Sterilization of heat liable
sterile system designed for filtration of fluids stock solutions
needed in tissue culture; polystyrene, screw-cap
base for sterile storage; 200 mL; 47 mm
diameter/0.22 μm pore size nylon membrane;
12 units/case

3 Forceps, dressing; 10” length, serrated, stainless 30.75 Transferring tissue

steel

3 Forceps, bayonet; stainless steel 71.85 Transferring tissue

5 Forceps; stainless steel, very fine point, 6” length Transferring tissue

1 Graduated cylinders, glass or polypropylene, 10 3.50 Preparing stock solutions

mL

1 Graduated cylinders, glass or polypropylene, 100 6.25 Preparing stock solutions

mL

1 Graduated cylinders, glass or polypropylene, 20.25 Preparing stock solutions

1000 mL

1 pkg Lab markers, assorted colors (10) Labeling cultures

1 roll Parafilm (4” x 250 ft) 36.50 Wrapping culture Closure

100 Pipets, 1 mL, graduated, sterile, disposable, 14.50 Measuring out stock solutions
individually wrapped

100 Pipets, 5 mL, graduated, sterile, disposable, 26.25 Measuring out stock solutions
individually wrapped

100 Pipets, 10 mL, graduated, sterile, disposable, 24.95 Measuring out stock solutions
ea individually wrapped

100 Pipets, 25 mL, graduated, sterile, disposable, 58.25 Measuring out stock solutions
ea individually wrapped

1 Pipet pump, electric 190.00 For safely measuring liquids

1 pkg Gloves, hot or autoclave; provides protection up 6.00 Safely removing hot items
to 3500 F from autoclave

2 ea Scalpel handle; No 3: 5” length; stainless steel 69.75 Cutting explants

2 ea Scalpel handle; No 3L: 8” length; stainless steel 83.75 Cutting explants

1 box Scalpel blades; No 10; stainless steel; 56.25 Cutting explants

individually wrapped; sterile; 100/box

1 Scoop, large plastic 5-3/4” x 9” bowl 11.00 Measuring large volumes of

biochemical’s

6
QTY ITEM DESCRIPTION APPROX. ITEM FUNCTION
COST
1 pkg Scoop, medium, 30 cm length, 2/pkg 34.65 Measuring small to medium
amounts of biochemical’s

2 ea Spatula, stainless steel blade, wooden handle, 4” 11.00 Measuring small to medium
wide x 19/32” thick amounts of biochemical’s

1 pkg Spatula, micro; 8” length, flat nickel stainless 25.25 Measuring small to medium
steel amounts rounded ends, 2”
end length; of biochemical’s

1 ea Sterilizer, pressure cooker; operates between 116- 500.00 Sterilizing media and
1260 C; 10-20 psi; aluminum sterilizer has a 30 x instrument
32.2 cm chamber; is supplied with chamber,
lid with pressure gauge, immersion heater and
safety valve, electric

1 ea Sterilizer, Autoclave; operates at 1210 C with dial 6500.00 Sterilizing media and
for fast or slow exhaust; 0-60 minute timer; stain- instruments
less unit with 66 41 cm chamber

1 ea Sterilizer, dry heat with glass beads 120 V (S636) 395.00 Sterilizes instruments in hood
or 240 V (S637) between transfers

1 pkg Stirring bars, magnetic; Teflon covered; 58.00 Used for mixing stock
leakproof, seamless and contain permanent solutions and in media
Alnico V magnets preparation

1 ea Stir bar retriever; contains an Alnico V magnet 9.00 Retrieving stir bars from
sealed in polyethylene; 18” length mixing vessel

1 roll Tape, autoclave indicator; impregnated to show 3.95 Identifying autoclaved media
the word “Autoclaved” after 15 minutes of
exposure at 1210 C

1 roll Tape, Label; all purpose, self adhesive tape can Labeling cultures, storage
be written on with pen or permanent marker bottles, media vessels, etc.

2 ea Thermometer; -20-1500 C temperature range 40.00 Measuring temperature of

liquids and culture room

1 Timer; electronic, countdown timer alarm, 30.00 Timing sterilization and

stopwatch feature general lab use

1 case Towels; commercial, single fold 13.00 Can be sterilized to provide

sterile work surface for
cutting explants, gen lab use

500 Weighing boats, plastic; can be utilized for liquid 26.50 Measuring chemicals
ea or solid samples; 1-5/8” x 1-5/8” x 5/16”

500 Weighing boats, plastic; can be utilized for liquid 32.75 Measuring chemicals
ea or solid samples; 3-5/16” x 3-5/16” x 1”

7
QTY ITEM DESCRIPTION APPROX. ITEM FUNCTION
COST
500 Weighing boats, plastic; can be utilized for liquid 75.75 Measuring chemicals
ea or solid samples; 5-1/2” x 5-1/2” x 1”

The glassware used in tissue culture can generally be found in most laboratories. The
glassware, particularly the culture vessels, should be made of Pyrex or borosilicate glass.
Due to the increasing expense of this type of glass, many laboratories are successfully
converting to soda glass, which may be seven to eight times cheaper. Wide-neck
Erlenmeyer flasks (50-, 125-, 250-ml capacity) are commonly used as culture vessels;
large volume Erlenmeyer flasks are required for media preparation. Test tubes, petri
dishes, mason jars, baby food jars, and other glassware can also be adapted to tissue
culture. Since all new glass may release substances that affect the composition of the
medium, it is recommended that all new glassware be filled with water, autoclaved twice
with detergent, washed, and rinsed between washes before being used for tissue culture.
Other glassware commonly required in a tissue culture facility includes beakers,
volumetric flasks, pipettes, and graduated cylinders.

8
 Designing a Plant Micropropagation Laboratory

Mark P. Bridgen and John W. Bartok, Jr.

Departments of Plant Science and Agricultural Engineering
University of Connecticut
Storrs, Connecticut 06268
Originally published in the Proceedings of the International Plant Propagators Society, Vol. 37 (1987), pp.
462-467. Reprinted here and distributed by permission of the senior author. March 4, 1996.
http://aggie-horticulture.tamu.edu/tisscult/microprop/facilities/microlab.html

Tissue culture is rapidly becoming a commercial method for propagating new cultivars,
rare species, and difficult-to-propagate plants. From a few research laboratories several
years ago, a whole new industry is emerging. Currently, the demand for micropropagated
plants is greater than the supply with some plants. Some growers specialize in only the
micropropagation of plantlets, leaving the growing-on to others; many growers are
integrating a tissue culture laboratory into their overall operation.

In designing any laboratory, big or small, certain elements are essential for a successful
operation. The correct design of a laboratory will not only help maintain asepsis, but it
will also achieve a high standard of work.

FACILITIES

Careful planning is an important first step when considering the size and location of a
laboratory. It is recommended that visits be make to several other facilities to view their
arrangement and operation. A small lab should be set up first until the proper techniques
and markets are developed.

A convenient location for a small lab is a room or part of the basement of a house, a
garage, a remodeled office or a room in the headhouse. The minimum area required for
media preparation, transfer and primary growth shelves is about 150 sq ft. Walls may
have to be installed to separate different areas.

A good location includes the following:

1. Isolation from foot traffic.

2. No contamination from adjacent rooms.
3. Thermostatically controlled heat.
4. Water and drains for a sink.
5. Adequate electrical service.
6. Provisions for a fan and intake blower for ventilation.
7. Good lighting.

Larger labs are frequently built as free-standing buildings. Although more expensive to
build, the added isolation form adjacent activities will keep the laboratory cleaner.

Prefabricated buildings make convenient low cost laboratories. They are readily available

9
in many sizes in most parts of the country. Built-in-place frame buildings can also be
used. Consideration should be given to the following:

1. Check with local authorities about zoning and building permits.

2. Locate the building away from sources of contamination such as a gravel
driveway or parking lot, soil mixing area, shipping dock, pesticide storage, or dust
and chemicals from fields.
3. A clear span building allows for a flexible arrangement of walls.
4. The floor should be concrete or capable of carrying 50 pounds per square foot.
5. Walls and ceiling should be insulated to at least R-15 and be covered inside with a
water-resistant material.
6. Windows, if desired, may be placed wherever convenient in the media preparation
and glassware washing rooms.
7. The heating system should be capable of maintaining a room temperature at 70 F
in the coldest part of winter.
8. A minimum 3/4 in. water service is needed.
9. Connection to a septic system or sanitary sewer should be provided.
10. Air conditioning for summer cooling may be necessary.
11. Electric service capacity for equipment, lights and future expansion should be
calculated. A minimum 100 amp service is recommended.

GENERAL LABORATORY DESIGN

Cleanliness is the major consideration when designing a plant tissue culture laboratory.
Most companies are not aware of their losses from contamination, but estimates run from
less than 1% up to 50%. When you consider the high value of the product, no losses from
contamination are acceptable. Routine cleaning and aseptic procedures can decrease your
losses to less than 1%. Laboratories should have easy to wash walls and floors. Acrylic or
urethane epoxy wall paints can be used; cement floors can be painted with an epoxy or
urethane floor enamel or have an inlaid linoleum installed. High efficiency particulate air
(HEPA) filters or regular furnace filters can be installed over air intakes to the laboratory
or on furnaces. If possible, an enclosed entrance should precede the laboratory; sticky
mats can be laid there to help collect dirt from the outside, or shoes can be removed.

The traffic pattern and work flow in a laboratory must be considered in order to
maximize cleanliness. The cleanest rooms or areas are the culture room, i.e. primary
growth room, and the aseptic transfer area. It is best to design these rooms so they are not
entered directly from the outside of a building. The media preparation area, glassware
washing area, or storage area should be located outside these rooms. The primary growth
room and aseptic transfer room should be enclosed with doors leading to each. Traffic
through these areas can be minimized by installing pass-through windows. Ideally, the
media preparation area would lead to the sterilization area, which would lead to the
aseptic transfer room and eventually the primary growth room.

Unusual requirements for electricity and fire safety dictate that power installation be done
by professional electricians. Most wiring will require 110 volts. Temperature and fire

10
alarms are to be connected directly to telephone lines to give fast warnings of problems.
An emergency generator should be available to operate essential equipment during power
outages.

GLASSWARE WASHING AND STORAGE AREA

The glassware washing area should be located near the sterilization and media
preparation areas. When culture vessels are removed from the growth area, they are often
autoclaved to kill contaminants or to soften semi-solid media. The vessels can be easily
moved to the washing area if the autoclave or pressure cooker is nearby. Locate the
glassware storage area close to the wash area to expedite storage; these areas also need to
be accessible to the media preparation area.

The glassware area should be equipped with at least one large sink; two sinks are
preferable. Adequate work space is required on both sides of the sink; this space will be
used for glassware soaking tubs and drainage trays. Plastic netting can be placed on
surfaces near the sink to reduce glassware breakage and enhance water drainage. The
pipes leading from the sink can be PVC to resist damage from acids and alkalis. Both hot
and cold water should be available with water distillation and/or deionization devices
nearby. Mobile drying racks can be stored nearby and lined with cheesecloth to prevent
water dripping and loss of small objects. Locate ovens or hot air cabinets (75 C) close to
the glassware washing and storage area. Dust-proof cabinets, low enough to allow easy
access, can be used in the storage area.

MEDIA PREPARATION AND STERILIZATION AREA

The water source and glassware storage area should be convenient to the media
preparation area. Benches, suitable for comfortable working while standing (34 to 36in.)
and deep enough (24 in.) to hold equipment listed below are essential. Their tops should
be made with molded plastic laminate surfaces that can tolerate frequent cleanings.

There is a variety of equipment available for micropropagation laboratories; this

equipment is generally located in the media preparation area. The equipment budget will
determine the type and amount purchased. All laboratories need the following basics:

1. Refrigerator/freezer-- This is needed to store chemicals and stock solutions. Small

laboratories may find it adequate to use countertop refrigerators.
2. High quality water--Bottled water can be purchased inexpensively and placed in
the media preparation area. Larger businesses may find it economical to obtain
distillation or deionization devices; these would normally be located in the
glassware washing area. Small, inexpensive, low production Pyrex distillation
devices can be purchased by small businesses that want the convenience of a still,
but not the cost.
3. Balances--High quality balances are essential for a micropropagation laboratory;
this is one area where it is difficult to find an inexpensive substitute. A triple
beam balance is useful for large amounts over 10 grams, but a balance that can

11
 measure down to 2 mg is essential. Most laboratories have both a microbalance
and a less sensitive top loading balance; the latter can be used more quickly and
efficiently for less sensitive quantities.
4. Hot plate/stirrer--At least one hot plate with an automatic stirrer is needed to
make semi-solid media. This purchase can be eliminated by using a stove and
hand stirring the media while it heats; however, the time saved by using a stirring
hot plate is worth the money spent.
5. pH meter--This is needed to measure media pH. Some laboratories use pH
indicator paper, however this method is considerably less accurate and could
severely affect the results.
6. Aspirator or vacuum pump--Aspirators can be easily attached to a water source
and used for filter sterilization of chemicals. They are also used to disinfest plant
material. Vacuum pumps are faster and more efficient, but also more expensive.
7. Autoclave--An autoclave or pressure cooker is a vital part of a micropropagation
laboratory. High pressure heat is needed to sterilize media, water, glassware, and
utensils. Certain spores from fungi and bacteria will only be killed at a
temperature of 121 F and 15 pounds per square inch (psi). Self generating steam
autoclaves are more dependable and faster to operate.
8. Optional equipment--A variety of non-essential equipment is available for tissue
culture laboratories; individual needs and equipment cost will determine what can
be purchased. Microwave ovens are convenient for defrosting frozen stocks and
heating agar media. Dissecting microscopes are useful to have in the laboratory
for meristeming, dissecting floral and shoot apices, and observing plant culture
growth. Labwashers, or regular dishwashers, can be useful. Automatic media
dispensers are helpful when pipetting large volumes of media.

PRIMARY GROWTH ROOM

Temperature, relative humidity, lighting units, and shelves need to be considered in the
culture room. All of these environmental considerations will vary depending on the size
of the growth room, its location, and the type of plants grown within it. For example, a
small primary growth room located in a cool, North American climate, can be placed in
an unheated or minimally heated basement. The ballasts from the fluorescent lights do
not need to be separated; rather they can be used as a heating source. Excess heat can be
blown out of the growth room and used to heat other parts of the basement or building. In
this case, solid wood shelves with air spaces located between shelves are recommended
to prevent the cultures on shelves above lights from becoming over-heated. A larger
growth room located in an above-ground location may need to have remote ballast and/or
a heat pump installed. Shelves in a larger growth room could then be glass or expanded
metal.

Temperature is the primary concern in culture rooms; it affects decisions on lights,

relative humidity, and shelving. Generally, temperatures are kept 76 +/-2 F. Heating can
be accomplished by traditional heating systems supplemented with heat from light
ballasts or space heaters. Cooling the room is usually a greater problem than heating;
cooler temperatures can be obtained by installing heat pumps, air conditioners, or exhaust

12
fans. Using outside windows to cool culture rooms invites contamination problems in the
summer and humidity problems in the winter.

Some plant cultures can be kept in complete darkness; however, most culture rooms are
lighted at 1 klux (approximately 100ft-c) with some going up to 5 to 10 klux. The plant
species being micropropagated will determine the intensity used. The developmental
stage of the plants will also help determine if wide spectrum or cool white fluorescent
lights are used. Rooting has been shown to increase with far-red light; therefore, wide
spectrum lights should be used during stage III and cool-white lights can be used during
Stages I and II. Automatic timers are needed to maintain desired photoperiods. Reflectors
can be placed over bulbs to direct their light. Heat generated by the lights may cause
condensation and temperature problems. In addition to using procedures previously
mentioned, small fans with or without polyethylene tubes attached, can be placed at the
ends of shelves to increase air flow and decrease heat accumulation.

Relative humidity (RH) is difficult to control inside growing vessels, but fluctuations in
the culture room may have a deleterious effect. Cultures can dry out if the room's RH is
less than 50%; humidifiers can be used to correct this problem. If the RH becomes too
high, a dehumidifier is recommended.

Shelving within primary growth rooms can vary depending upon the situation and the
plants grown. Wood is recommended for inexpensive, easy-to-build shelves. The wood
for shelves should be exterior particleboard or plywood and should be painted white to
reflect the room's light. Expanded metal is more expensive than wood, but provides better
air circulation; wire mesh of 1/4 or 1/2 in. hardware cloth can be used but tends to sag
under load. Tempered glass is sometimes used for shelves to increase light penetration,
but it is more prone to breaking. Air spaces, 2 to 4 in., between the lights and shelves will
decrease bottom heat on upper shelves and condensation in culture vessels. A room that
is 8 ft high will accommodate 5 shelves, each 18 in. apart, when the bottom shelf is 4 in.
off the floor. The top and bottom shelves may be difficult to work.

ASEPTIC TRANSFER AREA

In addition to the primary growth room, the aseptic transfer area needs to be as clean as
possible. It is preferable to have a separate room for aseptic transfer; this decreases spore
circulation and allows personnel to leave shoes outside the room. Special laboratory
shoes and coats should be worn in this area. Laminar flow hoods or still-air boxes can be
placed in this room and used for all aseptic work. Ultraviolet (UV) lights are sometimes
installed in transfer areas to disinfect the room; these lights should only be used when
people and plant material are not in the room. Safety switches can be installed to shut off
the UV lights when regular room lights are turned on. Surfaces inside the aseptic transfer
area should be smooth to minimize the amount of dust that settles. Several electric outlets
are to be installed to accommodate balances, flow hoods, bacti-cinerators, and
microscopes.

13