plant tissue culture designing a PTC lab

1. Designing a Plant Tissue
Culture Laboratory
Dr Fatimah Kayat

2. Outline
 Introduction
 General laboratory design
 Washing & Storage
 Media Preparation
 Instrumentation
 Aseptic transfer area
 Primary growth room

3. INTRODUCTION
 Certain elements are essential for a
successful operation
 The correct and strategic design helps to
achieve a high standard work.
 Careful planning is an important step
considering the size and location.
 A small lab should be set up first until the
proper techniques and markets are
developed.
 STRATEGIC PLANNING

5.  Good locations
Isolation from traffic.
No contamination from adjacent rooms.
 Access to water & electricity supply
 Connection to a septic system or sanitary
sewer
CONSIDER

6. CONSIDER
 Water service
 Air conditioning
 maintain constant temperature
 Electrical service capacity
 equipment, lights and future expansion
 An emergency generator
 to operate essential equipment during
power breakdown

7. PRIORITY
 Cleanliness
 No contamination.
 Easy to wash walls and floors.
 High efficiency particulate air (HEPA) filters.
 An enclosed entrance, sticky mats, no shoes.

8.  Traffic pattern and work flow
 The cleanest rooms or areas
 Aseptic transfer area
 Primary growth room
 Could not enter directly from outside.
 Should be enclosed with doors leading to each other
 Media preparation area, washing area, storage
area should be outside these rooms.
 Media preparation area should lead to the
sterilization area
TO CONSIDER

9.  Washing area located near sterilization and media
preparation area.
 should be equipped with at least one large sink.
 The pipes can be PVC to resist damage from acids
and alkaline
 Hot/cold water sources
 Water distillation and/or deionization devices
 Ovens or hot air cabinets (75 oC) close to the washing
and storage area
WASHING & STORAGE AREA

10.  Storage area should be close to the washing
area for storage
 Need to be accessible to the media preparation
area
 Dust-proof cabinets can be used in the storage
area
WASHING & STORAGE AREA

11.  Glassware
Good quality of borosilicate glass
Highly resistant to autoclaving, breakage and
scratch proof
E.g: Pyrex, Corning, Kimble, Schott

12.  Water source and storage area should be
convenient to the media preparation area.
 A variety of equipment for media preparation are
generally located in the media preparation area.
MEDIA PREPARATION &
STERILIZATION AREA

13. BASICS EQUIPMENT
 Refrigerator/freezer
 to store chemicals and stock solutions
(usually in media preparation area)
 High quality water
 Purchasing bottled water is not economical for
mass production
 Distilled water, double distilled water devices
(usually in washing area)

14.  A triple beam balance
 Useful for large amounts over 10 grams
 Can measure down to 2 mg is essential.
 Balances
 a microbalance
 a less sensitive top loading balance
BASICS EQUIPMENT

15.  Hot plate/stirrer
 At least one hot plate with an automatic
stirrer is needed to make semi-solid media.
BASICS EQUIPMENT

16.  pH meter
 to measure media pH.
 Some laboratories use pH indicator paper.
BASICS EQUIPMENT

17.  Aspirator or vacuum pump
 to filter sterilization of chemicals
 also used to disinfest plant material
Other Equipment

18.  Autoclave or Pressure Cooker
 vital part of a laboratory
 to sterile media, apparatus, used vessel
 Self generating steam autoclaves are more
dependable and faster to operate.
BASICS EQUIPMENT

19.  Optional equipment
Microwave oven are convenient for defrosting
frozen stocks and heating agar media
Dissecting microscopes are useful for
dissecting floral and shoot apices, and
observing plant culture growth
Labwashers/dishwashers are useful for
washing and drying glassware (mass
production)
OTHER EQUIPMENT

20.  Laminar flow hood
 For sterile working procedure
With High Efficiency Particulate Air (HEPA)
filter – filter the air to high degree
 With UV germicidal lamp – sterile the shell and
content when not in use
Horizontal laminar flow unit---An enclosed work area that has sterile
air moving across it. The air moves with uniform velocity along
parallel flow lines. Room air is pulled into the unit and forced through
a HEPA (High Energy Particulate Air) filter, which removes particles
0.3 μm and larger.
BASICS EQUIPMENT

21. LAMINAR FLOW HOOD

24.  Incubator
 To incubate the culture
 Controlled conditions (temperature, light,
humidity)
OTHER EQUIPMENT

25.  Need to be as clean as possible
 Preferably separated from other rooms
 to decreases spore circulation and allows personnel to
leave shoes outside the room.
 Laboratory shoes and lab coats should be worn
 Aseptic working area.
 Ultraviolet (UV) lights are sometimes installed to
disinfect the room (only switched on when people
and plant material are not in the room)
 Safety switches can be installed to shut off the UV
lights.
 air filters are sometimes installed to provide clean air
ASEPTIC TRANSFER AREA

26.  Smooth surfaces inside the aseptic transfer
area
 to minimize the amount of dust and easy to wipe.
 Several electric outlets are to be installed to
accommodate
 Flow hoods,
 Hot bead sterilizer
 Microscopes.
ASEPTIC TRANSFER AREA

27. ASEPTIC TRANSFER AREA –
commercial production

28. PRIMARY GROWTH
ROOM
 Should consider:
 temperature
 relative humidity
 lighting units and shelves.
 Varies depending on
 the size of the growth room
 its location
 the type of plants grown

29.  Temperature is the primary concern
 It affects decisions on lights, relative humidity, and
shelving.
 Generally, temperatures are kept at 24 ±1 oC.
 Cooling the room is usually a greater problem
than heating;
 cooler T can be obtained by installing heat pumps,
air conditioners or exhaust fans.
 Small fans can be placed at the ends of shelves
 to increase air flow and decrease heat accumulation.
PRIMARY GROWTH ROOM

30. PRIMARY GROWTH ROOM
 Shelving within primary growth rooms can vary
depending upon the situation and the plants
grown.
 Wood is inexpensive and easy-to-build shelves.
 It could be particleboard or plywood and painted
white to reflect the room's light.
 Expanded metal is more expensive but provides
better air circulation.

31.  Some plant cultures can /need to be kept in
complete darkness
 Most culture rooms are under light condition
 Depending on
 plant species
 developmental stage
 Automatic timers
 help to maintain desired photoperiods
 Reflectors can replaced bulbs
 reduce cost
 Heat generated by lights may cause
condensation and temperature problems.
PRIMARY GROWTH ROOM

32.  Relative humidity (RH) is difficult to control
inside growing vessels
 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.
PRIMARY GROWTH ROOM

33. PRIMARY GROWTH ROOM

34. PRIMARY GROWTH ROOM

35. PRIMARY GROWTH ROOM

36. Any questions?