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Setting Up a Tissue Culture Lab: Transfer Area

Under very clean and dry conditions, tissue culture techniques
Any laboratory, in which tissue culture techniques are can be successfully performed on an open laboratory bench.
performed, regardless of the specific purpose, must contain a However, it is advisable that a laminar flow hood or sterile
number of basic facilities. These usually include the following: transfer room be utilized for making transfers. Within the
transfer area there should be a source of electricity, gas,
 A general washing area compressed air, and vacuum.
 A media preparation, sterilization, and storage area The most desirable arrangement is a small dust-free room
 An aseptic transfer area equipped with an overhead ultraviolet light and a positive-
 Environmentally controlled incubators or culture pressure ventilation unit. The ventilation should be equipped
rooms with a high-efficiency particulate air (HEPA) filter. A 0.3-m
 An observation/data collection area. HEPA filter of 99.97-99.99% efficiency works well. All
surfaces in the room should be designed and constructed in such
Washing Area a manner that dust and microorganisms do not accumulate and
the surfaces can be thoroughly cleaned and disinfected. A room
The washing area should contain large sinks, some lead-lined to of such design is particularly useful if large numbers of cultures
resist acids and alkalis, draining boards, and racks, and have are being manipulated or large pieces of equipment are being
access to demineralized water, distilled water, and double- utilized.
distilled water. Space for drying ovens or racks, automated Another type of transfer area is a laminar flow hood. Air is
dishwashers, acid baths, pipette washers and driers, and storage forced into the unit through a dust filter then passed through a
cabinets should also be available in the washing area. HEPA filter. The air is then either directed downward (vertical
flow unit) or outward (horizontal flow unit) over the working
Media Preparation Area surface. The constant flow of bacteria-free filtered air prevents
nonfiltered air and particulate matter from settling on the
The media preparation area should have ample storage space for working surface.
the chemicals, culture vessels and closures, and glassware The simplest type of transfer area suitable for tissue culture
required for media preparation and dispensing. Bench space for work is an enclosed plastic box commonly called a glove box.
hot plates/stirrers, pH meters, balances, water baths, and media- This type of culture hood is sterilized by an ultraviolet light and
dispensing equipment should be available. Other necessary wiped down periodically with 95% ethyl alcohol when in use.
equipment may include air and vacuum sources, distilled and This type of unit is used when relatively few transfers are
double-distilled water, Bunsen burners with a gas source, required.
refrigerators and freezers for storing stock solutions and
chemicals, a microwave or a convection oven, and an autoclave Culture Room
or domestic pressure cooker for sterilizing media, glassware, and
instruments. All types of tissue cultures should be incubated under conditions
In preparing culture media, analytical grade chemicals should of well-controlled temperature, humidity, air circulation, and
be used and good weighing habits practiced. To insure accuracy, light quality and duration. These environmental factors may
and exact step-by-step routine should be developed for media influence the growth and differentiation process directly during
preparation and a complete checklist required of all media culture or indirectly by affecting their response in subsequent
preparers even for the simplest media. generations. Protoplast cultures, low-density cell suspension
The water used in preparing media must be of the utmost purity cultures, and anther cultures are particularly sensitive to
and highest quality. Tap water is unsuitable because it may environmental cultural condition.
contain cations (ammonium, calcium, iron, magnesium, sodium, Typically, the culture room for growth of plant tissue cultures
etc.), anions (bicarbonates, chlorides, fluorides, phosphates, should have a temperature between 15 and 30 C, with a
etc.), microorganisms (algae, fungi, bacterial), gases (oxygen, temperature fluctuation of less than 0.5C; however, a wider
carbon dioxide, nitrogen), and particulate matter (silt, oils, range in temperature may be required for specific experiments.
organic matter, etc.) Water used for plant tissue culture should It is also recommended that the room have an alarm system to
meet, at a minimum, the standards for type II reagent grade indicate when the temperature has reached preset high or low
water, i.e., be free of pyrogens, gases, and organic matter and temperature limits, as well as continuous temperature recorder to
have an electrical conductivity less than 1.0 mho/cm. monitor temperature fluctuations. The temperature should be
The most common and preferred method of purifying water to constant throughout the entire culture room (i.e., no hot or cold
type II standards is a deionization treatment followed by one or spots). The culture room should have enough fluorescent
two glass distillations. The deionization treatment removes most lighting to reach the 10,000 lux; the lighting should be
ionic impurities, and the distillation process removes large adjustable in terms of quantity and photoperiod duration. Both
organic molecules, microorganisms, and pyrogens. Three other light and temperature should be programmable for a 24-hr
methods that will produce type II purity water are absorption period. The culture room should have fairly uniform forced-air
filtration, which uses activated carbon to remove organic ventilation, and a humidity range of 20-98% controllable to 3
contaminants and free chlorine; membrane filtration, which percent. Many incubators, large growth chambers, and walk-in
removes particulate matter and most bacterial contamination; environmental chambers meet these specifications.
and reverse osmosis, which removes approximately 9% of the
bacterial, organic, and particulate matter as well as about 90% of
the ionized impurities.

non-sterile 1 case Bottle. Water.99% of transfer cultures particles larger that 0.BASIC LABORATORY EQUIPMENT Many tissue culture techniques require similar basic laboratory equipment. supplied with 33-340 black polypropylene distilled water.75 Mixing solutions 12 Beakers.00 Provide a sterile atmosphere to HEPA filtered to remove 99. supplied with 33-340 black polypropylene distilled water. B 974 6. (18” x100 ft roll) NA 2.20% at static pressures of 0.0 micromhos/cm 1 Electronic Balance (0. 1 Water Purification System.00 Storage of stock solutions. incoming air should be NA 3500. Type 1 borosilicate glass with volume 50. variable heating H 926 295. refrigerator temperature of 0-50 C with a freezer media.00 Measuring out biochemicals and capacity) media 1 pH meter (range 0-14 +/. 2000 mL B 939 32. flask or bottle Cleaning glassware .00 Mixing & heating media and range from ambient to 4000 C. 200 g minimum B 933 295. one or two point calibration) media pH 1 Hot Plate/Stirrer (7” x 7” ceramic top. pre-labeled for use in dispensing water. should meet or exceed the Class 100 Clean Standard 209D. 4000 mL B 960 46.0.00 Storage of stock solutions. The following items are commonly found in a laboratory for in vitro propagation of plant materials: Items Required to Start a Plant Cell Culture Laboratory QTY ITEM DESCRIPTION PHYTOTECH APPROX.50 Used to wrap instruments prior to sterilization. 500 mL.2” 4 liter Isopropyl alcohol NA 10.000 ohms-cm and a preparation conductivity 5. chemically resistant) 1 Refrigerator/freezer. sterile graduations.25 Mixing solutions and media 6 Beakers.00 Used to sterilize instruments and work areas 1 roll Aluminum foil. automatic temperature P 976 74. media cap with rubber liner. variable stirring speed stock from 50-150 rpm. capable of maintaining a NA 750.01. ITEM FUNCTION CATALOGUE COST NO. heavy duty.01 g readability. pre-labeled for use in B 987 6.25 Mixing solutions and media 4 ea Bottle.3m. maintain a flow of 90 fpm +/. 500 mL capacity transplants from tissue culture 4 ea Bottle. cover vessels 12 Beakers. beakers. Type 1 borosilicate glass with volume 24. 1000 mL B 931 13.75 Mixing solutions 6 Beakers.6-1. water should have a NA Purification of water for media resistivity of at least 200. 500 mL capacity surfaces 1 case Bottle.00 Rinsing instruments. sterile graduations. 250 mL B 910 7. non-sterile 2 Brush.00 Rinsing sterile hood work dispensing isopropyl alcohol.00 Storage of stock solutions.95 Measurement and adjustment of compensation 0-600 C. 100 mL. media cap with rubber liner. Isopropyl Alcohol. hormones temperature of approximately –200 C 1 Laminar Flow Transfer Hood.

Maintaining plant cultures 110 mm height. baby food jar.25 Preparing stock solutions 1 pkg Lab markers. fits both C 904 and C 900. glass culture vessel.50 Mixing media 1 Erlenmeyer flask. 25 mm culture tubes.22 m pore size nylon membrane. screw-cap base for sterile storage.50 Preparing stock solutions 1 Graduated cylinders. uses Magenta B Cap (C 903) as closure. plastic. glass or polypropylene. serrated. 4000 mL Mixing media 1 Erlenmeyer flask. disposable. C 900 32. sterile. glass or polypropylene. 25 x 150 mm.85 Transferring tissue 5 Forceps.75 Transferring tissue 3 Forceps. Items Required to Start a Plant Cell Culture Laboratory QTY ITEM DESCRIPTION PHYTOTECH APPROX. 1 case Culture tubes. 6000 mL Mixing media 1 case Filtration system. round vessel plant cultures measures.00 Sterilization of heat liable stock system designed for filtration of fluids solutions needed in tissue culture. glass culture vessel. C 904 42. assorted colors (10) NA Labeling cultures . wide mouth. stainless steel F 957 71. 100/case 2 cases Culture vessel. 500 each C 925 57. 1000 mL C 968 20. dressing. borosilicate glass. very fine point. 200 mL.50 Sealing culture tubes 1 case Culture vessel. C 908 90. autoclavable closure for baby food C 903 215. 1000 mL F 985 18. autoclavable culture vessel and lid C 913 Culture vessel for maintaining made from clear polypropylene. vacuum. ITEM FUNCTION CATALOGUE COST NO. 100 x 25 mm D 940 173. 100/case 1 case Magenta B Cap.25 Preparing stock solutions 1 Graduated cylinders. 10 mL C 962 3. 6” length Transferring tissue 1 Graduated cylinders. glass or polypropylene. uses Magenta B Cap (C 903) as closure. for 25 mm culture tubes. Maintaining plant cultures 72 mm height. 250/case 1 gal Detergent NA 5. stainless steel F 952 30.50 Culture vessel for autoclavable. bayonet.50 Closure for baby food culture jars. holds 40. disposable. 100 mL C 964 6. baby food jar.50 Mixing media 1 Erlenmeyer flask. 10” length.50 Starting cultures in Stage I tubes/case 1 case Culture tube racks. sterile NA 63. 100/case 1 case Culture vessel. polystyrene.75 Cleaning glassware 1 case Culture dishes. wide mouth.50 Holding culture tubes withstands temperatures up to 1210 C 500 Closure.00 Disinfects explants 1 Erlenmeyer flask. 500 C 930 146. commercial brand NA 2. stainless steel. 2000 mL F 986 25. clear polypropylene Vessel closure. 47 mm diameter/0. 12 units/case 3 Forceps.50 Culture vessel for autoclaveable.50 For Stage I cultures sterile surface for cutting explants 1 gal Disinfectant.

No 10.95 Identifying autoclaved media word “Autoclaved” after 15 minutes of exposure at 1210 C . instruments less unit with 66 41 cm chamber 1 ea Sterilizer. P995 58. 0-60 minute timer. stain. ITEM FUNCTION CATALOGUE COST NO. lid with pressure gauge. individually S970 56. magnetic.00 Measuring small to medium x 19/32” thick amounts of biochemical’s 1 pkg Spatula. large plastic 5-3/4” x 9” bowl NA 11. sterile. medium. 30 cm length.50 Wrapping culture Closure 100 Pipets. P994 24. 18” length vessel 1 roll Tape.00 Sterilizing media and instrument C.00 Safely removing hot items from 3500 F autoclave 2 ea Scalpel handle. 10 mL.00 Sterilizes instruments in hood 240 V (S637) between transfers 1 pkg Stirring bars. disposable.75 Cutting explants 1 box Scalpel blades. dry heat with glass beads 120 V (S636) or S636 395. stainless steel S963 69. 10-20 psi. hot or autoclave. P990 14. stainless steel. aluminum sterilizer has a 30 x 32.00 For safely measuring liquids 1 pkg Gloves. graduated. micro. disposable. Autoclave. 2” end length.95 Measuring out stock solutions individually wrapped 100 ea Pipets. 1 roll Parafilm (4” x 250 ft) S 911 36. stainless steel blade. graduated. 5 mL. operates at 1210 C with dial for NA 6500.00 Retrieving stir bars from mixing in polyethylene.00 Sterilizing media and fast or slow exhaust.00 Used for mixing stock solutions seamless and contain permanent Alnico V magnets and in media preparation 1 ea Stir bar retriever. 100/box 1 Scoop.25 Measuring small to medium amounts rounded ends. No 3L: 8” length. autoclave indicator.2 cm chamber. wooden handle.25 Measuring out stock solutions individually wrapped 1 Pipet pump. disposable. contains an Alnico V magnet sealed NA 9. is supplied with chamber. impregnated to show the T 998 3. sterile. graduated. P993 26. sterile.50 Measuring out stock solutions individually wrapped 100 Pipets. electric NA 190. sterile.25 Measuring out stock solutions individually wrapped 100 ea Pipets. No 3: 5” length. pressure cooker. operates between 116-1260 NA 500. disposable. 1 mL. provides protection up to NA 6. electric 1 ea Sterilizer. NA 58. graduated.25 Cutting explants wrapped. Teflon covered. sterile. immersion heater and safety valve.75 Cutting explants 2 ea Scalpel handle. 8” length.00 Measuring large volumes of biochemical’s 1 pkg Scoop. 25 mL. flat nickel stainless steel NA 25. Items Required to Start a Plant Cell Culture Laboratory QTY ITEM DESCRIPTION PHYTOTECH APPROX. of biochemical’s 1 ea Sterilizer. 2/pkg NA 34. leakproof.65 Measuring small to medium amounts of biochemical’s 2 ea Spatula. 4” wide NA 11. stainless steel S973 83.

particularly the culture vessels. The glassware. Label.75 Measuring chemicals solid samples. and rinsed between washes before being used for tissue culture. . many laboratories are successfully converting to soda glass. 3-5/16” x 3-5/16” x 1” 500 ea Weighing boats. electronic. media vessels. 1 roll Tape. washed. baby food jars. 5-1/2” x 5-1/2” x 1” The glassware used in tissue culture can generally be found in most laboratories. large volume Erlenmeyer flasks are required for media preparation. petri dishes. 2 ea Thermometer.50 Measuring chemicals solid samples. -20-1500 C temperature range NA 40.00 Measuring temperature of liquids and culture room 1 Timer. should be made of Pyrex or borosilicate glass. which may be seven to eight times cheaper. autoclaved twice with detergent.75 Measuring chemicals solid samples.00 Timing sterilization and general feature lab use 1 case Towels. Since all new glass may release substances that affect the composition of the medium. pipettes. stopwatch NA 30. ITEM FUNCTION CATALOGUE COST NO. can be utilized for liquid or W 881 75. storage written on with pen or permanent marker bottles. countdown timer alarm.00 Can be sterilized to provide sterile work surface for cutting explants. 125-. all purpose. gen lab use 500 ea Weighing boats. volumetric flasks. single fold 13. plastic. Test tubes. can be utilized for liquid or W 880 32. 250-ml capacity) are commonly used as culture vessels. 1-5/8” x 1-5/8” x 5/16” 500 ea Weighing boats. and graduated cylinders. plastic. etc. commercial. Wide-neck Erlenmeyer flasks (50-. Other glassware commonly required in a tissue culture facility includes beakers. and other glassware can also be adapted to tissue culture. Due to the increasing expense of this type of glass. can be utilized for liquid or W 879 26. Items Required to Start a Plant Cell Culture Laboratory QTY ITEM DESCRIPTION PHYTOTECH APPROX. self adhesive tape can be Labeling cultures. plastic. mason jars. it is recommended that all new glassware be filled with water.