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					T3: Getting started with tissue culture-media preparation, sterile technique, and laboratory

Chapter 3 gives us the proper tools to use in laboratory.

Equipment and supplies:
Lots of equipment is needed in the laboratory, but there always needs to be enough space to store and
perform your experiments. Tubes, culture containers, various glassware able to withstand autoclaving,
transport trays, tube racks, are some the things needed in a laboratory. Common science lab equipment
to weigh, heat, stir, sterilize, etc will be needed as well.

High-quality water is absolutely necessary for tissue culture. Tap contains lots of unnecessary particles
that had not been filtered out and can change the result of an experiment. There are many different
ways that water can be purified, but the most reliable for tissue culture is deionization and distillation.
(in most cases both are used in the lab).

Culture Room:
This is the room where new cultures are placed. It has white fluorescent lights with shelves underneath
them so cultures can grow. Constant airflow, and temperature control is important. Usually 26 to 28
degrees Celsius is the best for most cultures.
This reminds me of my internship at Twixwood where I used a seeding chamber. It’s basically the same
concept. It was small room with lights and an air conditioner. All our planted seeds would go into there
until the seeds germinated.

Common tissue culture media:
The main types of medium that are used in tissue culture are:
-Murashige and Skoog: most commonly used for basic tissue and callus
-Linsmaier and Skoog: difference from MS is the organic components (except inositol and thiamine HCL)
-Gamborg’s B5: for soybean callus culture, but happens to be a good basal medium
-Schenk and Hildebrandt: for callus culture of monocots and dicots
-Lloyd and McCown: for woody plants (less salt)
-White: for tomato roots
-Nitsch: for anther culture

Components of tissue culture medium
Inorganic mineral elements:
Macrosalts contain: Nitrogen, potassium, magnesium, calcium, phosphorus, and sulfer.
Microsalts contain: Boron, cobalt, iron, manganese, molybdenum, copper, and zinc.

Organic compounds:
Sugars- L sucrose is most common. Glucose, fructose, sorbitol, and maltose are some other possibilities.
Vitamins- Thiamine (Vitamin B1) is the only essential one. Others are nicotinic acid (niniacin),vitamins
B2,B3, B6, B5, E, M, C, are just a few others that are added to the medium sometimes.
Inositol- necessary for some monocots, dicots, and gymnosperms.
Amino acids, complex organic compounds, Polyamines, and activated charcoal, and antioxidants are
some other organic compounds that are sometimes included in media.

Plant growth regulators:
Auuxins- induce cell elongation, swelling of tissue, apical dominance, adventitious root formation, and
somatic embryogenesis
Cytokinins- promote cell division, axillary shoot formation, and stimulate initiation and growth of shoots.
Gibberellins- stimulate elongation of internodes and said to be useful for meristematic growth.
Abscisic acid: can promote maturation and germination of somatic embryos in woody plants.
2,4-D is mentioned in this section. According to our book, 2,4-D can induce ethylene formation. 2,4-D is
quite an interesting chemical because it acts as an herbicide that was once used in what they call Agent
Orange. To defoliate the vast jungles of Vietnam(during the Vietnam war) to spot hiding soldiers, this
orange chemical (hence the name ‘Orange’) was used. It wasn’t until years later they found how harmful
the chemical was on humans.

Culture support systems:
Agar is a gelling agent that is the most common in tissue culture for mediums. It is important to get one
that is high quality, and make sure the gel is not too hard or soft. Agarose, which is the purified version
of agar, is used for protoplast/single cell culturing. Gellan gums are alternate gelling agents that are nice
to detect contamination (because it is clear), but re-liquefied and re-gelled again like agar.

Procedure of making stock solutions:
Here discussed, are the many steps needed to make a stock solution. Proper containers, substance
concentrations, pH levels, and proper mixing of mineral salts, PGRs, and vitamins are explained clearly in
the book.

Sterilizing equipment and media:
Because we are creating an optimal environment for plants, unwanted fungi and bacteria also thrive in
such environment. This calls for the importance for everything to be sterilized. Autoclaving at 121
degrees Celsius with a pressure of 15 psi for at least 15 minutes (more for larger things) is recommended.
Media should also be autoclaved, where it is to be put in a vessel with a closure at the top so it doesn’t
spill over. Some components that cannot be autoclaved are filtered through a bacteria-proof

Sterile Transfer Hood:
It is has what is called positive-pressure ventilation and a HEPA filter. A constant flow of air goes into the
filter and lets out clean air on the working area. This makes it so that bacterial and fungal spores cannot
rest on the material at the work table. The surface area must be sterile, so sterilized petri dishes,
laboratory-grade paper towels, and ceramic tiles are some types that can be used.
Storage of culture media:
After being sterilized, media can be stored by keeping it sealed up, placed in a dark, cool place. Storing
it for 5-7 days, you can detect any contamination. However, some tissue culture call for fresh medium.

Surface disinfesting plant tissues:
It is important that the tissue be clean from anything. There are many different ways of disinfesting
tissues and depending on the sensitivity of plant, different methods are used.

F4-Laboratory Design and Layout
A laboratory should be designed to create a sterile and functional environment. These are a few
considerations that are listed:

-Ventilation: A balance in pressure to keep contaminated air outside of the lab.
-Accommodation: Being able to fit in every workspace in the right place
-Renovations: Location of facilities is of big importance
-Access: Doorways and ceilings must be wide/high enough to allow easy access.
-Quarantine: Must have a location because some cells are a biohazard risk.
Room should have filtered air, be easy to clean, and have easy access to places. Keep in mind of the
services you will need like power (outlets), gas, electricity, and possibly carbon dioxide, compressed air,
and a vacuum.

A laboratory must have six main functions:
1) Sterile Handling- still/quiet area only for tissue culture, have a laminar flow, quarantine containment
area (preferably in a different room), and a service bench.
2) Incubation- Can be done in either an incubator or a hot room. Hot rooms require certain
specification, mainly keeping the room evenly warm, yet keeping it from overheating.
3) Preparation- area must be large enough to do all the prep work. This area can take up a bit because
it includes things like pH meter, scale, space for mixing solutions, and places to store the media (fridge).
4) Washup- best if located outside of tissue culture lab to keep the humidity levels constant. Should be
enough space for placement of washed material, and possibly a deep enough sink for washing larger
5) Sterilization-( Is it just me, or did they not include this section? ) Sterilizing the lab would be
important. Maintaining its cleanliness is quite essential.
6) Storage- must store sterile and nonsterile should be kept apart. The refrigerator and freezer should
be located on the nonsterile side of the room. There should be plenty of room for storage for later

A tissue culture lab must have priorities in purchasing equipment. Deciding if they are essential,
beneficial, or useful will help you decide what equipment you should get.

Aseptic Area
Laminar-Flow Hood: possible to perform without one. However, if it is a busy lab, keeping everything
sterile is difficult. Considering the space and comfort level of the hood, there are many questions to
ask yourself when buying one.
Pipette Cylinders: made of polypropylene and freestanding.
Aspiration Pump: Used to suction out used or unneeded substance in flasks. A vacuum pump can be
used instead.
Service carts: used for easy access to other material, and relocating them around the lab.
Inverted Microscope: easier than using a regular microscope, you can put your culture right on the
tray and not have to take it out of the bottle/dish/etc.
Centrifuge: Required for some cell suspensions and get the cells to get closer to each other.
Sterile Liquid Handling-Pipetting and Dispensing: With a vacuum pump/line, ease of pipetting is nice.
There are many different kinds of ways to dispense fluids and different volume sizes. Some are even
Cell Counter: Used for precise quantitative growth kinetics.
CCD Camera and Monitor: For observing living cultures.

Incubator: For keeping temperatures warm at a constant degree.
Humid CO2 Incubator: for petri dishes and multi well plates. They require high humidity and a certain
CO2 level.
Tempreature Recorder: To monitor the environment of the incubator.
RollerRacks: Used to scale up monolayer culture. (the culture will have more surface area this way)
Magnetic Stirrer: used to rapidly dissolve chemicals.

Preparation and Sterilization
Washup: There needs to be a place where you can soak glassware to make sure they are clean. For
pipetts, they have special washer and driers.
Water Purifier: Used to rinse glassware, dissolving media, and diluting concentrates. There are many
techniques and filters.
Sterlizing and Drying Ovens: It is preferred to sterilize glassware. However autoclave will do.
Steam Sterilizer (Autoclave): It’s basically a pressure cooker they use in the cafeteria to steam foods. It
helps sterilize all the material. There are different procedures for a wet cycle and one for a dry cycle.
Balances: getting one that can weigh 10mg to 100g or even a kilogram is recommended.
pH Meter: Phenol red can be used instead, but there are some chemicals that cannot be read with it.
Using a meter is more precise.
Hot Plate Magnetic Stirrer: Mainly used for stable solutions that should be quickly mixed at a higher
Automatic Dispenser: like a soap dispenser at home, except that it measures up volumes of substance.
Conductivity Meter: used to quickly check an ionic concentration.
Osmometer: checks solutions so that you can know how to mix solutions more accurately.

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