General introduction of Plant Tissue Culture.
General history of Plant Tissue Culture.
Glass wares cleaning.
Glassware cleaners and its procedures.
Chromic acid.
Safe use of chromic acid.
Rinsing of glasswares.
Sterilization of contaminated glasswares for Plant Tissue Culture.
Cleaning specific type of glasswares for Plant Tissue Culture.
Hot-air oven.
Auto clave
Handling and storing of glasswares for Plant Tissue Culture.
Basic laboratory equipments and apparatus for Plant Tissue Culture.
In our laboratory different type of rooms are available for different types work.
Points of works in our laboratory.
Importance of tissue culture with cleaning laboratory and glasswares.
Conclusion.
References.
INTRODUCTION OF PTC
The field of plant tissue culture is based on the fact that plants can be separated into their component parts( organs, tissue or cells), which can be manipulated in vitro and then grown back into complete plants.
The term tissue culture is commonly used in a very wide sense, to include in vitro culture of plant cells, tissue as well as organs.
Plant tissue culture techniques have played a pivotal role in addressing many questions of basic and applied fields of plant science. Initially, this technique was used for studying the roles of hormones in cytodifferentiation and organogenesis.
HISTORY
The in vitro techniques were developed initially to demonstrate the totipotency of plant cells predicted by Haberlandt in 1902.
Totipotency is the ability of a plant cell to generate in a whole plant and perform all the functions of development, which are characteristic of zygote, i.e., its ability to develop into a complete plant.
CLEANING OF GLASSWARE
Wash labware as quickly as possible after use. If a thorough cleaning is not possible immediately, put glassware to soak in water.
If labware is not cleaned immediately, it may become impossible to remove the residue.
Most new glassware is slightly alkaline in reaction. For precision chemical tests, new glassware should be soaked several hours.
In acid water (a 1% solution of hydrochloric or nitric acid) before washing.
Brushes with wooden or plastic handles are recommended as they will not scratch or abrade the glass surface.
PROCEDURE FOR CLEANING OF. GLASSWARES
Soak glassware in 10℅soap water (labolene) for overnight.
Transfer glassware to conc. HCl and keep for 2 hours.
Properly rinse glassware in tap water.
Wash the glassware at least twice with distilled water.
Keep glassware for drying in oven at 70-80°c for 1-2 hours.
Autoclave keep glassware in oven at 140-160°c for 2 hours.
WASHING AREA
The washing area is one of the most important places in a laboratory as its use starts even before the actual start of an experiment and continues till the experiment gets completely over.
This area is mainly used for cleaning and washing of glassware and other items used in actual experiments, therefore maintenance of its cleanliness is equally important.
The washing area should consist of sinks and proper draining boards. It should also have provision for production of deionized/double-distilled water.
Moreover, it must also have a specific place to dry the washed glassware/vessels and a storage cabinet to store cleaned and dried culture vessels.
GLASSWARE CLEANERS
When washing, soap,labolene, detergent, or cleaning powder (with or without an abrasive) may be used. Cleaners for glassware include.
The water should be hot. For glassware that is exceptionally dirty, a cleaning
powder with a mild abrasive action will give more satisfactory results. The abrasive should not scratch the glass. During the washing, all parts of the glassware should be thoroughly scrubbed with a brush.
This means that a full set of brushes must be at hand-brushes to fit large and small test tubes, funnels, graduates and various sizes of flasks and bottles.
CHROMIC ACID
The term chromic acid is usually used for a mixture made by adding concentrated sulfuric acid to a dichromate, which may contain a variety of compounds, including solid chromium trioxide.
This kind of chromic acid may be used as a cleaning mixture for glass.
Chemical formula - H2CrO4 OR H2Cr2O7
IUPAC name - Chromic acid
Appearance – Dark red crystals.
Melting point – 197°C
Boiling point – 250°C
Solubility in water - 169 g/100mL
Acidity - ( -0.8 to 1.6)
SAFE USE OF CHROMIC ACID
If glassware becomes unduly clouded or dirty or contains coagulated organic matter, it must be cleansed with chromic acid cleaning solution1.
When chromic acid solution is used the item may be rinsed with the cleaning solution or it may be filled and allowed to stand.
The length of time it is allowed to stand depends on the amount of contamination on the glassware.
Relatively clean glassware May require only a few minutes of exposure; if debris is present, it may be necessary to let the glassware stand all night.
Extra care must be taken to be sure chromic acid solution is disposed of properly.
RINSING OF GLASSWARES
It is imperative that all soap,labolene detergents and other cleaning fluids be removed from glassware before use.
After cleaning, rinse the glassware with running tap water. When test tubes, graduates, flasks and similar containers are rinsed with tap water, allow the water to run into and over them for a short time, then partly fill each piece with water, thoroughly shake and empty at least six times.
Rinse with distilled water.
To conserve distilled water, use a five gallon bottle as a reservoir.
Store it on a shelf near your clean-up area.
STERILLIZING CONTAMINATED. GLASSWARES
Glassware which is contaminated with microbes in the container, test tubes, culture media, petri dishes, etc., must be sterilized before cleaning.
It can best be processed in the laboratory by placing it in a large bucket or boiler filled with water, to which 1-2% soft soap or detergent like that labolene has been added, and boiled for 30 minutes.
The glassware can then be rinsed in tap water, scrubbed with detergent, rinsed again.
You may autoclave glassware or sterilize it in large steam ovens or similar apparatus.
If Fungus or spore-bearing bacteria are present, autoclaving is absolutely necessary.
CLEANING SPECIFIC TYPE OF. GLASSWARES
Hot-air oven.
Specially use for drying of glasswares at 70-85ºc temp.
Auto clave.
The purpose of aseptic technique in plant tissue cultures is the elimination of microorganisms during experimental procedures.
Media and other apparatus are made sterile by autoclaving them at 15 lbs/square intch (121°C) for 15 minutes.
The use of disposable sterile plastic ware reduces the need for autoclaving, however, augments the total cost of the experiment.
HANDLING OF GLASSWARES
Never use broken glassware.
Remove broken glassware immediately.
If you connect tubes to glassware be aware of breaking/cutting danger. To avoid this wrap the part you handle with a sturdy towel.
Never put big forces on glass because it will break.
Never put pressure on glass because it could explode violently.
If you put pressure (column, nitrogen) always leave a way out to avoid overpressure.
If you handle reaction with develop pressure, leave the vessel open.
HANDLING AND STORING OF GLASSWARES
To prevent breakage when rinsing or washing pipets, cylinders , be careful not to let tips hit the sink or the water tap.
Dry test tubes, culture tubes, flasks and other labware by hanging them on wooden pegs or placing them in baskets with their Mouths downward and allowing them to dry in the air; or place them in baskets to dry in an oven.
Drying temperatures should not exceed 140°C. Line the drying basket with a clean cloth to keep the vessel mouths clean.
Dry pipets and cylinders by standing them on a folded towel. Protect clean glassware from dust. This is done best by plugging with cotton, corking, taping a heavy piece of paper over the mouth or placing the glassware in a dust-free cabinet.
Store glassware in specially designed racks. Avoid breakage by keeping pieces separated.
BASIC LABORATORY EQUIPMENTS AND APPARATUS
The following points enlists the items that are commonly required in a laboratory for in vitro propagation of plant materials:
Lab wares
Water purification system – provides purified water to make stock solution and other medium.
Weighing balance – measurements of biochemical and media components.
pH meter – for measurement and adjustment of pH of the prepared medium.
Refrigerator and freezer – for the storage of stock solutions, hormones and prepared medium.
Laminor air-flow – provides a sterile atmosphere during transfer of the cultures.
Orbital shaking incubator – To properly shake the mixture.
Micro-wave oven – To homogenize or properly boiled the medium.
Forceps – Transferring tissue.
Scalpel handles – Cutting explants.
Scalpel blades - Cutting explants.
Sterilizer, pressure cooker – Sterilizing media and instruments.
PLASTIC WARES
Beakers – For preparation of media like 1 litre, 2 litre, 5 litre. Specially small size beakers like 500ml,are used for the pouring of homogenized media.
Measuring cylinders – Preparing stocks solutions.
Spatula – To measure small to medium amount of biochemical.
Detergent (Labolene) – To clean glasswares.
Brushes – To clean glassware’s.
Role tap – Labeling cultures, storage bottles, media vessels etc.
Gloves – Safely removing hot items from autoclave.
Parafilm – Wrapping culture closures.
Lab markers – Labeling cultures.
Tray – Transportation of media, glass wares, cultures etc. one place to another place.
GLASSWARE'S
Conical flasks - Mixing of stock solutions during media preparation and its subsequent storage.
Wash bottles – Rinsing instruments, beakers, transplants from tissue culture.
Bottles – To store stock solutions, sterile distilled water and autoclaved medium.
Culture tubes – For starting culture in stage 1.
Culture tube racks – Holding culture tubes.
Measuring cylinders – Preparing stocks solutions.
Glass pipettes – Measuring out stocks solutions.
Forceps – Transferring tissue.
Petri plate – For use inoculation and culturing etc.
IN OUR LABORATORY DIFFERENT TYPE OF ROOMS ARE AVAILABLE FOR DIFFERENT TYPES WORKS
POINTS OF WORKS IN OUR LABORATORY
Autoclaving of contaminated cultures.
Discarding of contaminated cultures.
Deeping of discarded glass wares in the plastic tub with labolene(detergent) for over night.
Washing of contaminated glass wares.
Cleaned glass wares has to be rinse with 2-3 times of tap water and one time of rinse only distilled water.
After cleaning of glass wares we put on the Hot-Air oven for the dryness.
Then we can use glass wares for preparation of medium.
We can Autoclave prepared medium for 15 min(15 lb/squ.int.)121°c.
Autoclaved medium are stored at culture room for the further use.
After 2-3 days we can use medium for the Inoculation of buds, seeds.
After 15 days or 20 days of inoculation we can subculture of inoculated buds etc.
IMPORTANCE OF TISSUE CULTURE WITH CLEANING LABORATORY AND GLASSWARES.
Should clean our laboratory and our laboratory glassware.
For in vitro conditions, there is requirement of sterilized glasswares because when the culture vessel were not properly cleaned, some pathogens (bacteria and fungi) are easily grow on the medium and they infect the explants also inhibit their growth.
The large scale production of ornamental plants.
Conservation of endangered plant species.
It helps in rapid multiplication of plants.
A large number of plantlets are obtained within a short period.
Plants are obtained through the year under controlled conditions, independent of seasons.
It is an easy, safe and economical method for plant propagation.
Genetically similar plants are formed by this method.
The rare plant and species are multiplied by this method and such plants are saved.
CONCLUSION
There have been instances in my experience where experiments didn’t work because the glassware was not washed properly.
If you carry through a solvent, an agent, or a chemical from one experiment to the next you can not expect your experiment to work properly.
REFERENCES
https://www.sigmaladrich.com>labwares.
https://alconox.com>laboratory.
B.D.Singh Biotechnology: Expanding Horizons.
Fundamentals of Life-Science, Part-2:Pranav Kumar|Usha Mina.
Advancing Science: Technical Bulletin-ALDRICH.
Murashige T and Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15(3): 473-497.
http://www.uni-bonn.de/~ulp50b/tissue_culture.htm.
SOURCE OF MATERIALS REQUIRED IN PLANT TISSUE CULTURE WORK
Chemicals required for media preparation were of analytical grade obtained from Sigma, Hi-media and LOBA chemical companies.
Sugar used as a source of sucrose.
Sucrose was obtained from local market.
