The first thing to consider in the construction of the group culture room is what equipment is needed and what kind of cultivation conditions are needed in the group culture room. After a comprehensive understanding of these, we can be adapted to the new or use the existing housing construction laboratory. In the design of the group culture room, the general situation should be in accordance with the process of group culture to design, and at the same time to consider the character of smooth flow, the rational use of space. Plant tissue culture requires strict aseptic conditions, requires certain professional instrumentation, equipment and appliances, but also need to artificially control the temperature, light, humidity and other culture conditions.
First, the laboratory
A standard tissue culture laboratory should include a washing room,? Configuration room, inoculation room, incubation room, observation room (cooling room), sterilization room, washing seedling room. In practice, it can be combined with the feasible conditions, combined part.
(a) washing room
The washing room to complete the cleaning, drying and storage of glassware and other instruments. The room should be equipped with a large sink for the washing of culture vessels, preferably a concrete tank lined with white tiles. In order to prevent the glassware from being touched, rubber sheets can be laid, and the upper and lower watercourses should be unobstructed. Have large plastic baskets available for placing culture vessels. They can be placed directly on the shelves to save space and facilitate transportation work. There is an empty drying rack to empty dry clean culture vessels.
(B) configuration room, sterilization room (can be separated)
Preparation room requires bright, ventilated. In the preparation room to complete the preparation of culture medium and test tube seedlings out of the bottle, cleaning and organizing work. If there are more rooms, the preparation room can be divided into two parts: the washing room and the configuration room. The washing room is specifically responsible for the test tube seedlings out of the bottle and the cleaning of culture vessels; configuration room is responsible for the preparation of culture medium, packaging, packaging and autoclaving and other work.
In order to complete the preparation of culture medium, the preparation room should also be equipped with the following instruments and equipment:
1. workbench
Its height should be convenient for preparation.
2. Medicine cabinet
For placing commonly used medicines.
3. General refrigerator
Mainly used for storage of mother liquor, various perishable, easily decomposed chemicals and plant materials.
4. Electronic analytical balance and tray balance
Electronic analytical balance with an accuracy of 0.001g, used for weighing large amounts of elements, trace elements, vitamins, hormones, and other micro-pharmaceuticals; tray balance with an accuracy of 0.1g, used for weighing a large amount of sugar and agar and so on. The balance should be placed in a dry, vibration-free fixed operating table.
5. Water purifier or electric distiller
Water purifier to provide pure water, water quality. Electric distiller, made of hard glass or metal. Distilled water is used to prepare the mother liquor or culture medium, with culture medium can be replaced by tap water, if the experimental requirements are strict, then distilled water must be used.
6. Magnetic stirrer
Magnetic stirrer is used to accelerate the stirring of insoluble substances, such as a variety of chemicals, agar powder and so on. Magnetic stirrers can also be heated to make it more favorable for dissolution.
7. Thermostatic water bath or thermostatic oscillating water bath
The water bath is used for the dissolution of insoluble drugs, agar dissolution. If there is no water bath, can also be used 1500 or 2000W electric stove or rice cooker instead. Electric stove should be equipped with aluminum pot.
8. Acidimeter
Tissue culture in the medium pH accuracy is very important, should use an acidimeter, if there is no acidimeter, you can also use pH test paper for rough measurement. Before using the acidimeter for the first time, the standard solution should be adjusted for positioning and then fixed. When measuring pH, the solution to be measured must be well mixed. If the temperature of the medium is too high, adjust the temperature twist on the pH meter to make it equal to the temperature of the medium when measuring. Pay attention to protect the glass electrode, the electrode should be rinsed with distilled water after use, cover the electrode cap.
9. Medium dispensing equipment
Small tissue culture laboratories can use beakers, funnels, etc. as a tool for dispensing medium. Can also be used as a medical "under the mouth of the cup" as a packaging tool, in the "under the mouth of the cup" on the lower mouth tube over a section of flexible hose, plus a spring stop clamp, the use of the very fit. Larger scale or require a higher rate, can consider the use of liquid automatic quantitative filling equipment.
10. Autoclave
Used for sterilization of culture media and instruments. Small-scale laboratories can choose a small portable autoclave. If it is a continuous large-scale production, you should choose a large vertical or horizontal autoclave. Usually use electricity as an energy source.
11. oven
For drying clean glassware, can also be used for dry heat sterilization and determination of dry weight. For drying need to maintain 80 ~ 100 ℃; dry heat sterilization need to maintain 150 ℃, up to 1 ~ 3 hours; if the determination of dry weight, the temperature should be controlled at 80 ℃ drying.
12. Constant temperature incubator
Within the temperature regulator, biochemical incubator is also equipped with a light source for the cultivation of plant materials.
(C) inoculation room
Inoculation room is an important room for the separation of plant material inoculation and culture transfer. Its aseptic conditions play an important role in the success of tissue culture.
Under the premise of work convenience, the aseptic operation room should be small rather than large, generally 7 ~ 8m2; the requirements of the floor, ceiling and walls as close as possible to the clean, easy to clean and disinfected. The aseptic operation room should be equipped with a pull door to reduce the air disturbance when opening and closing the door.
Aseptic operation room requires dry and quiet, clean and bright, in the appropriate location of the hanging 1 ~ 2 ultraviolet sterilization lamps, used to frequently irradiate sterilization. It is best to place a small air-conditioning, so that the room temperature can be controlled, so that the doors and windows can be closed to reduce the air convection with the outside world.
The aseptic operating room should have a buffer room, the area of 2 m2 is appropriate. Before entering the aseptic operating room in this change of shoes, in order to reduce the entry and exit with stray bacteria. Buffer room is also best to install a UV sterilization lamp, used to irradiate sterilization.
The main equipment of the aseptic operation room and the use of:
1. Inoculation box
In the case of less investment, you can use the inoculation box instead of the ultra-clean table. Inoculation box rely on airtight, pharmaceutical fumigation and ultraviolet light to ensure that the internal space is sterile. However, the operation activities are restricted, long preparation time, low efficiency.
2. Ultra-clean bench
The advantages of the ultra-clean bench is easy to operate, more comfortable, high efficiency, short preparation time. Power on 10 minutes to operate, can be used for a long time. And can ensure that the sterile material is not contaminated during the transfer of inoculation.
3. Instruments for aseptic operation.
According to the dosage of a single ultra-clean table: 1 alcohol lamp; 20-25cm long medical tweezers 1; No. 4 dissecting knife 1, a number of anatomical blades; 15cm medical scissors 1; 500ml wide-mouth bottle 1, put the alcohol inside, the modern professional histocultivation room with inoculation instruments sterilizer and container mouth sterilizer, used to soak the tweezers, knives, scissors, etc.; used to put a small shelf for the cauterization of knives and tweezers. The small frame.
4. shelves.
Storage of autoclaved media awaiting inoculation.
(D) culture room
is the inoculation of the material for the culture of the growth of the place. The size of the culture room can be determined by the size and number of culture racks and other ancillary equipment needed. The design is based on the principle of utilizing space and saving energy. The height is slightly higher than the culture frame is appropriate, the surrounding walls are required to have adiabatic fire performance.
Cultivation materials on the culture frame culture. Most of the culture frame is made of metal, generally set up five layers, the lowest layer is about 10 centimeters above the ground. The lowest layer is about 10 centimeters above the ground, and the other layers are spaced about 30 centimeters apart, and the culture frame is about 1.8 meters high. Culture frame length, because generally in each layer of the upper end of the installation of fluorescent lamps for lighting, so are based on the length of fluorescent lamps and design, such as the use of 28W fluorescent lamps, the length of 1.25m, 21 W length of 1 meter can be. Width is generally 50cm.
The most important factor in the culture room is the temperature, generally maintained at about 20 to 27 ℃, can be installed window or vertical air-conditioning units. Required temperature varies depending on the species of tropical plants and frigid plants, etc. It is best to have different culture rooms for different species.
Indoor humidity is also required to be constant, relative humidity to maintain at 70 ~ 80% is good, can be installed humidifier.
When the culture frame is equipped with fluorescent lamps, a timer switch clock can be installed to control the lighting time, the light intensity of plant tissue culture is generally in the range of 1000 to 4000 lux (lux), 10 to 16 hours of lighting per day, there is also a need for continuous lighting. Short sunlight plants need short sunlight conditions, long sunlight plants need long sunlight conditions.
Most modern histoculture laboratories are designed to use natural sunlight as a source of energy, which not only saves energy, but also accepts the sun's seedlings to grow well, domestication easy to survive. In the rainy days with light as a supplement.
Supplemental light source is currently used in the professional culture room with full-spectrum cold light or led special lamps for culture.
Secondly, ? Equipment and devices
(a) glassware
In tissue culture in the preparation of medium and culture requires a large number of glassware. Requirements by the alkaline solubility of small hard glass made to ensure long-term storage of drugs and the effect of culture; culture also requires good light transmission, can withstand high pressure and high temperature, can be convenient to put into the medium and the culture of the material of the vessel, according to the purpose of the cultivation and the requirements of the different types of specifications can be used in the glassware. Among them to test tubes, triangular bottles, Petri dishes, etc. are used more.
The most commonly used is the triangular flask, specifications for 100, 250, 500 ml, etc., generally use 100 ml triangular flasks, regardless of the static or oscillating culture are applicable. Its culture area is large, conducive to the growth of tissue, light is also better than the test tube. Because of the small mouth of the bottle, it is not easy to contaminate.
Petri dishes are commonly used 9, 12 cm in diameter and other specifications, the requirements of the upper and lower can be closely matched. This is in the free cells, protoplasts, pollen and other static culture, caretaker culture, germination of sterile seeds, separation of plant material, etc. need to be used.
Test tubes commonly used caliber 18 × 180 mm or 20 × 200 mm specifications. It can be used to cultivate higher test tube seedlings, and in addition, it is not easy to contaminate.
Culture vessels can also be locally sourced, the use of some substitutes. Factory production can be used in wide-mouth 200ml or so canning jars, plus translucent plastic cover, because the bottle mouth is large, so a large number of propagation when the operation is convenient, high efficiency, but also reduces the damage of the culture material. But the disadvantage is easy to cause pollution.
Currently, culture containers and glassware required for the preparation of culture medium are gradually replaced by plastic containers. Plastic containers are lightweight, transparent, unbreakable, low-cost advantages, such as culture containers are mostly flat-bottomed square box shape, can increase the number of cultivated plants, and can be stacked one layer on top of the other, thus saving space. These plastic products are mostly made of polypropylene materials, can withstand high temperatures, can be autoclaved. Some products are disposable consumables, which not only saves washing labor, but also saves time and raises the rate. Disposable plastic containers or glass bottles with screw caps, without the need for additional lids, is very convenient to use.
Bottle cork can be used in a variety of ways, to have a certain degree of aeration and airtight, in order to prevent the medium from drying and contamination of stray bacteria. Previously, the closure of commonly used cotton plugs. However, this sealing method is very easy to contaminate in the summer, and it is not easy to maintain the humidity of the culture medium. Now more polypropylene plastic film as a seal, with a string ligation or rubber band band. In order to increase the aeration, can be lined with a layer of sulfate paper or kraft paper. This is economical and convenient, and good ventilation. Specialized sealing materials such as sealing film can also be used.
In tissue culture, the preparation of medium, storage of mother liquor, sterilization of materials, etc. need a variety of chemical experiments with glassware, including 100, 250, 1000 ml beaker; 100, 1000 ml measuring cylinder; 100, 1000 ml reagent bottles (brown) and so on.
(B) instrumentation
1. tweezers
The main use of medical tweezers. According to the needs of the operation there are various types, if you use 100 ml triangular bottle as a culture bottle, can be 220 cm long forceps. The tweezers are too short. It is easy for the hand to touch the mouth of the bottle, resulting in contamination. Tweezers that are too long are not flexible. Some small parts of the fine operation with clock tweezers, such as in the separation of young leaves at the tip of the stem, because of its sharp tip, so almost instead of scissors.
2. scissors
Can be used in the Department of Pentacology with medium-sized scissors. Mainly used to cut off stem segments, leaves and so on. You can also use curved scissors, because of its head bend, you can go deep into the mouth of the bottle to cut.
3. Scalpel
Cutting smaller materials can be used scalpel. Separation of stem tip meristematic tissue, sometimes also use a scalpel. Commonly used dissecting knife blade can be frequently switched, the knife should be kept sharp, otherwise the cutting will cause extrusion, causing a large number of deaths of the surrounding cells and tissues, affecting the culture effect.
4. Dissecting needle
Dissecting needle is used to penetrate into the culture bottle, transfer cells or healing tissue. It can also be used to isolate young leaves from the tip of a microstem.
Histoculture room design? The construction of histoculture room, histoculture instrument and equipment--Jinan Tenghao Scientific Instrument Co.