ACTIVITY 2
MAKING MEDIA
A. Background
In addition to tissue culture equipment, media is one major factor in the success of the culture. Plant tissue culture media should contain all the substances necessary to ensure the growth of explants grown.Tissue culture media have the characteristics of each.
This means that not all media can be used on all plant culture. Because some media have different knadungan and concentration of substances that are needed or used in the culture.
B. PurposeThis lab aims to:
Acknowledge and practice how to make a stock solution
Knowing and practicing how to make MS medium (Murashige and Skoog)
Sterilization of the mediumII. REVIEW REFERENCESPlanting medium must contain all the substances necessary to ensure the needs of the explants. These ingredients are mixed salts contain a mixture of mineral resources element of the macro and micro elements, sugar, protein, vitamins, and plant hormones. The success of many tissue culture aseptic conditions also determined other than by the growing media. A mixture of media, can be suitable for certain plants, but can be less suitable for other crops.In tissue culture, the elements are not given in the form of a pure element, but a salt-shaped compound. Before mixed into the growing medium, mineral salts, it must be more dahulul dissolved in a certain concentration, so that in future the amount of growing medium per gram is in accordance with the provisions of distilled water as the solvent used (Rahardja, 1995)To meet the plant growth factor, tissue culture media containing either (Anonimous, 2009):A. Inorganic nutrients. There are 12 essential mineral nutrients for plant growth and some nutrients that affect the reported growth in vitro. For normal growth in tissue culture, the elements - these essential elements must be included in the culture medium.2. Organic nutrients. Plants growing under normal conditions are autotrophs and can synthesize all the organic material needs. Although plants in vitro can synthesize these compounds, suggesting that they do not produce sufficient amounts of vitamins for healthy growth and one or more vitamins must be added to the media. Thiamin is an essential vitamin, besides nicotine acid, pyridoxine and inositol is usually added.3. Carbon source. Plants grown in tissue culture heterotrophs and because they are not sufficient to synthesize the needs of its carbon, the sucrose must be added to the media. Carbon source provides energy for plant growth as well as the building blocks to produce larger molecules needed for growth.4. Order. Networks generally cultured on solid media such as a gel made by using the order or substitute for Gelrite or Phytagel sperti. Concentration used to range between 0.7 - 1.0%. At high concentrations to be very loud, very little water available, so the diffusion of nutrients to the plants is very bad. To overcome this problem, new products have been manufactured ole bernaman Agargel Sigma. This product is a mixture of agar and synthetic gels and offers the advantages of both products while reducing the problems of vitrification. This product can be created in the lab by mixing 1 g Gelrite (Phytagel) with 4 g of order as a thickening agent for 1 L media.5. pH. media are usually set in the range 5.6 - 5.8 but different plants may require a different pH for optimum growth. If the pH is higher than 6.0, media may be too hard and if the pH is less than 5.2, so as not to be compact.6. Growing an organizing principle. In the media generally added growth regulators. Plant growth regulators will be discussed separately at week 13.7. Water. distilata typically used in tissue culture, and many labs use aquabides (double destilata water). Some of the lab, with economic reasons, use of rain water, but this cause is difficult to control the content of organic matter and non-organic media.8. Selection of Media. If there is no initial information, usually starting with MS medium (Murashige and Skoog 1962). This medium contains salt and nitrate concentrations are higher than other media, and has been successfully used on various crops dikotil.III. Practical MethodsA. Tools and MaterialsEquipment used in pickle making this medium is magnetic Stirrer, analytical scales, Erlenmeyer tubes, measuring cups, beakers, pipettes macro, micro pipettes and pipette drops, stirrer, heating stoves, pH meter, culture bottles, autoclave, aluminum foil, plastic seal. While the materials used include MS media containing elements of the macro, micro elements, iron, vitamins, ZPT of Kinetin and IAA, a compactor material for "Swallow", Sucrose, 1M KOH or NaOH, and 1M HCL.B. Work ProceduresA. Preparation of stock solutionsa. A stock solution is a macro nutrient solution, made 10 times dissolved in 1000 ml distilled water.b. Stock solution B is a micro nutrient solution, made 1000 times dissolved in 100 ml distilled water.c. C stock solution was mixed and Na2-EDTA FeSO4.7H2O. Made 100 times and dissolved into 200 ml distilled water.d. Stock solution is a solution of vitamin D than mio-inositol, made 100 times into 200 ml of distilled water.e. Stock solution of E is a solution of mio-inositol, made 100 times and dissolved into 100 ml of distilled water.f. Stock solution F is a solution of ZPT, made 100 times into 500 ml of distilled water.2. Preparation of culture mediaa. A total of 500 ml distilled water was prepared in 1000 ml Erlenmeyer size. stock solution A, B, C, D, E, and F incorporated into the Erlenmeyer as required. For the manufacture of medium 1L, then stock A is taken as 100 ml, 5 ml of stock B is taken, taken stock of C 5 ml, 50 ml stock of D is taken, stock auxin and cytokinin E for each 1 ml. arbitrarily materials are mixed until uniform.b. b.30 gr sucrosa added into the measuring cup and let it homogeneous.c. Distilled water was added into the measuring cup until its volume reached 1000 ml.d. d.pH media was measured with pH meter by inserting the sensor into the solution media. If less than 5.6 to 5.8 was added a solution of NaOH, and if more is added a solution of HCL.e. Each solution was cooked medium with a stove and add 350 grams of agar-agar.f. Media is inserted into the culture bottles Approximately 2-3 cm high, then the culture bottle was closed again.3. media sterilizationa. bottles already containing medium, put into the autoclave to be sterilized at a temperature of 1210C with a pressure of 15 to 17.5 psi for 20 minutes.b. After completion of sterilization, the bottle kept the room cool.IV. OBSERVATION RESULTS AND DISCUSSIONA. Observations
Murashige and Skoog solid medium.B. DiscussionPreparation of media should be based on the calculation of the exact concentration. Because it will affect the success of growing explants. The medium used was MS medium (Murashige and Skoog). In the manufacturing process, the macro elements were diluted by 5 times, 100 times the micro elements, Fe stock 200 times, 10 times the vitamin, ZPT 100 times. Ditambakan sucrose which also aims to provide raw materials for the metabolism of explants explants asimilat does not yet capable of producing such plants in general. A compactor is then added to "swallow" to solidify the media.Laritan stock made with dissolved Fe using absolute alcohol or 90-96% alcohol. Because the materials used for membuatan laruten poorly soluble Fe stock. Or to facilitate can also be burned or heated.Preparation of stock solutions is basically intended to provide the materials needed to manufacture the media with the appropriate concentration. Because the media used in tissue culture with the necessary elements at very low concentrations. Because it is not possible to weigh the elements with very small concentrations, the stock solution was prepared by using the concept of calibration, so that in making the media, these elements can be used the same age as the desired concentration (Sriyanti, 2002).As with any culture equipment, media used is also necessary to sterilization to create an aseptic environment for the explants. By Anonymous (2009) there are several ways of sterilization media are:A. Media Using a Portable autoclave sterilization, the procedure can be performed with sebaga following:(Heating using heat)A. Fill outer pan with water, if possible with distilled water to avoid precipitation of Ca are commonly found in tap water, as much as 1 liter for a small autoclave, and 1.5-liter autoclave for a large2. Media bottles to be sterilized, put in a panel-in. Arrange the bottles to reach the surface of the panel.3. Set the pan position with respect to the flow channel where the steam contained in the lid and the outer circular surface of the pan-4. Close tightly. (Tighten the lock without using tools)5. Let the steam valve in the open state spending.6. Place the autoclave on the gas stove or a Bunsen burner.7. Heat to boiling water in the autoclave and the steam coming out of the steam valve spending.8. Let the steam out for 5 minutes (minimum), to remove trapped air out the air in the autoclave.9. Close the steam valve expenditure.10. Observe the increase in temperature and pressure.11. Once the pressure reaches 15 psi, the flame diminished.12. Keep the pressure of 15 psi this condition by adjusting the size of the burner manually. During sterilization, do not leave the autoclave and the other room doing something else, because the pressure can be increased up to cross the line. This situation is dangerous and can cause damage to equipment.13. After the sterilization time is reached, turn off the stove.14. Steam released bit by bit by adjusting the steam valve expenses (open a little). Never open the valve and let the steam out at once. This condition causes the media or the water bubble up.15. Once the pressure drops to 0, open the lock and remove the pan containing the media.B. Using distilled water sterilization and autoclave Media System (Digital or Non-Digital)In distilled water sterilization, is more effective when used in containers having a volume between 300-500 ml. Fill the container to 80% by volume, and cover with paper, and fasten with a rubber band. Media sterilized in an autoclave. To distilled water should be included in a small container such as erlemeyer 250 ml with a maximum content of 100 ml, so that more effective sterilization. Sterilization time equal to the time for the sterilization of equipment within 30 minutes at a pressure of 15 psi. or 1 atm. For culture media that do not contain ingredients that heat-labile, with autoclave sterilization performed on 121Oc temperature, pressure of 15 psi or 1 atm with a time of between 20-25 minutes depending on the volume of the container and the volume of media. To 15-50 ml of medium in test tubes or small bottles of 50-100 ml size, sterilization performed at a pressure of 15 psi with a time of 20 minutes. For 20 bottles of 1 liter volume requires a longer time is 34 minutes, 10 bottles of 2 liter volume takes 37 minutes, 5 bottles of 4 liters of the time used 52 minutes. With a longer time.In distilled water and sterilization of media, after sterilization of the desired time is reached, the autoclave should not be abruptly reduced pressure. When the pressure suddenly lowered, the liquid inside to boil and overflow (bubbled up). For materials that are heat-labile in solution, sterilization is done by filtering the solution through a filter having a pore size of um 0:20 to 0:22. Diameter filters vary depending on the volume of solution to be sterilized. To a solution of 10 ml volume, used filter mounted on the tip of the syringe. Heat labile materials such as: GA3, Thiamin-HCL, Ca-panthothenate, Antibiotics: carbenocilin (Anonimous, 2009).V. CONCLUSIONBased on observations, it can be concluded that:A. Manufacturing performed by diluting the stock solution using distilled water. macro elements were diluted by 5 times, 100 times the micro elements, Fe stock 200 times, 10 times the vitamin, ZPT form of IAA and Kinetin 100 times.2. Preparation of medium MS carried out by mixing a stock that has been made. For the manufacture of 1L medium, then the stock of the macro element is taken as 100 ml, taken stock of the micro elements of 5 ml, 5 ml of stock Fe taken, taken 50 ml vitamin stock, stock of auxin and cytokinin ZPT for each 1 ml.3. Medium sterilizations carried out using an autoclave at 121 degrees Celsius at a pressure of 15 to 17.5 psi for 20 minutes.
In addition to tissue culture equipment, media is one major factor in the success of the culture. Plant tissue culture media should contain all the substances necessary to ensure the growth of explants grown.Tissue culture media have the characteristics of each.
This means that not all media can be used on all plant culture. Because some media have different knadungan and concentration of substances that are needed or used in the culture.
B. PurposeThis lab aims to:
Acknowledge and practice how to make a stock solution
Knowing and practicing how to make MS medium (Murashige and Skoog)
Sterilization of the mediumII. REVIEW REFERENCESPlanting medium must contain all the substances necessary to ensure the needs of the explants. These ingredients are mixed salts contain a mixture of mineral resources element of the macro and micro elements, sugar, protein, vitamins, and plant hormones. The success of many tissue culture aseptic conditions also determined other than by the growing media. A mixture of media, can be suitable for certain plants, but can be less suitable for other crops.In tissue culture, the elements are not given in the form of a pure element, but a salt-shaped compound. Before mixed into the growing medium, mineral salts, it must be more dahulul dissolved in a certain concentration, so that in future the amount of growing medium per gram is in accordance with the provisions of distilled water as the solvent used (Rahardja, 1995)To meet the plant growth factor, tissue culture media containing either (Anonimous, 2009):A. Inorganic nutrients. There are 12 essential mineral nutrients for plant growth and some nutrients that affect the reported growth in vitro. For normal growth in tissue culture, the elements - these essential elements must be included in the culture medium.2. Organic nutrients. Plants growing under normal conditions are autotrophs and can synthesize all the organic material needs. Although plants in vitro can synthesize these compounds, suggesting that they do not produce sufficient amounts of vitamins for healthy growth and one or more vitamins must be added to the media. Thiamin is an essential vitamin, besides nicotine acid, pyridoxine and inositol is usually added.3. Carbon source. Plants grown in tissue culture heterotrophs and because they are not sufficient to synthesize the needs of its carbon, the sucrose must be added to the media. Carbon source provides energy for plant growth as well as the building blocks to produce larger molecules needed for growth.4. Order. Networks generally cultured on solid media such as a gel made by using the order or substitute for Gelrite or Phytagel sperti. Concentration used to range between 0.7 - 1.0%. At high concentrations to be very loud, very little water available, so the diffusion of nutrients to the plants is very bad. To overcome this problem, new products have been manufactured ole bernaman Agargel Sigma. This product is a mixture of agar and synthetic gels and offers the advantages of both products while reducing the problems of vitrification. This product can be created in the lab by mixing 1 g Gelrite (Phytagel) with 4 g of order as a thickening agent for 1 L media.5. pH. media are usually set in the range 5.6 - 5.8 but different plants may require a different pH for optimum growth. If the pH is higher than 6.0, media may be too hard and if the pH is less than 5.2, so as not to be compact.6. Growing an organizing principle. In the media generally added growth regulators. Plant growth regulators will be discussed separately at week 13.7. Water. distilata typically used in tissue culture, and many labs use aquabides (double destilata water). Some of the lab, with economic reasons, use of rain water, but this cause is difficult to control the content of organic matter and non-organic media.8. Selection of Media. If there is no initial information, usually starting with MS medium (Murashige and Skoog 1962). This medium contains salt and nitrate concentrations are higher than other media, and has been successfully used on various crops dikotil.III. Practical MethodsA. Tools and MaterialsEquipment used in pickle making this medium is magnetic Stirrer, analytical scales, Erlenmeyer tubes, measuring cups, beakers, pipettes macro, micro pipettes and pipette drops, stirrer, heating stoves, pH meter, culture bottles, autoclave, aluminum foil, plastic seal. While the materials used include MS media containing elements of the macro, micro elements, iron, vitamins, ZPT of Kinetin and IAA, a compactor material for "Swallow", Sucrose, 1M KOH or NaOH, and 1M HCL.B. Work ProceduresA. Preparation of stock solutionsa. A stock solution is a macro nutrient solution, made 10 times dissolved in 1000 ml distilled water.b. Stock solution B is a micro nutrient solution, made 1000 times dissolved in 100 ml distilled water.c. C stock solution was mixed and Na2-EDTA FeSO4.7H2O. Made 100 times and dissolved into 200 ml distilled water.d. Stock solution is a solution of vitamin D than mio-inositol, made 100 times into 200 ml of distilled water.e. Stock solution of E is a solution of mio-inositol, made 100 times and dissolved into 100 ml of distilled water.f. Stock solution F is a solution of ZPT, made 100 times into 500 ml of distilled water.2. Preparation of culture mediaa. A total of 500 ml distilled water was prepared in 1000 ml Erlenmeyer size. stock solution A, B, C, D, E, and F incorporated into the Erlenmeyer as required. For the manufacture of medium 1L, then stock A is taken as 100 ml, 5 ml of stock B is taken, taken stock of C 5 ml, 50 ml stock of D is taken, stock auxin and cytokinin E for each 1 ml. arbitrarily materials are mixed until uniform.b. b.30 gr sucrosa added into the measuring cup and let it homogeneous.c. Distilled water was added into the measuring cup until its volume reached 1000 ml.d. d.pH media was measured with pH meter by inserting the sensor into the solution media. If less than 5.6 to 5.8 was added a solution of NaOH, and if more is added a solution of HCL.e. Each solution was cooked medium with a stove and add 350 grams of agar-agar.f. Media is inserted into the culture bottles Approximately 2-3 cm high, then the culture bottle was closed again.3. media sterilizationa. bottles already containing medium, put into the autoclave to be sterilized at a temperature of 1210C with a pressure of 15 to 17.5 psi for 20 minutes.b. After completion of sterilization, the bottle kept the room cool.IV. OBSERVATION RESULTS AND DISCUSSIONA. Observations
Murashige and Skoog solid medium.B. DiscussionPreparation of media should be based on the calculation of the exact concentration. Because it will affect the success of growing explants. The medium used was MS medium (Murashige and Skoog). In the manufacturing process, the macro elements were diluted by 5 times, 100 times the micro elements, Fe stock 200 times, 10 times the vitamin, ZPT 100 times. Ditambakan sucrose which also aims to provide raw materials for the metabolism of explants explants asimilat does not yet capable of producing such plants in general. A compactor is then added to "swallow" to solidify the media.Laritan stock made with dissolved Fe using absolute alcohol or 90-96% alcohol. Because the materials used for membuatan laruten poorly soluble Fe stock. Or to facilitate can also be burned or heated.Preparation of stock solutions is basically intended to provide the materials needed to manufacture the media with the appropriate concentration. Because the media used in tissue culture with the necessary elements at very low concentrations. Because it is not possible to weigh the elements with very small concentrations, the stock solution was prepared by using the concept of calibration, so that in making the media, these elements can be used the same age as the desired concentration (Sriyanti, 2002).As with any culture equipment, media used is also necessary to sterilization to create an aseptic environment for the explants. By Anonymous (2009) there are several ways of sterilization media are:A. Media Using a Portable autoclave sterilization, the procedure can be performed with sebaga following:(Heating using heat)A. Fill outer pan with water, if possible with distilled water to avoid precipitation of Ca are commonly found in tap water, as much as 1 liter for a small autoclave, and 1.5-liter autoclave for a large2. Media bottles to be sterilized, put in a panel-in. Arrange the bottles to reach the surface of the panel.3. Set the pan position with respect to the flow channel where the steam contained in the lid and the outer circular surface of the pan-4. Close tightly. (Tighten the lock without using tools)5. Let the steam valve in the open state spending.6. Place the autoclave on the gas stove or a Bunsen burner.7. Heat to boiling water in the autoclave and the steam coming out of the steam valve spending.8. Let the steam out for 5 minutes (minimum), to remove trapped air out the air in the autoclave.9. Close the steam valve expenditure.10. Observe the increase in temperature and pressure.11. Once the pressure reaches 15 psi, the flame diminished.12. Keep the pressure of 15 psi this condition by adjusting the size of the burner manually. During sterilization, do not leave the autoclave and the other room doing something else, because the pressure can be increased up to cross the line. This situation is dangerous and can cause damage to equipment.13. After the sterilization time is reached, turn off the stove.14. Steam released bit by bit by adjusting the steam valve expenses (open a little). Never open the valve and let the steam out at once. This condition causes the media or the water bubble up.15. Once the pressure drops to 0, open the lock and remove the pan containing the media.B. Using distilled water sterilization and autoclave Media System (Digital or Non-Digital)In distilled water sterilization, is more effective when used in containers having a volume between 300-500 ml. Fill the container to 80% by volume, and cover with paper, and fasten with a rubber band. Media sterilized in an autoclave. To distilled water should be included in a small container such as erlemeyer 250 ml with a maximum content of 100 ml, so that more effective sterilization. Sterilization time equal to the time for the sterilization of equipment within 30 minutes at a pressure of 15 psi. or 1 atm. For culture media that do not contain ingredients that heat-labile, with autoclave sterilization performed on 121Oc temperature, pressure of 15 psi or 1 atm with a time of between 20-25 minutes depending on the volume of the container and the volume of media. To 15-50 ml of medium in test tubes or small bottles of 50-100 ml size, sterilization performed at a pressure of 15 psi with a time of 20 minutes. For 20 bottles of 1 liter volume requires a longer time is 34 minutes, 10 bottles of 2 liter volume takes 37 minutes, 5 bottles of 4 liters of the time used 52 minutes. With a longer time.In distilled water and sterilization of media, after sterilization of the desired time is reached, the autoclave should not be abruptly reduced pressure. When the pressure suddenly lowered, the liquid inside to boil and overflow (bubbled up). For materials that are heat-labile in solution, sterilization is done by filtering the solution through a filter having a pore size of um 0:20 to 0:22. Diameter filters vary depending on the volume of solution to be sterilized. To a solution of 10 ml volume, used filter mounted on the tip of the syringe. Heat labile materials such as: GA3, Thiamin-HCL, Ca-panthothenate, Antibiotics: carbenocilin (Anonimous, 2009).V. CONCLUSIONBased on observations, it can be concluded that:A. Manufacturing performed by diluting the stock solution using distilled water. macro elements were diluted by 5 times, 100 times the micro elements, Fe stock 200 times, 10 times the vitamin, ZPT form of IAA and Kinetin 100 times.2. Preparation of medium MS carried out by mixing a stock that has been made. For the manufacture of 1L medium, then the stock of the macro element is taken as 100 ml, taken stock of the micro elements of 5 ml, 5 ml of stock Fe taken, taken 50 ml vitamin stock, stock of auxin and cytokinin ZPT for each 1 ml.3. Medium sterilizations carried out using an autoclave at 121 degrees Celsius at a pressure of 15 to 17.5 psi for 20 minutes.
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