ACTIVITY 1
Sterilization of equipment
I. INTRODUCTION
A. Background
Tissue culture is a method to isolate parts of plants such as protoplasm, cells, a group of cells, tissues or organs, as well as growing them in an aseptic, so that these parts can multiply and regenerate a whole plant back (Sany, 2007).
The initial concept of culture is learned kemempuan jarngan totipotensi of plant cells. Totipotensi cells (Total Genetic Potential), meaning that each cell has the genetic potential of such zygotes are able to reproduce themselves and berediferensiasi into complete plants. Aseptic environment as one of the main conditions of tissue culture success of the activities to be implemented in
earnest. For businesses that need the sterilization of equipment that will be used in the culture process.Not limited to equipment, but the room must be used in aseptic conditions. The main purpose of the sterilization room and the equipment is basically culture to avoid contamination by micro-organisms that exist in the equipment or in the air around the room. The treatment is to be conducted at room terutapa sower or place used for the cultivation of the explants.B. PurposeThe purpose of this event is to improve student skills in performing sterilization equipment using an autoclave.II. REVIEW REFERENCESAs a prerequisite to success of plant tissue culture, usually done by using autoclave sterilization. Even autoclave sterilization can also be used to grow tissue culture media. Type that can be used for autoclave sterilization is very diverse kinds, ranging from simple to digital (programmable) (Gunawan, 1988).Simple autoclave uses steam from the heating source of water was added to the autoclave. Water heating can use a stove or a Bunsen flame. With this simple autoclave, pressure and temperature is set by the amount of heat from the fire. The downside of this autoclave is that the need for preservation and heat settings and manually controlled, during the sterilization done. But the autoclave has the advantage, namely: simplified simple, relatively inexpensive price, regardless of power flow is often a problem for countries that are developing, as well as autoclave faster than the size and comparable power.Autoclave a more complete use of electrical energy sources. Appliance is equipped with a timer and thermostat. When the automatic regulator is going well. Then the autoclave can be run while doing other work. The disadvantage is that if one controller does not work, then the media preparatory work to be futile and possibly lead to total failure in the autoclave. As a source of steam, also derived from water is added into the autoclave and bring to a boil.Usually for commercial laboratories, according to Gunawan (1988), required a large capacity autoclave with steam and the source is usually from a separate boiler. Autoclave is very fast and can diprogam sterilization time and cooling time. After sterilization of materials or equipment is completed, the autoclave temperature and pressure is slowly reduced within 15-20 minutes. On a programmable autoclave (a program that can be arranged), the heat is regulated by atomatis. But on this simple autoclave must be set manually.III. Practical MethodsA. Tools and Materials
Materials and equipment used at the lab include an autoclave, as well as gas stove, utensils glass / Glass ware (such as culture bottles, Erlenmeyer, petridish, glass cup), planting tools / dissecting kit (such as tweezers, scalpel), aluminum foil, paying paper, rubber bands, paper straw, paper wrappers, plastic seal.B. Work ProceduresA. Glass ware and dissesting kit washed with soap, rinsed with water and then dried. Set; ah dry mouth bottle covered with aluminum foil and seal with plastic dieratkan (plastic seal). Tweezers and a scalpel wrapped in aluminum foil.2. Glass ware and sterilized by autoclaving dissesting kit at a temperature of 120oC at a pressure of 17.5 psi for 30 minutes.3. During the sterilization process takes place, the autoclave was closed tightly so that the pressure within the autoclave increased. High pressure was maintained for 30 minutes with heat shrink, conducted over three times.4. The stove is turned off after the sterilization process is complete the valve is opened to remove the water vapor pressure to 0 psi.5. Autoclave was opened and the sterilized equipment was taken.6. Equipment is stored in a clean place.IV. OBSERVATION RESULTS AND DISCUSSIONA. Observations
Some equipment is sterilized using an autoclave are:A. Petridish: used as a groundbreaking piece of explants in the LAF before planting.2. Culture bottles: bottles used as explants planted where.3. Erlenmeyer: used to store the stock solution.4. Scalpel: used to cut explants.5. Tweezers: used for explant menjapit6. Aluminum Foil: used to close the bottle culture and to protect stock solution of direct sunlight.B. DiscussionThe tools used must be in a sterile condition. Because the conditions that will determine success or failure seteril a tissue culture activities. Because if the conditions are not sterile, it will be susceptible to contamination, so the ability of the cells is inhibited ttipotensi. Metal tools and glass are used at the time of planting can be sterilized in an autoclave. Cropping tools such as tweezers and scissors can also be sterilized by burning or by heating in a Bunsen burner or bacticinerator.By Anonymous (2009), specifically for the scalpel, the handle can be sterilized by heating, but the blade (blade) can be dull when heated in a high temperature. It is therefore recommended for the blade to sterilization by immersion in alcohol or chlorine solution.In principle, using autoclave sterilization of heat and pressure of water vapor. Temperature sterilasi typically 1210C, pressures between 15 to 17.5 commonly used psi (pounds per square inch) or 1 atm. The duration of sterilization depends on the volume and type. Tools and sterilized water for 1 hour, but the media between 20-40 minutes depending on the volume of sterilized materials.Sterilization of media that are too long can cause:A. Sugars.2. Degradation of vitamins and amino acids.3. Inactivation of cytokinin zeatin riboside.4. Depolymerization berakibatkan pH change that.Autoclave portable gas or electricity in general use steam from the heating source of water is added into the autoclave, while the large autoclave at commercial laboratories generally use steam from a central boiler.Parts of the autoclave:A. Pan out.2. Put the pan in place the bottle with a groove where the steam channel.3. Close the bookmark along with pressure and steam vents.4. Vaporization valve.5. Locking or clamping.Culture equipment sterilizationA. Clean bottles are given a few drops of distilled water and cover with paper or aluminum foil (not too tight when using aluminum foil). For bottles that have a lid that autoclaveable, do not close too fast, because the expansion occurs during heating.2. Tools that need to be sterilized before planting are: tweezers, scissors, scalpel handles, filter paper, petri-dish, empty bottles, needles and pipettes.3. These tools and the filter paper wrapped with heavy paper or placed in a stainless steel tray and the tray covered with a thick cloth before being put in an autoclave. Aluminum foil is not recommended as a wrapper, because the steam can not enter into a bundle. Sektio tools such as tweezers, scissors, scalpel handle and needle, wrapped in paper or a paper coffee straw. Avoid the use of Al-foil as vapor into the package so hard to be less effective sterilization.4. Petri-dish to be sterilized, well wrapped in paper or a paper coffee straw.5. Temperatures used for sterilization of the culture bottle is empty and the tools that will be used to plant explants, was 1210C at a pressure of 15 psi (pounds per square inch) or 1 atm for 30-60 minutes. Sterilization timing begins after the desired pressure and temperature is reached.V. CONCLUSIONBased on data from observation, it can be concluded that the culture of sterilization equipment is done by using an autoclave at a temperature of 1210C at a pressure of 15 to 17.5 psi. for 30 minutes. Some of the equipment in use autoclave sterilization, among others, the culture bottles, petridish, Erlenmeyer, aluminum foil, a scalpel, and tweezers.
Tissue culture is a method to isolate parts of plants such as protoplasm, cells, a group of cells, tissues or organs, as well as growing them in an aseptic, so that these parts can multiply and regenerate a whole plant back (Sany, 2007).
The initial concept of culture is learned kemempuan jarngan totipotensi of plant cells. Totipotensi cells (Total Genetic Potential), meaning that each cell has the genetic potential of such zygotes are able to reproduce themselves and berediferensiasi into complete plants. Aseptic environment as one of the main conditions of tissue culture success of the activities to be implemented in
earnest. For businesses that need the sterilization of equipment that will be used in the culture process.Not limited to equipment, but the room must be used in aseptic conditions. The main purpose of the sterilization room and the equipment is basically culture to avoid contamination by micro-organisms that exist in the equipment or in the air around the room. The treatment is to be conducted at room terutapa sower or place used for the cultivation of the explants.B. PurposeThe purpose of this event is to improve student skills in performing sterilization equipment using an autoclave.II. REVIEW REFERENCESAs a prerequisite to success of plant tissue culture, usually done by using autoclave sterilization. Even autoclave sterilization can also be used to grow tissue culture media. Type that can be used for autoclave sterilization is very diverse kinds, ranging from simple to digital (programmable) (Gunawan, 1988).Simple autoclave uses steam from the heating source of water was added to the autoclave. Water heating can use a stove or a Bunsen flame. With this simple autoclave, pressure and temperature is set by the amount of heat from the fire. The downside of this autoclave is that the need for preservation and heat settings and manually controlled, during the sterilization done. But the autoclave has the advantage, namely: simplified simple, relatively inexpensive price, regardless of power flow is often a problem for countries that are developing, as well as autoclave faster than the size and comparable power.Autoclave a more complete use of electrical energy sources. Appliance is equipped with a timer and thermostat. When the automatic regulator is going well. Then the autoclave can be run while doing other work. The disadvantage is that if one controller does not work, then the media preparatory work to be futile and possibly lead to total failure in the autoclave. As a source of steam, also derived from water is added into the autoclave and bring to a boil.Usually for commercial laboratories, according to Gunawan (1988), required a large capacity autoclave with steam and the source is usually from a separate boiler. Autoclave is very fast and can diprogam sterilization time and cooling time. After sterilization of materials or equipment is completed, the autoclave temperature and pressure is slowly reduced within 15-20 minutes. On a programmable autoclave (a program that can be arranged), the heat is regulated by atomatis. But on this simple autoclave must be set manually.III. Practical MethodsA. Tools and Materials
Materials and equipment used at the lab include an autoclave, as well as gas stove, utensils glass / Glass ware (such as culture bottles, Erlenmeyer, petridish, glass cup), planting tools / dissecting kit (such as tweezers, scalpel), aluminum foil, paying paper, rubber bands, paper straw, paper wrappers, plastic seal.B. Work ProceduresA. Glass ware and dissesting kit washed with soap, rinsed with water and then dried. Set; ah dry mouth bottle covered with aluminum foil and seal with plastic dieratkan (plastic seal). Tweezers and a scalpel wrapped in aluminum foil.2. Glass ware and sterilized by autoclaving dissesting kit at a temperature of 120oC at a pressure of 17.5 psi for 30 minutes.3. During the sterilization process takes place, the autoclave was closed tightly so that the pressure within the autoclave increased. High pressure was maintained for 30 minutes with heat shrink, conducted over three times.4. The stove is turned off after the sterilization process is complete the valve is opened to remove the water vapor pressure to 0 psi.5. Autoclave was opened and the sterilized equipment was taken.6. Equipment is stored in a clean place.IV. OBSERVATION RESULTS AND DISCUSSIONA. Observations
Some equipment is sterilized using an autoclave are:A. Petridish: used as a groundbreaking piece of explants in the LAF before planting.2. Culture bottles: bottles used as explants planted where.3. Erlenmeyer: used to store the stock solution.4. Scalpel: used to cut explants.5. Tweezers: used for explant menjapit6. Aluminum Foil: used to close the bottle culture and to protect stock solution of direct sunlight.B. DiscussionThe tools used must be in a sterile condition. Because the conditions that will determine success or failure seteril a tissue culture activities. Because if the conditions are not sterile, it will be susceptible to contamination, so the ability of the cells is inhibited ttipotensi. Metal tools and glass are used at the time of planting can be sterilized in an autoclave. Cropping tools such as tweezers and scissors can also be sterilized by burning or by heating in a Bunsen burner or bacticinerator.By Anonymous (2009), specifically for the scalpel, the handle can be sterilized by heating, but the blade (blade) can be dull when heated in a high temperature. It is therefore recommended for the blade to sterilization by immersion in alcohol or chlorine solution.In principle, using autoclave sterilization of heat and pressure of water vapor. Temperature sterilasi typically 1210C, pressures between 15 to 17.5 commonly used psi (pounds per square inch) or 1 atm. The duration of sterilization depends on the volume and type. Tools and sterilized water for 1 hour, but the media between 20-40 minutes depending on the volume of sterilized materials.Sterilization of media that are too long can cause:A. Sugars.2. Degradation of vitamins and amino acids.3. Inactivation of cytokinin zeatin riboside.4. Depolymerization berakibatkan pH change that.Autoclave portable gas or electricity in general use steam from the heating source of water is added into the autoclave, while the large autoclave at commercial laboratories generally use steam from a central boiler.Parts of the autoclave:A. Pan out.2. Put the pan in place the bottle with a groove where the steam channel.3. Close the bookmark along with pressure and steam vents.4. Vaporization valve.5. Locking or clamping.Culture equipment sterilizationA. Clean bottles are given a few drops of distilled water and cover with paper or aluminum foil (not too tight when using aluminum foil). For bottles that have a lid that autoclaveable, do not close too fast, because the expansion occurs during heating.2. Tools that need to be sterilized before planting are: tweezers, scissors, scalpel handles, filter paper, petri-dish, empty bottles, needles and pipettes.3. These tools and the filter paper wrapped with heavy paper or placed in a stainless steel tray and the tray covered with a thick cloth before being put in an autoclave. Aluminum foil is not recommended as a wrapper, because the steam can not enter into a bundle. Sektio tools such as tweezers, scissors, scalpel handle and needle, wrapped in paper or a paper coffee straw. Avoid the use of Al-foil as vapor into the package so hard to be less effective sterilization.4. Petri-dish to be sterilized, well wrapped in paper or a paper coffee straw.5. Temperatures used for sterilization of the culture bottle is empty and the tools that will be used to plant explants, was 1210C at a pressure of 15 psi (pounds per square inch) or 1 atm for 30-60 minutes. Sterilization timing begins after the desired pressure and temperature is reached.V. CONCLUSIONBased on data from observation, it can be concluded that the culture of sterilization equipment is done by using an autoclave at a temperature of 1210C at a pressure of 15 to 17.5 psi. for 30 minutes. Some of the equipment in use autoclave sterilization, among others, the culture bottles, petridish, Erlenmeyer, aluminum foil, a scalpel, and tweezers.
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