0 evaluări0% au considerat acest document util (0 voturi)
63 vizualizări11 pagini
This document discusses sterile (aseptic) technique which is necessary for successful plant cell, tissue and organ cultures. The main points are:
1. Contamination from microorganisms like bacteria, fungi and insects is a major risk for plant cultures. Proper aseptic technique and environmental controls are needed to minimize contamination.
2. Common sources of contamination include microbes on plant materials, airborne spores, improper sterilization, and dirty lab conditions. Contamination can kill cultures or spread between them.
3. Maintaining sterile conditions requires sterilizing equipment, media, and supplies through autoclaving or filtration. Laminar flow hoods provide sterile air. Careful technique and monitoring are
This document discusses sterile (aseptic) technique which is necessary for successful plant cell, tissue and organ cultures. The main points are:
1. Contamination from microorganisms like bacteria, fungi and insects is a major risk for plant cultures. Proper aseptic technique and environmental controls are needed to minimize contamination.
2. Common sources of contamination include microbes on plant materials, airborne spores, improper sterilization, and dirty lab conditions. Contamination can kill cultures or spread between them.
3. Maintaining sterile conditions requires sterilizing equipment, media, and supplies through autoclaving or filtration. Laminar flow hoods provide sterile air. Careful technique and monitoring are
This document discusses sterile (aseptic) technique which is necessary for successful plant cell, tissue and organ cultures. The main points are:
1. Contamination from microorganisms like bacteria, fungi and insects is a major risk for plant cultures. Proper aseptic technique and environmental controls are needed to minimize contamination.
2. Common sources of contamination include microbes on plant materials, airborne spores, improper sterilization, and dirty lab conditions. Contamination can kill cultures or spread between them.
3. Maintaining sterile conditions requires sterilizing equipment, media, and supplies through autoclaving or filtration. Laminar flow hoods provide sterile air. Careful technique and monitoring are
Aseptic technique is absolutely necessary for the successful establishment and
maintenance of plant cell, tissue and organ cultures. The in vitro environment in which the plant material is grown is also ideal for the proliferation of microorganisms. In most cases the microorganisms outgrow the plant tissues, resulting in their death. Contamination can also spread from culture to culture. The purpose of aseptic technique is minimize the possibility that microorganisms remain in or enter the cultures. The environmental control of air is also of concern because room air may be highly contaminated. Eample! "neezing produces #$$,$$$ % &$$,$$$ aerosol droplets which can then attach to dust particles. These contaminated particles may be present in the air for wee's. ()ave you ever viewed the air around you when you open the curtains on a sunny day*+. Air may also contain bacterial and fungal spores, as do we. I. Contaminants A. Bacteria, fungi, and insects 1. Bacteria ,acteria are the most frequent contaminants. They are usually introduced with the eplant and may survive surface sterilization of the eplant because they are in interior tissues. "o, bacterial contamination can first become apparent long after a culture has been initiated (see below+. "ome bacterial spores can also survive the sterilization procedure even if they are on the tissue surface. -any 'inds of bacteria have been found in plant tissue cultures including Agrobacterium, Bacillus, Corynebacterium, Enterobacter, Lactobacillus, Pseudomanas, Staphylococcus, and Xanthomonas. ,acteria can be recognized by a characteristic .ooze./ the ooze can be many colors including white, cream, pin', and yellow. There is also often a distinctive odor. 2. Fungi 0ungi may enter cultures on eplants or spores may be airborne. 0ungi are frequently present as plant pathogens and in soil. They may be recognized by their .fuzzy. appearance, and occur in a multitude of colors. 3. Yeast 1east is a common contaminant of plant cultures. 1easts live on the eternal surfaces of plants and are often present in the air. 4. iruses, etc. 2iruses, mycoplasma%li'e organisms, spiroplasmas, and ric'ettsias are etremely small organisms that are not easily detected. Thus, plant culture is not necessarily pathogen%free even if microorganisms are not detected, and this can influence culture success. "pecial measures such as meristem culture are often necessary to eradicate such contaminants. !. "nsects The insects that are most troublesome in plant cultures include ants, thrips, and mites. Thrips often enter cultures as eggs present on the eplants. Ants and mites, however, usually infest already established cultures. -ites feed on fungus and mite infestations are often first detected by observing lines of fungal infection that lead from the edge of the culture vessel to the plant tissue, having been introduced by the insect. It is very difficult to eradicate insect infestations. Careful lab practices and cleanliness should prevent most infestations. B. "nitial #$nta%inants -ost contamination is introduced with the eplant because of inadequate sterilization or 3ust very dirty material. It can be fungal or bacterial. This 'ind of contamination can be a very difficult problem when the plant eplant material is harvested from the field or greenhouse. Initial contamination is obvious within a few days after cultures are initiated. ,acteria produce 4ooze5 on solid medium and turbidity in liquid cultures. 0ungi loo' 4furry5 on solid medium and often accumulate in little balls in liquid medium. #. &atent #$nta%inati$n This 'ind of contamination is usually bacterial and is often observed long after cultures are initiated. Apparently the bacteria are present endogenously in the initial plant material and are not obviously pathogenic in situ. 6nce in vitro, however, they increase in titer and overrun the cultures. 7atent contamination is particularly dangerous because it can easily be transferred among cultures. '. "ntr$duced #$nta%inati$n Contamination can also occur as a result of poor sterile technique or dirty lab conditions. This 'ind of contamination is largely preventable with proper care. (. 'etecti$n $f #$nta%inants Contamination is usually detected by the .eyeball. method in research labs. )owever, indeing is possible, and is frequently done in commercial settings. This involves ta'ing a part of the plant tissue and culturing it in media that are specific for bacteria and fungi. -edia that have been used for this purpose include 89A (potato detrose agar+ and :, broth (with salts, yeast etract and glucose+. This is the most reliable method for detecting bacteria and fungi, but, as indicated above, there may be infecting organisms that won;t be detected. II. The Transfer Hood 7aminar airflow hoods are used in commercial and research tissue culture settings. A horizontal laminar flow unit is designed to remove particles from the air. <oom air is pulled into the unit and pushed through a )E8A ()igh Energy 8article Air+ filter with a uniform velocity of =$ ft>min across the wor' surface. The air is filtered by a )E8A (high efficiency particulate air+ filter so nothing larger than $.? micrometer, which includes bacterial and fungal spores, can pass through. This renders the air sterile. The positive pressure of the air flow from the unit also discourages any fungal spores or bacteria from entering. 9epending on the design of the hood, the filters are located at the bac' or in the top of the bo. III. Sterilization and Use of Supplies and Equipment: A. Sterili)ing t$$ls, %edia, *essels etc. 1. Aut$cla*ing Autoclaving is the method most often used for sterilizing heat%resistant items and our usual method for sterilizing items. In order to be sterilized, the item must be held at #&#C, #@ psi, for at least #@ minutes. It is important that items reach this temperature before timing begins. Therefore time in the autoclave will vary, depending on volume in individual vessels and number of vessels in the autoclave. -ost autoclaves automatically ad3ust time when temperature and psi are set, and include time in the cycle for a slow decrease in pressure. There are tape indicators that can be affied to vessels, but they may not reflect the temperature of liquid within them. There are also 4test 'its5 of microorganisms that can be run through the autoclave cycle and then cultured. Empty vessels, bea'ers, graduated cylinders, etc., should be closed with a cap or aluminum foil. Tools should also be wrapped in foil or paper or put in a covered sterilization tray. It is critical that the steam penetrate the items in order for sterilization to be successful. 2. Aut$cla*ing and Filter+sterili)ing ,edia and -ther &iquids Two methods (autoclaving and membrane filtration under positive pressure+ are commonly used to sterilize culture media. Culture media, distilled water, and other heat stable mitures can be autoclaved in glass containers that are sealed with cotton plugs, aluminum foil, or plastic closures. )owever, solutions that contain heat%labile components must be filter%sterilized. 0or small volumes of liquids (#$$ ml or less+, the time required for autoclaving is #@%&$ min, but for larger quantities (&%A liter+, ?$%A$ min is required to complete the cycle. The pressure should not eceed &$ psi, as higher pressures may lead to the decomposition of carbohydrates and other components of a medium. Too high temperatures or too long cycles can also result in changes in properties of the medium. 6rganic compounds such as some growth regulators, amino acids, and vitamins may be degraded during autoclaving. These compounds require filter sterilization through a $.&& Bm membrane. "everal manufacturers ma'e nitrocellulose membranes that can be sterilized by autoclaving. They are placed between sections of a filter unit and sterilized as one piece. 6ther filters (the 'ind we use+ come pre%sterilized. 7arger ones can be set over a sterile flas' and a vacuum is applied to pull the compound dissolved in liquid through the membrane and into the sterile flas'. "maller membranes fit on the end of a sterile syringe and liquid is pushed through by depressing the top of the syringe. The size of the filter selected depends on the volume of the solution to be sterilized and the components of the solution. :utrient media that contain thermo labile components are typically prepared in several steps. A solution of the heat%stable components is sterilized in the usual way by autoclaving and then cooled to ?@C%@$C C under sterile conditions. "olutions of the thermo labile components are filter%sterilized. The sterilized solutions are then combined under aseptic conditions to give the complete medium. In spite of possible degradation, however, some compounds that are thought to be heat labile are generally autoclaved if results are found to be reliable and reproducible. These compounds include A,A, IAA, I,A, 'inetin, pyridoine, &%ip and thiamine. 3. (th.lene -/ide 0as 8lastic containers that cannot be heated are sterilized commercially by ethylene oide gas. These items are sold already sterile and cannot be resterilized. Eamples of such items are plastic petri dishes, plastic centrifuge tubes etc. 4. 1 2adiati$n It is possible to use germicidal lamps to sterilize items in the transfer hood when no one is wor'ing there. De do not do this. E2 lamps should not be used when people are present because the light is damaging to eyes and s'in. 8lants left under E2 lamps will die. !. ,icr$3a*e It is also possible to sterilize items in the microwave/ we do not do this. 4. ,$re #$%%ents Fnow which of your implements, flas's, etc. are sterile and which are not. "terile things will have been autoclaved and should be wrapped with some 'ind of protective covering, e.g. foil, for transport from the autoclave to the hood. 6ur usual autoclave time of &$ minutes is intended for relatively small volumes. 7arge flas's of media, water, etc. may require longer autoclaving periods. It is preferable to put no more than one liter of liquid in a container to be autoclaved. Also, be sure to leave enough room in the container so that the liquid does not boil over. "terilized items should be used within a short time (a few days at most+. Items that come pac'aged sterile, e.g. plastic petri plates, should be eamined carefully for damage before use. If part of a pac'age is used, seal up the remainder and date and label. Ese up these items unless there is some question about their sterility/ they are epensive. De use :algene vessels for some purposes in the lab. These will melt if eposed to high heat. IV. Working in the Transfer Hood: The hoods in our lab run continuously. If for some reason one has been turned off, turn it on and let it run for at least #@ minutes before using. -a'e sure that everything needed for the wor' is in the hood and all unnecessary things are removed. As few things as possible should be stored in the hood. Chec' the bottom of the hood to ma'e sure there is no paper or other debris bloc'ing air inta'e. <emove watches, etc., roll up long sleeves, and wash hands thoroughly with soap (preferably bactericidal+ and water. "pray or wipe the inside of the transfer hood (bottom and sides, not directly on the filters+ with G$H Et6). 6thers use disinfectants such as 7ysolI. Dipe the wor' area and let the spray dry. Dipe hands and lower arms with G$H Et6). It is not necessary to flame them (This is a 3o'e.+. "pray everything going into the sterile area with G$H ethanol. 0or eample, spray bags of petri dishes with G$ H alcohol before you open them and place the desired number of unopened dishes in the sterile area. Dor' well bac' in the transfer hood (behind the line+. Especially 'eep all flas's as far bac' to the bac' of the hood as possible. -ovements in the hood should be contained to small areas. A line drawn across the distance behind which one should wor' is a useful reminder. -a'e sure that materials in use are to the side of your wor' area, so that airflow from the hood is not bloc'ed. 9on;t touch any surface that is supposed to remain sterile with your hands. Ese forceps, etc. Instruments (scalpels, forceps+ can be sterilized by flaming % dipping them in =@H Et6) and then immediately placing them in the flame of an alcohol lamp or gas burner. This can 5e danger$us if the *essel h$lding the alc$h$l tips $*er and an alc$h$l fire results. A fairly deep container, li'e a coplin%staining 3ar, should be used to hold the ethanol. Ese enough ethanol to submerge the business ends of the instruments but not so much that you burn your hands. "ome people wear gloves in the hood for certain procedures. If you do this, be very careful not to get them near the flame. 6ther methods of sterilization that do not require alcohol are with a bacticinerator or glass bead sterilizer. There is not as much ris' from fire with these, but the instruments can still get etremely hot, causing burns. Arrange tools and other items in the hood so that your hands do not have to cross over each other while wor'ing. 0or a right%handed person, it is best that sterilizing implements and tools be placed on the right. The plant material should be placed to the left. All other items in the hood should be arranged so that your wor' area is directly in front of you, and between J and #$ inches in from the front edge. :o materials should be placed between the actual wor' area and the filter. Feep as little in the hood as possible. 8lant material should be placed on a sterile surface when manipulating it in the hood. "terile petri dishes (epensive+, sterile paper towels, or sterile paper plates wor' fine. 8re%sterilized plastic dishes have two sterile surfaces%the inside top and inside bottom. "terilize your instruments often, especially in between individual petri plates, flas's, etc. The tools should be placed on a holder in the hood to cool or should be cooled by dipping in sterile water or medium before handling plant tissues. Dipe up any spills quic'ly/ use G$H Et6) for cleaning. Clean hood surface periodically while wor'ing. Ese of glass or plastic pipettes! Klass pipettes are put into containers or wrapped and then autoclaved. 8lastic pipettes are purchased presterilized in individual wrappers. To use a pipette, remove it from its wrapper or container by the end opposite the tip. 9o not touch the lower two%thirds of the pipette. 9o not allow the pipette to touch any laboratory surface. Insert only the untouched lower portion of the pipette into a sterile container. "terilize culture tubes with lids or caps on. Dhen you open a sterile tube, touch only the outside of the cap, and do not set the cap on any laboratory surface. Instead, hold the cap with one or two fingers while you complete the operation, and then replace it on the tube. This technique usually requires some practice, especially if you are simultaneously opening tubes and operating a sterile pipette. After you remove the cap from the test tube, pass the mouth of the tube through a flame. If possible, hold the open tube at an angle. 8ut only sterile ob3ects into the tube. Complete the operation as quic'ly as you reasonably can, and then flame the mouth of the tube again. <eplace the lid. Inoculating loops and needles are the primary tools for transferring microbial cultures. De use plastic ones that come sterile. If you are moving organisms from an agar plate, touch an isolated colony with the transfer loop. <eplace the plate lid. 6pen and flame the culture tube, and inoculate the medium in it by stirring the end of the transfer tool in the medium. If you are removing cells from a liquid culture, insert the loop into the culture. Even if you cannot see any liquid in the loop, there will be enough cells there to inoculate a plate or a new liquid culture. If you donLt have to be careful about the volume you transfer, a pure culture or sterile solution can be transferred to a sterile container or new sterile medium by pouring. 0or eample, we do not measure a specific volume of medium when we pour culture plates, although after you have done it for a while, you become pretty consistent. <emove the cap or lid from the solution to be transferred. Thoroughly flame the mouth of the container, holding it at an angle as you do so. <emove the lid from the target container. )old the container at an angle. Muic'ly and neatly pour the contents from the first container into the second. <eplace the lid. If you must transfer an eact volume of liquid, use a sterile pipette or a sterile graduated cylinder. Dhen using a sterile graduated cylinder, complete the transfer as quic'ly as you reasonably can to minimize the time the sterile liquid is eposed to the air. <emove items from the hood as soon as they are no longer needed. All cultures must be sealed before leaving the hood. Dhen transferring plant cultures, do contaminated cultures last. "ituate the cultures so that the contaminated part is closest to the front of the hood (so contaminants blow outward away from the interior of the hood. 8lace waste in the proper containers! Empty (e.g. after transfer+ or old petri plates used in transformation eperiments go in the big bag to be autoclaved, as do other disposables that were in contact with recombinant bacterial or plant material. All needles go in the sharps bo, needles used with bacteria get autoclaved. "mall bags used in the hood for waste go in the big bag to be autoclaved/ do not overfill the small bags or leave full bags in or on the hood for someone else to dispose of. Klassware that comes in contact with bacteria is placed in a separate pan to be autoclaved. Dhen finished in the hood, clean up after yourself. <emove all unnecessary materials and wipe the hood down with G$H Et6). It is pointless to practice good sterile technique in a dirty lab. "pecial problems are contaminated cultures, dirty dishes and solutions where microorganisms can grow. "tore cultures in a sequestered area. De will discuss this area later. Chec' cultures every ?%@ days for contamination. V. Surfae!sterilizing "lant #aterial 1. 6reparati$n $f St$c7 6lants 8rior good care of stoc' plants may lessen the amount of contamination that is present on eplants. 8lants grown in the field are typically more 4dirty5 than those grown in a greenhouse or growth chamber, particularly in humid areas li'e 0lorida. 6verhead watering increases contamination of initial eplants. 7i'ewise, splashing soil on the plant during watering will increase initial contamination. Treatment of stoc' plants with fungicides and>or bacteriocides is sometimes helpful. It is sometimes possible to harvest shoots and force buds from them in clean conditions. The forced shoots may then be free of contaminants when surface%sterilized in a normal manner. "eeds may be sterilized and germinated in vitro to provide clean material. Covering growing shoots for several days or wee's prior to harvesting tissue for culture may supply cleaner material. Eplants or material from which material will be cut can be washed in soapy water and then placed under running water for # to & hours. 2. (than$l ($r "s$pr$p.l Alc$h$l) Ethanol is a powerful sterilizing agent but also etremely phytotoic. Therefore, plant material is typically eposed to it for only seconds or minutes. The more tender the tissue, the more it will be damaged by alcohol. Tissues such as dormant buds, seeds, or unopened flower buds can be treated for longer periods of time since the tissue that will be eplanted or that will develop is actually within the structure that is being surface% sterilized. Kenerally G$H ethanol is used prior to treatment with other compounds. 3. S$diu% 8.p$chl$rite "odium hypochlorite, usually purchased as laundry bleach, is the most frequent choice for surface sterilization. It is readily available and can be diluted to proper concentrations. Commercial laundry bleach is @.&@H sodium hypochlorite. It is usually diluted to #$H % &$H of the original concentration, resulting in a final concentration of $.@ % #.$H sodium hypchlorite. 8lant material is usually immersed in this solution for #$ % &$ minutes. A balance between concentration and time must be determined empirically for each type of eplant, because of phytotoicity. 4. #alciu% 8.p$chl$rite Calcium hypochlorite is used more in Europe than in the E.". It is obtained as a powder and must be dissolved in water. The concentration that is generally used is ?.&@ H. The solution must be filtered prior to use since not all of the compound goes into solution. Calcium hypochlorite may be less in3urious to plant tissues than sodium hypochlorite. !. ,ercuric #hl$ride -ercuric chloride is used only as a last resort in the E.". It is etremely toic to both plants and humans and must be disposed of with care. "ince mercury is so phytotoic, it is critical that many rinses be used to remove all traces of the mineral from the plant material. 4. 8.dr$gen 6er$/ide The concentration of hydrogen peroide used for surface sterilization of plant material is ?$H, ten times stronger than that obtained in a pharmacy. "ome researchers have found that hydrogen peroide is useful for surface%sterilizing material while in the field. 9. (nhancing (ffecti*eness $f Sterili)ati$n 6r$cedure "urfactant (e.g.Tween &$+ is frequently added to the sodium hypochlorite. A mild vacuum may be used during the procedure. The solutions that the eplants are in are often sha'en or continuously stirred. :. 2insing After plant material is sterilized with one of the above compounds, it must be rinsed thoroughly with sterile water. Typically three to four separate rinses are done. ;. 1se $f Anti5i$tics and Fungicides in itr$ De have found that the use of antibiotics and fungicides in vitro is not very effective in eliminating microorganisms and these compounds are often quite phytotoic. ,ut there are compounds that could be further evaluated. 1<. 6lant 6reser*ati*e ,i/ture 88-N is a proprietary broad%spectrum biocide, which can be used to control contamination in plant cell cultures, either during the sterilization procedure, or as a medium component. 88-N comes in an acidic liquid solution (p) ?.J+. The recommended dose is $.@O&.$ m7 of 88-N per liter of medium. )igher doses are required to treat endogenous contamination and for Agrobacterium. Its ma'ers say that 88-N has several advantages over antibiotics! It is effective against fungi as well as bacteria, thus it can be substituted for a coc'tail of antibiotics and fungicides. 88-N is less epensive than antibiotics, which ma'es it affordable for wide and routine use. The formation of resistant mutants toward 88-N is very unli'ely because it targets and inhibits multiple enzymes. -any antibiotics adversely affect plant materials. If used as recommended, 88-N does not adversely affect in vitro seed germination, callous proliferation, or callous regeneration. "eeds and eplants with endogenous contamination can be sterilized at doses of @%&$ m7>7 of 88-N. This is useful when routine surface sterilization is insufficient.